METHOD DETAILS

The injectable solution of (Z)-4-Hydroxytamoxifen (4HT, Sigma-Aldrich, Cat# H7904) was prepared as described before (Kang et al., 2010). Briefly, 4HT was dissolved in 100% ethanol (20 mg/ml) with bath sonication, and aliquots of 1 mg (in 50 μl) were prepared and kept at −80°C until use. For each application, aliquots were emulsified after adding 250 μl of sunflower seed oil (Sigma-Aldrich, Cat# 88921), and the ethanol was evaporated for 30 min using a vacuum centrifuge. The total 4HT dose and number of injections for each set of experiments are stated in either results or figure legends. For example, a single dose of 4HT (0.2 mg s.c. per injection) was injected to Cspg4-CreER; Ai14; R26-EGFP-Gria2 mouse pups at P3 before the H/I injury was applied.

For P51 or older Cspg4-CreER; Ai14; R26-EGFP-Gria2 mice, tamoxifen (Sigma-Aldrich, Cat# T5648) was administered. After tamoxifen was dissolved (20 mg/ml) in a mixture of sunflower seed oil-ethanol (10:1), ethanol was evaporated for 30 min using a vacuum concentrator. 40 mg/kg (b.w.) of tamoxifen was intraperitoneally (i.p.) administered twice a day with at least 6 hours interval between injections. A total of 10 doses was injected into the mice between P51 and P55.

The OPCs on coverslips were fixed with 4% paraformaldehyde (PFA; in 0.1M phosphate buffer, pH 7.4) for 20 min and immunostained in the absence of Triton X-100 throughout entire staining procedures until DAPI addition. OPCs were blocked with 10% normal donkey serum for 1 hour at RT and incubated with goat anti-GFP antibodies (Rockland, Cat# 600-101-215; 1:500) and rabbit anti-Olig2 antibodies (EMD Millipore, Cat# AB9610; 1:500) at 4°C overnight. After washing with PBS (each washing for 5 min, three times), OPCs were incubated with secondary antibodies for 2 hours at RT. After secondary antibody incubation, OPCs were exposed to 0.1% Triton X-100 solution containing DAPI for 20 min. As the experimental control, OPCs on a separate coverslip were permeabilized with 0.1% Triton X-100 from the blocking step, and subjected to the immunostaining for the detection of intracellular EGFP and Olig2.

To measure calcium responses to AMPA, cultured OPCs were loaded with Fura-2 acetoxymethyl ester (Fura-2 AM; Thermo Fisher Scientific, Cat# F1201; 2 μM) in cation-safe solution (107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl₂, 11.5 mM glucose, 20 mM HEPES-NaOH, 1 mM CaCl₂, pH 7.2) for 30 min at RT. Cells were washed and incubated for an additional 30 min for complete de-esterification of the dye. A Leica DMI 6000B fluorescence microscope controlled by SlideBook Software (Intelligent Imaging Innovations) was used to monitor intracellular calcium levels, assessed by fluorescence emission at 505 nm, while alternating excitation wavelengths between 340 and 380 nm. AMPA-dependent calcium responses in OPCs were induced by the addition of cyclothiazide (CTZ; Hello Bio, Cat# HB0221; 100 μM) and AMPA (Tocris, Cat# 0254; 20 μM). The peak values of CTZ-sensitized, AMPA-dependent calcium responses minus peak values of AMPA (with no CTZ) responses of all recorded cells were averaged for each coverslip, which was counted as one replicate.

The entire cerebra were isolated from P15 pups and chopped with a blade into small pieces. The brain cells were dissociated using the Neural Dissociation Kit (Miltenyi Biotec, Cat# 130-092-628) according to the manufacturer’s instruction. After enzymatic digestion, the cells were mechanically dissociated with gentle pipetting and suspended in Hank’s Balanced Salt Solution (HBSS; GIBCO, Cat# 14175-075). The cell suspension was passed through a 40-μm cell strainer (Corning, Cat# 352340). Cells were re-suspended in 0.5% FBS in HBSS. Cells were isolated with BD influx (BD Biosciences) at the Flow Core Facility of Temple University School of Medicine.

Total RNAs were extracted from the FACS-isolated cells with RNAeasy Plus Micro Kit (QIAGEN, Cat# 74034). cDNAs were generated using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Cat# 18080051). Quantitative PCR was performed using a SYBR Green PCR kit (QIAGEN, Cat# 204143) and StepOnePlus™ Real-Time PCR system (Applied Biosystems).

The mouse neonates underwent a surgical procedure for permanent unilateral common carotid artery (CCA) ligation and subsequent exposure to hypoxia (Levine, 1960; Rice et al., 1981) with minor modifications (Shen et al., 2012). After being anesthetized with isoflurane (4% for induction and 2.5% for maintenance), P7 pups were fixed on a surgical platform. After a midline incision of the skin was made in the upper anterior thorax region, the right CCA was identified by key anatomical landmarks. Fascia, the vagus nerve, and sympathetic ganglia were carefully moved aside, and the CCA was isolated using a small hook, and cauterized with a fine-tipped cauterizer (Fine Scientific Tools, Cat# 18000-00). After confirming lack of blood flow into the external and internal carotid artery, muscles and superficial fascia were gently moved back into the proper anatomical position, and the incision was closed using 3M Vetbond Tissue Adhesive (3M Science). After recovery from anesthesia, the pups were returned to the dam and kept with it for an additional 30 min. They were then moved to a sealed hypoxia chamber (BioSpherix) infused with nitrogen to maintain the oxygen level at 6.0% for 30 min at 35°C and returned to the dam.

After being anesthetized with a mixture of Ketamine (200 mg/kg) and Xylazine (15mg/kg) (Sigma-Aldrich, Cat# K4138), mice were fixed on a stereotaxic instrument (Stoelting). One μl of 1% L-α-Lysophosphatidylcholine (LPC or lysolecithin in physiological saline; Sigma-Aldrich, Cat# L4129) was injected into right CC (AP: +1.0 mm; ML: −0.8 mm; DV: 2.1 mm from bregma) with Hamilton syringe. The Hamilton syringe was equipped with a 33-gauge needle (45° beveled tip) attached to a motorized stereotaxic injector (Stoelting), and LPC was injected at a rate of 0.05 μl/min. After the injection, the needle was held in place for an additional 10 min and gently pulled out. The scalp was sutured, and the mice were allowed to recover from anesthesia.

Ethynyl-2′-deoxyuridine (EdU; Lumiprobe, Cat# 40540; 10 mg/kg per injection) or 5-bromo-2′-deoxyuridine (BrdU; Thermo Fisher, Cat# BP-2508-5; 50 mg/kg per injection) was injected intraperitoneally to analyze cell proliferation of OPCs. The number of doses and frequency of injections varied depending on the sampling age, and the details are described in figure legends. For example, EdU was injected daily at P8 and P9 (a total of two doses) for the sampling at P10, whereas it was injected three times between P22 and P23 for the sampling at P24. Seven doses of BrdU were injected daily to the mice that had received LPC between P68 and P74, and a total of 4 doses of EdU were injected between P73 and P74.

Mice were anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and subjected to trans-cardiac perfusion with PBS for 3 min and 4% PFA. Brains were removed from the crania and incubated in 4% PFA at 4°C for an additional 5 hours for post-fixation. The fixed brains were moved to and stored in 30% sucrose (in PBS) at 4°C for at least 36 hours for cryoprotection until sectioning. Brain samples were frozen in Tissue-Tek optimum cutting temperature (O.C.T.) compound (Sakura, Cat# 4586) with dry ice, and sectioned 35 μm thick using a cryostat (Leica). Brain sections were collected into 6-well plates in a stereological manner, and 4 or 5 free-floating sections were used for each set of immunostaining. The sections were first permeabilized with 0.3% Triton X-100, and then blocked with blocking solution (5% normal donkey serum, 0.3% Triton X-100) for 1 hour at RT. The sections were then incubated with primary antibodies in the same blocking solution at 4°C overnight. The primary antibodies used for immunofluorescence were rabbit anti-GFP (Proteintech, Cat#50430-2-AP; 1:500), goat anti-GFP (Rockland, Cat# 600-101-215; 1:500), mouse anti-APC (clone CC1, EMD Millipore, Cat#OP80; 1:60), rat anti-CD140a (PDGFRα, BD Biosciences, Cat# 558774; 1:500), rabbit anti-PDGFRα (a gift from Dr. William Stallcup, Sanford Burnham Prebys Medical Discovery Institute; 1:500), mouse anti-MBP (Covance, Cat#SMI-99P; 1:500), rabbit anti-MBP (Cell Signaling Technology, Cat# 78896; 1:500), guinea pig anti-NG2 (generated by Bergles’ Lab, Johns Hopkins University; 1:4,000), rabbit anti-Olig2 (EMD Millipore, Cat# AB9610; 1:500), rabbit anti-NeuN (Cell Signaling Technology, Cat# 24307; 1:500), rabbit anti-GFAP (Agilent, Cat# Z033429-2; 1:500), rat anti-BrdU (Abcam, Cat# ab6326; 1:500), mouse anti-GluT1 (Abcam, Cat# ab40084; 1:500) and goat anti-Sox9 (R & D Systems, Cat# AF3075, 1:500). After brief washing with PBS (for 5 min, three times), the sections were incubated with secondary antibodies for 2 hours at RT. The secondary antibodies used in this study were Alexa Fluor 488-, Cy3-, or Cy5-conjugated donkey IgG against rat, rabbit, mouse, goat, chicken, or guinea pig (Jackson Immuno Research; 1:500). Nuclei were co-stained with DAPI. After brief washing (for 5 min, three times), the sections were mounted on slide glasses with ProLong antifade reagents (Thermo Fisher Scientific, {"type":"entrez-protein","attrs":{"text":"P36970","term_id":"172045845","term_text":"P36970"}}P36970).

For anti-APC (CC1) immunostaining, brain sections were pretreated with 5 mM citrate buffer (5 mM citric acid, 0.05% Tween 20, pH 6.0) at 95°C for 1.5 min (for sections from P8 or P10 mice) or for 5 min (for sections from P35 mice) before permeabilization. For the detection of EGFP-GluA2 at P35 mice, sections were pretreated with 0.3% H₂O₂ for 30 min before permeabilization, and anti-EGFP signals were amplified using Tyramide Signal Amplification kit (TSA; PerkinElmer, Cat# NEL753001KT) according to the manufacturer’s instruction.

For BrdU immunostaining, sections were pretreated with 2 N HCl for 30 min at 37°C, followed by incubating with 0.1 M borate buffer (pH 8.5) twice (5 min each time) before the initial washing with 0.01 M PBS. In case of EGFP and BrdU co-immunostaining, EGFP immunopositive signals were amplified with the TSA kit before the sections are incubated with 2 N HCl for BrdU immunostaining.

Brains sections were treated with Alexa Fluor-647 Click-iT reaction cocktail solution (Thermo Fisher Scientific, Cat# {"type":"entrez-nucleotide","attrs":{"text":"C10340","term_id":"1535411","term_text":"C10340"}}C10340) according to manufacturer’s instructions before blocking. For assessing the co-localization of EGFP and EdU, GFP immunostaining was performed prior to the incubation of the brain sections with EdU detection reagents.

Widefield fluorescence images were collected with an epifluorescence microscope Axio-Imager M2 (Zeiss). Confocal images were obtained with a laser scanning microscope TCS SP8 (Leica), and processed with LAS X software (Leica).

Mice cortices were isolated, snap frozen in dry ice, and kept at −80°C until use. The cortices were homogenized in RIPA buffer (Cell Signaling Technology, Cat# 9806) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Cat# 1861281) and EDTA (Thermo Fisher Scientific, Cat# 1861274). 5 μg of protein lysate per each sample was mixed with an equal volume of 2X Laemmli Sample Buffer (Bio-Rad, Cat# 1610737), and resolved with a 12% Bis-Tris gel (Bio-Rad, Cat# 3450118). Proteins were transferred to a PVDF membrane (EMD Millipore, Cat# IPVH00010) by the electrophoretic transfer method. The blot membranes were blocked in 5% skim milk (in TBST) for 1 hour, and incubated with primary antibody for 1 hour at RT. The primary antibodies used in this study were rabbit anti-MBP (Cell Signaling Technology, Cat# 78896; 1:2,000), rabbit anti-CNPase (Phosphosolutions, Cat#325-CNP; 1:2,000), mouse anti-MAG (Santa Cruz Biotechnology, Cat# sc-166849; 1:4,000), mouse anti-MOG (Santa Cruz Biotechnology, Cat# sc-166172; 1:2,000), and mouse anti-β-actin (Santa Cruz Biotechnology, Cat# sc-47778; 1:2,000). After washing the blot membrane with TBST for 10 min (three times), the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (Jackson Immuno Research; 1:20,000) in 5% skim milk for 1 hour at RT. After washing with TBST for 10 min (four times), the membranes were subjected to chemiluminescence reaction using an ECL kit (Supersignal West Dura, Thermo-Fisher Scientific, Cat# 34075), and signal detection using X-ray film. Intensities of protein bands on X-ray films were quantified with ImageJ. For sequential probing with different antibodies, membranes were stripped with a stripping solution (2% SDS, 0.06 M Tris-HCl, pH 6.8,0.8% 2-mercaptoethanol) at 55°C for 10 min, and washed with TBST for 5 min (five times) before re-blocking.

Mice were deeply anesthetized with pentobarbital (70 mg/kg, i.p.) and perfused with PBS followed by 2.5% PFA, 2% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4). After brain isolation, samples were post-fixed in the same fixative for 18 hours at 4°C, transferred to, and kept in 0.1 M phosphate buffer at 4°C. Samples were treated with 2% OsO₄ for 1 hour, and 2% uranyl acetate for 30 min. Samples were sequentially dehydrated using 50%, 70%, 90%, 100% ethanol, and propylene oxide. Samples were embedded in Epon 812 resin (Ted Pella), and ultrathin sections were obtained using Ultracut UCT (Leica). Sections were stained with 2% uranyl acetate and lead citrate, and imaged with an H-7600 microscope (Hitachi, Tokyo, Japan). ImageJ was used to measure the calibers of axons and myelin sheaths.