Breeding and experimental procedures for transgenic mice
mice have been described previously (Fukushima et al., 2009
). In these mice, exons 19–20 (putative transmembrane segment M4) of the gene for the NR1 subunit (Grin1
) are flanked by LoxP sites. Olig1cre/+
mice were obtained from Dr. David Rowitch (Univeristy of California, San Francisco) and have been described previously (Lu et al., 2002
); in these mice Cre recombinase was knocked into one allele of the Olig1
gene locus. PDGFαR-CreER
BAC transgenic mice have been described previously (Kang et al., 2010
For inducible ablation of NMDAR within a cohort of OPCs and their progeny, NR1flox/flox
mice were bred to PDGFαR-CreER
as well as Z/EG
reporter mice (Novak et al., 2000
) which allows visualization of cells in which Cre was active. For electrophysiology experiments, PDGFαR-CreER;NR1+/+;Z/EG
) and PDGFαR-CreER;NR1flox/flox;Z/EG
) mice were given a single 1mg intraperitoneal (IP) injection of 4-hydroxy-Tamoxifen (4HT) during the 3rd
postnatal week and were analyzed 10–35 days post injection as indicated in the text. For analysis of cell morphology and differentiation capacity, P-NR1+/+
mice were given a single 0.1mg subcutaneous injection of 4HT at P4 and were analyzed at P30. Some mice in these experiments were also given IP injections of BrdU (10mg/kg) twice daily for two days at P20.
For constitutive ablation of NMDAR within the oligodendrocyte lineage, Olig1cre/cre;NR1flox/+
mice were bred to Olig1+/+;NR1flox/+
mice to obtain both Olig1cre/+; NR1flox/flox
) and Olig1cre/+; NR1+/+
) littermate offspring. To assess the extent and specificity of Cre activity, O-NR1+/+
mice were bred to Z/EG
reporter mice. For electrophysiology experiments, O-NR1+/+
mice were bred to NG2-DsRed
BAC transgenic mice (Ziskin et al., 2007
; Zhu et al., 2008
) to accurately identify OPCs in acute brain slices. For analysis of cell proliferation, O-NR1+/+
mice aged P7 and P14 were given a single IP injection of Bromodeoxyuridine (BrdU, 300mg/kg) two hours before perfusion.
In all experiments, both male and female mice were used and the number of males and females in each analysis group was balanced. All experiments were carried out in strict accordance with protocols approved by the Animal Care and Use Committee at Johns Hopkins University.
Acute brain slice preparation
Mice were anesthetized with isofluorane and decapitated; their brains were dissected into an ice-cold N-methyl-D-glucamine (NMDG)-based solution containing: 135 mM NMDG, 1 mM KCl, 1.2 mM KH2PO4, 20 mM choline bicarbonate, 10 mM glucose, 1.5 mM MgCl2, and 0.5 mM CaCl2 (pH 7.4, 310 mOsm). Coronal forebrain slices (250 μm thick) were prepared using a vibratome equipped with sapphire blade in ice cold NMDG-based cutting solution. Following sectioning, slices were transferred to artificial cerebral spinal fluid (ACSF) containing: 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose (290 mOsm) and were maintained at 37°C for 30 min, and at room temperature thereafter. Both NMDG solution and ACSF were bubbled continuously with 95% O2/5% CO2. All experiments were carried out at room temperature.
Oligodendrocyte precursor cells (OPCs), pre-oligodendrocytes (Pre-OLs), and oligodendrocytes (OLs) were visualized with an upright microscope (Zeiss Axioskop FS2) equipped with both DIC optics and filter sets for DsRed (HQ:TRITC 41002c, Chroma) and GFP (Brightline, GFP-ABasic-ZHE, Semrock). Whole-cell recordings from OPCs, Pre-OLs, and OLs were made under visual control using both GFP and DsRed fluorescence and IR-DIC as a guide. For hypertonic solution and UV-uncaging experiments, the electrode solution consisted of 100 mM CsCH3
H (cesium methanesulfonate), 20 mM tetraethylammonium (TEA) chloride, 20 mM HEPES, 1 mM MgCl2
, 10 mM EGTA, 2 mM sodium ATP and 0.2 mM sodium GTP (pH 7.3, 295 mOsm). For experiments performed in current-clamp mode, CsMeS was replaced with KCH3
H (potassium methanesulfonate) in the electrode solution and TEA was omitted. Pipette resistance was 3.4 to 4.2 MΩ and recordings were made without series resistance compensation. Hypertonic solution (HS; normal ACSF containing 500 mM sucrose; 850–900 mOsm) was focally delivered through a glass pipette (resistance 1–1.5 MΩ) using a pressure application system (Toohey Company, Pressure System IIc) as described previously (De Biase et al., 2010
). For glutamate uncaging experiments, MNI-L-glutamate (500 μM, Tocris) was locally perfused over the slice with a wide-bore glass pipette. Photolysis was accomplished with a 1 ms flash of UV light from a Stabilite 2017 argon laser (Spectra Physics) in a 100 μm circle centered on the cell body. D-serine (30 μM, Sigma Aldrich), a co-agonist at the NMDA receptor glycine binding site, was included in the ACSF during experiments aimed at detecting NMDAR-mediated currents. For AMPAR rectification experiments, spermine (Tocris, 100μM) was included in the recording pipette. The following agents were applied by addition to the superfusing ACSF: TTX (NaV antagonist; Ascent Scientific, 1 μM); gabazine (SR-95531; GABA receptor antagonist; Tocris, 5 μM); RS-CPP (NMDA receptor antagonist; Tocris, 5 μM or 20 μM for uncaging); 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; competitive AMPA/kainate receptor antagonist; Tocris, 5 μM or 50 μM for uncaging); GYKI 53655 (AMPA selective, non-competitive antagonist; IVAX 100 μM); 7-Chlorokynurenic acid (NMDAR antagonist acting at the glycine site; Tocris, 50μM).
Responses were recorded using a MultiClamp 700A amplifier (Axon Instruments), filtered at 3 kHz, digitized at 50 kHz and recorded to disk using pClamp9.2 software (Axon Instruments). Data were analyzed offline using Clampfit (Axon Instruments), Origin (OriginLab Corp.) and Mini analysis (Synaptosoft Inc.) software. Membrane resistance and membrane capacitance were calculated from a 10 mV depolarizing step (holding potential −80 mV). The amplitude of voltage-gated sodium current (NaV) was calculated from a 70 mV depolarizing step (holding potential −80 mV) performed before and after application of TTX and within 2 minutes of initiating whole cell recording. Resting membrane potential was measured within 30 seconds of establishing whole cell recording (KMs-based internal solution). The response to HS was quantified by counting the number of evoked mEPSCs (> 5 pA amplitude, <1 ms rise as measured in MiniAnalysis software, within 15s of stimulus onset) during 2 successive puffs (separated by at least 1.5 min). For clarity, slow alterations in holding current observed during HS application were subtracted from baseline using PClamp software. For experiments in P-NR1+/+
mice in which EGFP+
cells may be OPCs, Pre-OLs, or OLs, basic membrane properties were used to categorize the stage of cell differentiation as described previously (De Biase et al., 2010
). Specifically, cells with NaV current and <35 pF capacitance were considered to be OPCs, cells +/−NaV current with >35 pF capacitance and >500 MΩ membrane resistance were considered Pre-OLs, and cells that lacked NaV currents with < 500 MΩ membrane resistance were considered OLs.
Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg) and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Brain tissue was isolated and post-fixed in this solution for 6–18 h at 4°C, then washed in phosphate buffer, cryoprotected in 30% sucrose, and sectioned at 30–60 μm thickness on a cryostat (Microm). Free-floating sections were permeabilized/blocked with 0.3% Triton X-100 and 5% normal donkey serum in 0.1 M sodium phosphate buffer for 2 hrs at room temperature or 4°C overnight. Sections were incubated with primary antibodies prepared in permeabilizing/blocking solution for 4 hrs at room temperature or 4°C overnight. Sections were incubated with secondary antibodies in 5% normal donkey serum in 0.1 M sodium phosphate buffer for 2 hrs at room temperature. For immunolabeling with CC1 antibody, sections were treated with LAB solution (Polysciences) for 10–15 min prior to blocking. For visualization of cells with incorporated BrdU, sections were incubated with 2N HCl at 37°C for 30min, washed twice with 0.1M borate buffer for 5min, and were then permeabilized/immunostained as outlined above. Control sections incubated with secondary antibody alone did not result in labeling of cells. Primary antibodies used: Rabbit anti-NG2 (1:500; gift from Dr. Stallcup, Burnham Institute), Rabbit anti-GFP (1:500, gift from Dr. Huganir, Johns Hopkins), Chicken anti-GFP (1:500, Aves), Goat anti-GFP (1:500, Frontier Institute), Rabbit anti-PDGFαR (1:500; gift from Dr. Stallcup, Burnham Institute), Rat anti- PDGFαR (1:500, BD Pharmingen), Guinea Pig anti-GFAP (1:500, Advanced ImmunoChemical Inc.), Rabbit anti-GFAP (1:500, Sigma), Mouse anti-MBP (1:1000, Sternberger Monoclonals), ALDH1 (1:100, UC Davis/NeuroMab), Mouse anti-NeuN (1:500, Millipore), CC1 (1:50, Anti-APC/Ab-7, Calbiochem), Guinea pig anti-Olig2 (1:10,000, gift from Dr. Ben Novitch, UCLA), Rabbit anti-Olig2 (1:500, Millipore). Secondary antibodies (raised in donkey): Alexa 488- (Invitrogen), Cy2-, Cy3- or Cy5-conjugated secondary antibodies to rabbit, mouse, goat or guinea pig (1:500; Jackson ImmunoResearch).
Image Acquisition and Analysis
For evaluation of MBP immunostaining, quantification of oligodendrocyte lineage cell density, and cell proliferation analysis, fluorescence images were collected on a AxioImager M1 microscope (Zeiss). For analysis of cell differentiation capacity in P-NR1+/+/P-NR1fl/fl mice, and extent of Cre expression in O-NR1+/+/O-NR1fl/fl mice, fluorescence images were acquired with a LSM 510 Meta confocal microscope (Zeiss) or a LSM 710 Meta confocal microscope (Zeiss). For morphological analysis of OPCs in P-NR1+/+/P-NR1fl/fl mice, stacks of confocal images (0.3μm z-interval) of EGFP+PDGFαR+ cortical OPCs were imported into Imaris software for 3D morphological reconstruction and quantification. For analysis of Cre expression in O-NR1+/+/O-NR1fl/fl mice (bred to Z/EG reporter), 6 O-NR1+/+ mice (P30-55) and 1 O-NR1fl/fl (P55) mouse were examined. To evaluate colocalization between BrdU and PDGFαR or Olig2, 3 O-NR1fl/fl and 3 O-NR1+/+ mice were examined at P7 and 6 O-NR1fl/fl and 5 O-NR1+/+ mice were examined at P14. In the differentiation capacity analysis, to evaluate colocalization between EGFP and PDGFαR or CC1, 6 P-NR1fl/fl and 4 P-NR1+/+ mice were examined. In all histological analysis, at least 4 brain sections from each mouse were analyzed.
Western Blot Analysis
O-NR1fl/fl and O-NR1+/+ mice age P24-26 and P55-58 were deeply anesthetized with isofluorane and perfused transcardially with ice cold PBS. Mouse cortical tissue was carefully microdissected and flash frozen in 2-methyl butane chilled with dry ice. Tissue samples were then homogenized in RIPA buffer (Boston BioProducts) using a hand-held homogenizer. Protein concentration was determined using Bio-Rad protein assay (Bio-Rad). For MBP and CNPase immunoblots, protein samples were denatured in Laemmli’s SDS sample buffer containing β-mercaptoethanol. For detection of MAG, protein samples were prepared in a non-denaturing Laemmli’s sample buffer (Boston BioProducts). 20 μg of each sample was separated on a 12% Tris HCl gel (Bio-Rad) and transferred to a PVDF membrane. Membranes were blocked in 0.1% Tween-20 TBS containing 5% non-fat milk for 1 hour prior to incubation with primary antibodies. Primary antibodies were diluted in the same solution as follows: anti-CNPase (1:200, clone 11-5B, Millipore), anti-MBP (1:1000, clone smi99, Covance), anti-MAG (1:300, clone 513, Millipore), and anti-β-actin (1:1000, clone 20–33, Sigma). Anti-mouse or anti-rabbit secondary antibodies conjugated to IRDye® 680 or 800 were incubated for 1 hour prior to scanning the membrane on a LI-COR Odyssey® imager. Images were analyzed using the Odyssey® infrared imaging system application software version 3.0. Protein expression was normalized to β-actin.
Electron Microscopic Analysis
O-NR1fl/fl and O-NR1+/+ mice age P24-26 and P55-57 were anesthetized with pentobarbital and perfused transcardially with 4% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer. Brain tissue was isolated and post-fixed in this solution for 4 hrs at 4°C, then washed in phosphate buffer. Brains were treated with 2% OsO4 for 1 hour. After washes in water, samples were incubated in 2% uranyl acetate for 30 min. After dehydration using 50%, 70%, 90%, and 100% ethanol and 100% propylene oxide, samples were embedded in Epon 812 resin (Ted Pella). Ultrathin sections were obtained using a Leica Ultracut UCT and stained with 2% uranyl acetate and lead citrate. Electron micrographs were taken with an H-7100 electron microscope (Hitachi). NIH Image J software was used to measure the diameter of axons and their myelin sheaths.
Data are expressed as mean ± SEM throughout, and statistical significance was determined using the Mann-Whitney test with a cutoff value of 0.05 and Kruskal-Wallis ANOVA with sequential Bonferroni correction for multiple comparisons.