Animals and experimental treatment
Weanling male C57BL/6J mice (3-week-old) were randomly divided into GH and SI group. Five mice (for GH group) or a single mouse (for SI group) were housed in a plexiglass cage (31 cm × 22 cm ×15 cm) for 3 weeks as the social treatments (Fig. 1a); for resocialization, two SI mice were then put in a cage containing three GH mice for 4 weeks (Fig. 2a). For virus injection, 3-week-old weanling mice were injected with indicated viruses (AAV-Egr2-eGFP/AAV-Egr2-RNAi-eGFP or AAV-Control-eGFP) in the mPFC and then underwent the social treatments (Fig. 3a, Extended Data Fig. 7a). Mice were maintained on a 12-h light/dark cycle with ambient temperature (18-22°C) and humidity (30-50%), and having free access to food and water. All experiments were conducted in accordance with international standards on animal welfare and the guidelines of the Institute for Laboratory Animal Research of Nanjing Medical University.
Mouse activity in the following behavioral test was collected using a computer-connected digital video camera (TopScan, CleverSys, Inc., Reston, VA). Room lights, except for a dim light, were switched off during each experimental session for mouse comfort. Before each test, the apparatuses were cleaned with 70% alcohol to eliminate possible remaining olfactory cues. All tests were performed by two independent experimenters, who were each blind to the treatment schedule.
The cooperative ability of mice was evaluated by a cooperative drinking device developed by our laboratory (Fig. 1b-c, Supplemental video 1). The device was plexiglass cage (60 cm × 40 cm × 35 cm) equipped with two water valves from which drop shape water could be delivered by turning on two serial photoelectric switches. During the first phase of training, named the training period, one photoelectric switch was defaulted ON; a mouse was placed into the chamber, and, in a 5-min period, it can explore and turn on the other switch to get water-rewards. Training was conducted over 7 consecutive days, with 2 trials per day. The latency for the first successful drinking, as well as the times and time of drinking during each trial were collected. During the second phase, named the testing period, both switches were defaulted OFF. Two trained mice were placed in the chamber and allowed to move freely for 10 min. Only if they turn on both switches at the same time, they can get the water-reward together. The co-drinking latency, times and time of co-drinking were recorded. The test was carried out once a day for five days. The mice were returned to their cages after each trail. To motivate mice to drink from the device, water supply was suspended for six hours before each trial. To increase their incentive for getting drinking water during the training or testing period, we made water unavailable from 6 hours before each training or testing; this short-term water deprivation had no significant effect on mice’s exploratory activities, anxiety-like behavior, spatial working memory, or weight (Extended Data Fig. 1).
The open field test was used to evaluate the locomotion and anxiety-like behaviors34. The open field consisted of a square blue box (60 cm × 60 cm × 25 cm), with an outlined center area (30 cm × 30 cm). Each mouse was placed in the center of box and allowed to move freely for 5 min within the box. The time spent in the center area, times of entering the center area and total distance traveled were calculated during the test.
Elevated plus maze test
The anxiety-like behavior was also evaluated by the elevated plus maze that was composed of four arms (50 cm ×10 cm) connected by a central square (10 cm × 10 cm) and elevated 100 cm above the floor15. Two opposite arms were open, while the remaining were closed with 40 cm high walls. The mice were placed into the close arm and allowed to freely explore the maze for 5 min. The percentage of time spent and the frequency of entries into the open arm were calculated.
The spatial working memory of mice was examined using a Y-shaped maze15. The maze consisted of three arms (8× 30× 15 cm), with an angle of 120 degrees between each arm. The procedure included 5 min-training stage and 5 min-testing stage, with an interval of 2 h. During the first stage, one arm named novel arm (NA) was blocked by a baffle, allowing the mice to move freely in the other two arms for 5 min. During the second stage, the NA was opened and mice could freely explore throughout all 3 arms for 5 min. The percentage of time traveled in the NA arm, number of entries into the NA arm were analyzed.
Fear conditioning test
The mouse was placed in the conditioning chamber for 3 min as a habituation period followed by one tone-foot-shock (tone, 30 s, 70 dB, 1 kHz; foot-shock, 2 s, 0.8 mA)35. The stimulation was given every two min within six min. The mouse was placed in the chamber again with tone-shock pairing (tone, 30 s, 70 dB, 1kHz) for 3 min after 24 h. Freezing behavior, defined as the absence of all visible movement of the body except the movement necessitated by respiration, was scored.
Total RNA from the mPFC was extracted with RNAiso Plus (Takara, #9109) according to the manufacturer’s instructions. The RNA quality and quantity were measured using NanoDrop 2000 (Thermo Scientific). RNA library was constructed using the BGISEQ-500 platform according to the manufacturer's instruction; after that, adapters were ligated to each end of the RNAs and subsequently reverse transcribed to create single-stranded cDNA and sequenced on the BGISEQ-500 platform with a read length of 50 bps. Stringent criteria were set to determine significantly dysregulated genes: adjusted p value below 0.05 and log2FC of greater than 1. The mPFC samples from SI and GH mice were analyzed as two independent datasets. Heatmaps representing z-scores were generated using the seaborn package in python.
Plasmids and Adeno-associated virus
Egr2 and the scrambled control siRNA were purchased from RiboBio (Guangzhou, China) (Supplemental Fig. 5i-j). Egr2 overexpression plasmid, pcDNA3.1 vector, PGL3-basic vector, Arc luciferase reporter plasmid were obtained from GenScript (Nanjing, China). Adeno-associated viruses (AAV9s) for Egr2 overexpression (AAV-Egr2) or RNAi (AAV-Egr2-RNAi), and the control AAV-GFP virus were purchased from Genechem (Shanghai, China).
Deeply anesthetized mice were fixed in a stereotaxic instrument with the skull surface exposed. A total of 1.5 µl of AAV-Egr2, AAV-Egr2-RNAi (1 × 1013 µg) or control virus (AAV-GFP) were infused bilaterally into the mPFC at the following coordinates: +1.5 mm anterior/posterior, -0.75 mm dorsal/ventral and ±1.0 mm medial/lateral relative to Bregma use 33-gauge syringe needles (Hamilton)36. The infusion rate was 0.2 µl/min, and the cannula was left in the place for 5 min following completion of the infusion. Mice were allowed to recover for 2 days before isolated housing. Behavioral experiments were performed 21 days after the injection. Following the behavioral tests, mice were perfused and brain sections were examined by electron microscopy, qPCR, histology, or Western blot analysis.
Primary OPCs culture and differentiation
The cortices from postnatal 5-7-day old C57Bl/6J mouse pups were quickly dissected and diced with a scissors after removing the meninges and blood vessels. Tissue was digested in Neurobasal (Thermo Fisher, #21103049) with 20-30 U/ml Papain (Sangon Biotech, #A003124-0100) and 2500 U DNase I (Sigma, #D4513) at 37°C for 20 min; the digested cells were pelleted and resuspended in Neurobasal with 2% B27 (Gibco Thermo Fisher, 17504044), and then filtered through a 40 µm mesh filter (BD Falcon, #352340); the filtered cells were pelleted and resuspended again in PBS containing anti-O4 beads (Miltenyi Biotec, #130-094-543): 90 µl PBS and 10 µl beads per 1×107 cells; cells and beads were incubated at 4°C for 15 min, with the tube finger-tapped 4-5 times every 5 min; a column (Miltenyi Biotec, #130-042-201) was then used to capture the beads and thus enrich the O4-positive primary OPCs; the OPCs were resuspended in the proliferation medium consisted of DMEM-F12 (Thermo Fisher, #11320-033) with 1% N2 (Gibco thermofisher, #17502-048), 2% B27, 1% penicillin/streptomycin (Gibco Thermofisher, #15140-122), and 40 ng/ml PDGF-AA (R&D Systems, #1055-AA-050), and plated onto 24-well or 6-well plate covered with PDL (sigma, #P6407) and laminin (Sigma, #114956-81-9) at a density of 9000-15000 cells/cm2. The proliferation media was replaced completely by fresh one on the first day, and then half changed every other day. After 8-9 days of proliferation, the medium was replaced by the differentiation medium that is consisted of DMEM-F12 with 1% N2, 2% B27, 1% penicillin/streptomycin, 50 µg/ml insulin (sigma, #I-6634), 40 ng/ml triiodo tyrosine (Sigma, #T2877), and 1 ng/ml ciliary neurotrophic factor (R&D Systems, #557-NT) to differentiate oligodendrocyte.
Neuro-2a cell was a gift from Dr. Gang Hu (Nanjing Medical University) and was used for the ChIP, luciferase reporter assay and western blot analysis. For transfection, the cells were plated 24 hours before and then transfected with appropriate constructs using Lipofectamine 2000 (Invitrogen, #52887) according to the manufacturer’s instruction. Culture medium was replaced 5-6 hours after transfection; transfected cells were cultured for 24 additional hours for q-PCR or 48 h for Western blotting.
Luciferase reporter assay
The luciferase reporter assay for Arc/Egr1 promoter activity was performed according to the manufacturer’s instructions (Promega, E1910). Briefly, 0.05 μg luciferase reporter plasmid, 0.2 μg EGR2 expression plasmid and 1.25 ng Renilla were co-transfected into the 80-90% confluence Neuro-2a cells; 48 hours after the transfection, cells were harvested and resuspended in 50 μl passive lysis buffer, and then placed on a micro oscillator and split for 15 min. The supernatant was used to measure luciferase activity; the normalized values (Renilla/firefly activity) were used for analysis. Experiments were performed in triplicate.
ChIP and ChIP qPCR data analysis
We used a Magna ChIP Kit (Millipore, #17-10085) for the chromatin analysis. Briefly, Neuro-2a cells were cross-linked with 1% formaldehyde; the cells were collected by scratching, and then lysed and resuspended in nuclear buffer. 24 cycles of sonication (10 s for each, followed by a 20 s interval) were applied to break the chromatin into fragments between 200 and 1000 bp. The samples were incubated with anti-Egr2 antibody (Santa Cruz, #sc-293195) or anti-RNA polymerase Ⅱ antibody (Millipore, #17-10085) and magnetic beads overnight at 4°C. Normal IgG was used as a negative control. The immunoprecipitants were separated by magnetic rack and washed. The DNA fragments were released by incubation with proteinase K at 62°C for 3 h with continuous shaking, and isolated by filtration. qPCR was performed in a 20-μl reaction volume using the SYBR Green PCR master mix (Takara, #RR420B). For electrophoresis analysis, PCR was performed with a NEB Next High-Fidelity 2X PCR Master Mix (New England Biolabs) with product resolved by agarose electrophoresis, followed by gel imaging. ChIP-qPCR (qChIP) primers were designed in proximity to Arc binding motif sequences. For each gene of interest, separate primer pairs were designed. The PCR primers are listed in (Supplementary Table 1). qChIP results were presented as percent to input.
Orbit blood was collected from mice into ice-cooled centrifugal tubes and centrifuged (1700 ×g, 10 min, 4°C) to separate serum and stored below 80°C until the assay. Blood sampling took place between 09:00 and 12:00 h37. Corticosterone was quantified by the enzyme-linked immunosorbent assay (Parameter Corticosterone Assay R&D Systems, #KGE009) according to assay instructions. All samples were run in duplicate, counterbalanced across plates, and were within the standard curve. Final values were determined by averaging the results of duplicated samples.
Immunohistochemistry and immunofluorescence.
Immunohistochemical staining was performed as previously described38. Briefly, cardiac of the anesthetized mice was poured into 0.9% saline by perfusion pump for 2 min, followed by 4% paraformaldehyde for 7 min. Brain tissue were post-fixed overnight in 4% PFA. For immunohistochemical staining, brain tissues were dehydrated in a series of graded ethanol solutions, embedded in paraffin and serially cut at 5-μm, using a paraffin slicing machine (Leica, Germany). Brain tissue sections containing the mPFC or hippocampus were incubated with a primary antibody anti- Synaptophysin (Millipore, #MAB5258-I, 1:100 dilution), anti-PSD-95 (abcam, #ab18258, 1:200 dilution) or anti-MBP (abcam, #ab7349, 1:200 dilution) at 4°C overnight. For immunofluorescence staining, brain tissues were cryoprotected overnight in 30% sucrose in phosphate buffer solution (PBS); the tissue was then embedded in OCT compound (SUKURA, #4583), and cut on a cryostat (Leica, Germany) at a thickness of 15 μm, mounted on super frost plus slides and stored at -20°C. For the staining, the sections were air dried for 1 hour at room temperature and rinsed with 1× PBS, blocked and permeabilized in blocking solution (2.5% bovine serum albumin in PBS) containing 0.1% Triton X for 1 hour at room temperature, incubated overnight at 4°C with primary antibodies anti-Tuj1 (santa cruz, #sc-80005, 1:200 dilution), anti-O4 (R&D, #MAB1326, 1:200 dilution), anti-MBP (abcam, #ab7349, 1:200 dilution), anti-EGR2 (Novus, #NB100-92327, 1:200 dilution), anti-GR (santa cruz, #sc-393232, 1:50 dilution) or anti-P-GR (Technology, #4161S, 1:200 dilution), and then with the corresponding fluorescent probe-conjugated secondary antibodies (Thermo Fisher, #A21202, #A21206, #A31572, #A31570 and #A31571) for 2 h at room temperature. For primary oligodendrocyte staining, fixed coverslips were incubated with primary antibodies anti-O4 (R&D, #MAB1326, 1:200 dilution), anti-MBP (abcam, #ab7349, 1:200 dilution) and anti-Egr2 (Novus, # NB100-92327, 1:200 dilution). Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI) at a 1:1000 dilution. Pictures were taken with an LSM700 confocal microscope (Zeiss, Germany).
Cardiac of the anesthetized mice was poured into 0.9% saline by perfusion pump for 2 min, followed by 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M PB. The brain was rapidly removed and post-fixed (4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer containing 0.5% NaCl) for at least one week at 4°C. Following washing with distilled water, the sections were stained with 0.5% uranyl acetate in 70% ethanol for 1 h, dehydrated in a serial dilution of ethanol, and cleared in propylene oxide, embedded in Epon, and incubated at 60°C for 24 h. The tissues in Epon blocks were then trimmed and reoriented so that ultrathin (70 nm) cross sections were cut using the ultramicrotome (Leica EM UC7).
Quantitative real-time PCR
Total RNA from the mPFC were extracted with TRIzol according to the manufacturer’s instructions. cDNAs were synthesized from 1 µg total RNA using the Maxima First Strand Synthesis Kit for RT-qPCR (Takara, #RR047B). qPCR was performed by amplifying cDNA for 40 cycles using the SYBR Green PCR master mix. Relative expression of mRNA for the target genes was calculated by the comparative ∆∆Ct method using GAPDH as control reference genes. The primers for Egr2, Myoc, Rxrg, Adora2a, Lef1, Drd1, Nab2, Rarb2, Dusp10, Cldn11, Arc, Nr4a2, Myo3b, Drd2, Egr1, Cd24a, Sdk2, NTs, Cartpt, Slc10A4, Drd5, Zic1, Htr1d, Pcsk9, Oligo2, Sox10, ErbB3, PDGFa, Enpp6, MBP, CC1 and GAPDH were synthesized by TsingKe (Beijing, China). All primer information is listed in (Supplementary Table 3). Six (for RNA extraction) or four (for qPCR) mice per group in duplicate experiments were averaged to provide a mean value for each group.
For Western blot analyses, samples were lysed in the RIPA buffer (Beyotime, #P0013B) containing protease inhibitors (Beyotime, #st506) and phosphatase inhibitors (Roche, #04906837001). Cell lysates were cleared of cellular debris by centrifugation (12000 rpm for 15 min) and equal amounts of protein were loaded in the SDS-PAGE on 8-12% gels and transferred to PVDF membranes. After blocking for 1 h in 5% nonfat milk in TBST, the membranes were incubated at 4℃ overnight with one of the following primary antibodies: anti-Egr1 (santa cruz, #sc-189, 1:1000 dilution), anti-Egr2 (santa cruz, #sc-293195, 1:1000 dilution), anti-Arc (santa cruz, #sc-17839, 1:1000 dilution), anti-Lef1 (Proteintech, #14972-1-AP, 1:500 dilution), anti-Nab2 (Proteintech, #19601-1-AP, 1:1000 dilution), anti-NTs (Immunoway, #YT5535, 1:500 dilution), anti-Pcsk9 (Proteintech, #55206-1-AP, 1:500 dilution), anti-BDNF (abcam, #ab108319, 1:1000 dilution), anti-Trkb (santa cruz, #sc-12, 1:500 dilution), anti-Erk (Technology, #8201, 1:1000 dilution), anti-Slc10a4 (abcam, #ab107407, 1:500 dilution), anti-Htr1d (Novus, # NB100-56349SS, 1:250 dilution), anti-Synaptophysin (Millipore, #MAB5258-I, 1:1000 dilution), anti-PSD-95 (abcam, #ab18258, 1:1000 dilution), anti-MBP (abcam, #ab7349, 1:1000 dilution) and anti-GAPDH (Proteintech, #60004-1-Ig, 1:3000 dilution). HRP-conjugated secondary antibodies are from Vector Laboratories; bands were visualized using ECL plus detection system (Imagequant LAS4000 mini). Quantification was performed using Gelpro32.
Light and electron micrographs were analyzed with the aid of Image J (NIH, USA). The mean integrated optical density was measured to assess immunostaining intensity of MBP, Synaptophysin, PSD-95 and Egr2 in the mPFC, respectively. Three sections per mouse, and four mice per group, were averaged to provide a mean value for each group. A minimum of 15 electron micrographs per animal, taken at 10000×, were collected from the layer II/III pyramidal neurons in the anterior cingulate of mPFC. These micrographs were used to determine the extent of heterochromatin of individual oligodendrocyte lineage cells. The total nuclear area of each cell was calculated, and heterochromatin was selected using the threshold tool and reported as percentage of total nuclear area. In addition, the g ratio that used for determination of the extent of myelination was calculated as the diameter of the axon divided by the diameter of the entire myelinated fiber as previously described22. A minimum of 100 myelinated axons per area of interest, per animal, were analyzed. The synaptic structure was analyzed by measuring the thickness of the postsynaptic dense, synaptic cleft width, length of the active zones, synaptic curvature. An average of 10 measurements of synaptic cleft width evenly spaced across the synapse was taken as a reading for each synapse, where the ends of the synapse were defined as the ends of the electron-dense PSD. The length of the active zones was measured, and synaptic curvature was subsequently calculated from the ratio of the arc length of the active zone to its corresponding chord length39. To avoid bias, images were analyzed by an individual who was blind to the experimental condition.
Statistical analysis was performed with Prism 6.0 (GraphPad Software). The number of animals and culture used for the experiments are indicated in the bar graphs of the Fig.s or in the Fig. legends. The cooperative training and cooperative test date were analyzed by repeated-measures ANOVA. The comparisons between two groups were examined by two-tailed Student’s t-tests. The two-way ANOVA followed by Tukey’s multiple comparisons test was performed for multiple groups. Data shown are the mean ± SEM. with P < 0.05 considered statistically significant.
34 Wang, L. et al. Deep cervical lymph node ligation aggravates AD-like pathology of APP/PS1 mice. Brain Pathol 29, 176-192 (2019).
35 Gao, J. et al. A novel pathway regulates memory and plasticity via SIRT1 and miR-134. Nature 466, 1105-1109 (2010).
36 Matos, M. R. et al. Memory strength gates the involvement of a CREB-dependent cortical fear engram in remote memory. Nat Commun 10, 2315 (2019).
37 Kinn Rod, A. M., Harkestad, N., Jellestad, F. K. & Murison, R. Comparison of commercial ELISA assays for quantification of corticosterone in serum. Sci Rep 7, 6748 (2017).
38 Xu, Z. et al. Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Abeta accumulation and memory deficits. Mol Neurodegener 10, 58 (2015).
39 Jones, D. G. & Devon, R. M. An ultrastructural study into the effects of pentobarbitone on synaptic organization. Brain Res 147, 47-63 (1978).