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ANA_23626_sm_SuppFig1.tif2899KSupporting Information Figure 1. Supplementary Figure 1. Generation and characterization of conditional microglial NMDA receptor NR1 knockout mouse. Conditional microglial NR1 knockout (KO) mice were generated by breeding Nr1flox/flox mice with LysMCre/Cre mice (see Supplementary methods and Figure 2A, B for detail). In the (A) cortex and (B) white matter of KO mice, tomatolectin (Lectin)-positive microglia lacked NR1-immunopositivity, while neurons were NR1-immunopositive. P5 (C) and adult (D) KO mice had normal body weight. (E) P5 KO mice showed no gross brain malformations. (F) KO mice had normal blood counts as well as bone marrow phenotypes at P56. (G) In an in vivo excitotoxic brain damage model, a brain lesion was induced by intracerebral injection of 10 μg ibotenate into infant (P5) and adult (P56) mice. White matter lesion (L) size was assessed 1 day later (n=16-18) or 5 days later (n=18-19) for P5 mice and 5 days later for adult mice (n=9; unpaired two-tailed T-test). Conditional NR1 KO mice (NR1flox/floxLysMCre/-) lacking to a large extent the NMDAR subunit NR1 in microglia showed significantly reduced white matter lesion size as compared to their wild type littermates. One-way ANOVA; values represent mean ± S.E.M.; *p>0.05, **p>0.01, ***p>0.001. See Figure 1 for further detail.
ANA_23626_sm_SuppFig2.tif1310KSupporting Information Figure 2. Supplementary Figure 2. Microglia express NMDAR1 subunit in vivo. NR1 immunopositivity was detected on tomatolectin (lectin)-positive microglia in murine brain sections at postnatal day 5 (P5) and adult age (P56) (nuclei labeling with DAPI in blue). Fluorescence microscopy 40x, scale bar 20 μM. See Supplementary Table 1 for marker explanation, Figure 2E for higher magnification confocal images and Figure 1C, Supplementary Figures 1, 3, and 4 for controls.
ANA_23626_sm_SuppFig3.tif1031KSupporting Information Figure 3. Supplementary Figure 3. NR1 antibody specificity in vitro. (A) Specificity of immunolabeling with the anti-NR1 antibody was confirmed through incubation of microglia cultures with secondary antibodies red and green only as well as in control specimen immunolabeled with an anti-NR1 antibody (Abcam) pre-incubated with the corresponding antigenic peptide. Secondary antibodies did not produce a significant signal and a pre-incubation with the corresponding peptide inhibited the binding of the antibody to the sample NR1 peptide. (B) Colocalization between the signal obtained by immunostaining of microglia with an anti-NR1 antibody and the GFP-signal from microglia transfected with an NR1-EGFP plasmid was revealed by confocal microscopy (magnification 63x). See Figure 1C, Supplementary Figures 1 and 4 for further controls.
ANA_23626_sm_SuppFig4.tif560KSupporting Information Figure 4. Supplementary Figure 4. NR1 antibody specificity in vivo. Control immunohistological stainings were performed by applying the secondary antibody only to (A) human or (B) murine brain sections. A pre-incubation of the NR1-antibody (Chemicon) and its corresponding peptide inhibited antibody binding, as analyzed in (C) human and (D) murine tissue. See Figure 1C, Supplementary Figures 1 and 3 for further controls.
ANA_23626_sm_SuppFig5.tif400KSupporting Information Figure 5. Supplementary Figure 5. Expression of NMDA receptor subunits in murine and human spinal cords in vivo. (A) Immunohistological staining of murine spinal cord sections at P5 with antibodies directed against NMDA receptor subunits NR1, NR2B or NR2D and against Iba1 revealed the existence of these subunits on microglia. (B) The presence of the NR1 subunit was also detected on CD68-positive microglia in the spinal cord of a patient with ALS. Confocal microscopy 63x.
ANA_23626_sm_SuppFig6.tif1416KSupporting Information Figure 6. Supplementary Figure 6. Microglia and neurons express NMDA receptor subunit NR2B and NR2D in vitro. Immunocytogical staining of cultured cortical murine microglia (DIV15) with antibodies directed against (A) NMDA receptor subunit 2B (NR2B), tomatolectin (Lectin) or DAPI, and (B) NMDA receptor subunit 2D (NR2D), tomatolectin (Lectin) or DAPI. Immunocytological staining of cultured cortical neurons (DIV10) with antibodies directed against (C) NR2B or DAPI and (D) NR2D and DAPI. Fluorescence microscopy; magnification 63x.
ANA_23626_sm_SuppFig7.tif1279KSupporting Information Figure 7. Supplementary Figure 7. Microglia express NMDA receptor subunits NR2B and NR2D in infant murine brains in vivo. Immunohistological staining of murine brain sections at P5 with antibodies directed against NMDA receptor subunits NR2B or NR2D and with tomatolectin (Lectin) revealed the existence of these subunits on microglia (confocal microscopy 63x).
ANA_23626_sm_SuppFig8.tif275KSupporting Information Figure 8. Supplementary Figure 8. Cytokines and chemokine induction through NMDA in microglia from conditional Nr1 knockout mice. Interleukin (IL) 1a, IL1ß, IL3, IL4, IL5, IL6, IL9, IL10, IL12 (p70), IL13, IL17, granulocyte macrophage colony-stimulating factor (G-CSF), interferon ? (IFN?), monocyte chemotactic protein 1 (MCP1) and tumor necrosis factor alpha (TNFa) levels 12 hours after treatment initiation with PBS (control), NMDA 300 μM as well as LPS 10 μg/ml, respectively. Treatment of microglia from Nr1flox/flox LysMCre/- mice with NMDA did not significantly change the levels of these proteins while LPS as a positive control did (n=3-4; One-way ANOVA,, Bonferroni's multiple comparison test, * p>0.05, ** p>0.01, *** p>0.001, values represent mean ± S.E.M.). See Figure 5 for results using wild type microglia.
ANA_23626_sm_SuppInfo.doc81KSupporting Information
ANA_23626_sm_SuppTabs.doc61KSupporting Tables and Figure Captions.

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