Journal: eLife

Article Title: Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

doi: 10.7554/eLife.78982

Figure Lengend Snippet: Live-cell fluorescence resonance energy transfer (FRET) titration experiment data and statistics. Table 1—source data 1. Source data for Table 1 .

Article Snippet: Chemical compound, drug , Ro 64–5229 , Tocris , Cat # 2,913 , .

Techniques: Fluorescence, Förster Resonance Energy Transfer, Titration, Concentration Assay

Journal: eLife

Article Title: Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

doi: 10.7554/eLife.78982

Figure Lengend Snippet: Live-cell fluorescence resonance energy transfer (FRET) max normalization experiment data and statistics. Table 2—source data 1. Source data for Table 2 .

Article Snippet: Chemical compound, drug , Ro 64–5229 , Tocris , Cat # 2,913 , .

Techniques: Fluorescence, Förster Resonance Energy Transfer

Journal: eLife

Article Title: Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

doi: 10.7554/eLife.78982

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Ro 64–5229 , Tocris , Cat # 2,913 , .

Techniques: Transfection, Construct, Modification, Plasmid Preparation, Software

Journal: Journal of Neuroinflammation

Article Title: mGlu3 receptor regulates microglial cell reactivity in neonatal rats

doi: 10.1186/s12974-020-02049-z

Figure Lengend Snippet: Developmental expression of mGlu3 receptors following the LPD/IL-1β double hit in rat. a , b Grm3 mRNA expression in cortex ( a ) and in microglia ( b ) sorted from CTRL and LPD/IL-1β animals. Data (mean ± SEM) are relative to Grm3 expression in sorted cells of CTRL cortex and CTRL microglia at PND1. Two-way ANOVA followed by the Newman-Keuls multiple comparison test; ** p < 0.01, **** p < 0.0001 vs respective P1; && p < 0.01, &&&& p < 0.0001 vs respective P4; $$ p < 0.01, $$$ p < 0.001 vs respective P7; xxx p < 0.001 vs respective P10; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 effect of LPD/IL-1β. c – f mGlu3 protein quantification in cortex and primary microglia cells at P4 ( c , e ) and P7 ( d , f ) in CTRL and LPD/IL-1β pups. P4 cortex and microglia cells sorted from three animals were pooled. Data (mean ± SEM). Quantification of mGlu3 receptor expression in tissue or cells from LPD/IL-1β pups was performed relative to that of CTRL pups. The optical density (O.D.) for mGlu3 receptors was normalized to the O.D. of Ponceau S staining. Unpaired t test; # p < 0.05, ## p < 0.01

Article Snippet: LY 2389575 hydrochloride, a selective negative allosteric modulator of mGlu3 receptors (Tocris), and Ro 64-5229, a selective, non-competitive mGlu2 receptor antagonist (Tocris), were dissolved in DMSO.

Techniques: Expressing, Comparison, Staining

Journal: Journal of Neuroinflammation

Article Title: mGlu3 receptor regulates microglial cell reactivity in neonatal rats

doi: 10.1186/s12974-020-02049-z

Figure Lengend Snippet: mGlu3 receptor activation and microglial reactivity in CTRL and LPD/IL-1β cultured microglia at P4. a , b Microglial cells were stained with Iba1 (green) and DAPI (blue) under basal and challenged conditions (IL-1β + IFNγ) ± LY 379268 (1 μM) + Ro 64-5229 (25 μM). Representative photomicrographs at × 40 magnification are shown in a (scale bar = 50 μm). Four cell-culture wells for each condition were analyzed (mean cell number, 165 ± 1 7) and the cell area, cell perimeter, and cell circularity were assessed in b . Data (mean ± SEM). Two-way ANOVA followed by the Newman-Keuls multiple comparison test; * p < 0.05, ** p < 0.01, *** p < 0.001 effect of LY379268 + Ro 64-5229; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 effect of IL-1β + IFNγ; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 effect of LPD/IL-1β. c , d mRNA expression of pro-inflammatory ( c ) and anti-inflammatory/immune-regulatory ( d ) markers under basal and pro-inflammatory conditions ± LY 379268 (1 μM) + Ro 64-5229 (25 μM). Data (mean ± SEM) are relative to the gene expression under basal CTRL conditions. Two-way ANOVA followed by the Newman-Keuls multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.001 effect of LY 379268 + Ro 64-5229; ## p < 0.01, ### p < 0.001, #### p < 0.0001 effect of LPD/IL-1β; $ p < 0.05, $$$ p < 0.001; $$$$ p < 0.0001 effect of IL-1β + IFNγ

Article Snippet: LY 2389575 hydrochloride, a selective negative allosteric modulator of mGlu3 receptors (Tocris), and Ro 64-5229, a selective, non-competitive mGlu2 receptor antagonist (Tocris), were dissolved in DMSO.

Techniques: Activation Assay, Cell Culture, Staining, Comparison, Expressing, Gene Expression

Journal: Journal of Neuroinflammation

Article Title: mGlu3 receptor regulates microglial cell reactivity in neonatal rats

doi: 10.1186/s12974-020-02049-z

Figure Lengend Snippet: Pharmacological mGlu3 receptor blockade and microglial reactivity in response to inflammatory stimulation in vitro. a , b Microglial cells sorted from P4 pups were stained with Iba1 (green) and DAPI (blue) under basal conditions ± LY 2389575 (5 μM). Representative photomicrographs at × 40 magnification are shown in a (scale bar = 50 μm). The cell area, cell perimeter, and cell circularity were assessed in b (mean cell number, 162 ± 10). Data (mean ± SEM). Unpaired t test; ** p < 0.01. c , d mRNA expression of pro-inflammatory ( c ) and anti-inflammatory/immune-regulatory ( d ) markers in microglial cells sorted from P4 pups cultured under basal or pro-inflammatory (IL-1β + INFγ) conditions ± LY 2389575 (5 μM). The data (mean ± SEM) are relative to the gene expression under basal CTRL conditions. One-way ANOVA followed by the Newman-Keuls multiple comparison test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 effect of LY 2389575; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, $$$$ p < 0.0001 effect of IL-1β + INFγ. e , f Microglial cells sorted from P7 pups were stained with Iba1 (green) and DAPI (blue) under basal conditions ± LY 2389575 (5 μM). Representative photomicrographs at × 40 magnification are shown in e (scale bar = 50 μm). The cell area, cell perimeter, and cell circularity were assessed in f (mean cell number, 162 ± 10). Data (mean ± SEM). Unpaired t test * p < 0.01, ** p < 0.01. g , h mRNA expression of pro-inflammatory ( g ) and anti-inflammatory/immune-regulatory ( h ) markers in microglial cells sorted from P7 pups and cultured under basal and pro-inflammatory (IL-1β + INFγ) conditions ± LY 2389575 (5 μM). The data (mean ± SEM) are relative to the gene expression under basal CTRL conditions. One-way ANOVA followed by the Newman-Keuls multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001 effect of LY 2389575; $$ p < 0.01, $$$ p < 0.001, $$$$ p < 0.0001 effect of IL-1β + INFγ

Article Snippet: LY 2389575 hydrochloride, a selective negative allosteric modulator of mGlu3 receptors (Tocris), and Ro 64-5229, a selective, non-competitive mGlu2 receptor antagonist (Tocris), were dissolved in DMSO.

Techniques: In Vitro, Staining, Expressing, Cell Culture, Gene Expression, Comparison

Journal: Journal of Neuroinflammation

Article Title: mGlu3 receptor regulates microglial cell reactivity in neonatal rats

doi: 10.1186/s12974-020-02049-z

Figure Lengend Snippet: Effect of Grm3 genetic deletion in vivo and knock-down in vitro on microglial reactivity. a , b Iba1 + cells (red)/DAPI (blue) within Cingular white matter of P4 WT and Grm3 −/− mice under basal conditions and after IL-1β injections. Representative photomicrographs at × 10 magnification are shown in a (scale bar = 50 μm). Data (mean ± SEM) are expressed as number of Iba1 + /mm 2 in b . Two-way ANOVA followed by the Newman-Keuls multiple comparison; $ p < 0.05, $$ p < 0.01 effect of IL-1β + INFγ; # p < 0.05, ## p < 0.01 effect of Grm3 −/− . c , d Iba1 + cells within Cingular white matter of P4 WT and Grm3 −/− mice under basal conditions and after IL-1β injections. Representative photomicrographs at × 40 magnification are shown in c (scale bar = 25 μm). The cell area, cell perimeter, and cell circularity were assessed in d . Data (mean ± SEM). Two-way ANOVA followed by the Newman-Keuls multiple comparison; $$ p < 0.01, $$$ p < 0.001 effect of IL-1β + INFγ; # p < 0.05, ## p < 0.01, ### p < 0.001 effect of Grm3 −/− . e , f mRNA expression of pro-inflammatory ( e ) and anti-inflammatory/immune-regulatory ( f ) markers in Prefrontal/Cingular cortex isolated from P4 WT and Grm3 −/− mice under basal conditions and after IL-1β injections. Data (mean ± SEM) are relative to the mRNA expression of WT mice. Two-way ANOVA followed by the Newman-Keuls multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.01 effect of IL-1β + INFγ; # p < 0.05, ## p < 0.01, ### p < 0.001 effect of Grm3 −/− . g Grm3 mRNA expression in primary microglia cultures 48 h after transfection with Grm3 siRNA (25 nM) and with Grm3 siRNA (50 nM). Data (mean ± SEM) are relative to Grm3 expression in the scramble group (SCR). Unpaired t test; **** p < 0.0001. h mGlu3 receptor protein expression in primary microglia cultures 48 h after transfection with Grm3 siRNA (50 nM). Data (mean ± SEM) are relative to mGlu3 receptor protein expression in the scramble group (SCR). Three wells per condition pooled. Unpaired t test; ** p < 0.01. i , j mRNA expression of pro-inflammatory ( i ) and anti-inflammatory/immune-regulatory ( j ) markers in response to SCR and Grm3 siRNA (50 nM). Data (mean ± SEM) are relative to the mRNA expression for the respective SCR. Unpaired t test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: LY 2389575 hydrochloride, a selective negative allosteric modulator of mGlu3 receptors (Tocris), and Ro 64-5229, a selective, non-competitive mGlu2 receptor antagonist (Tocris), were dissolved in DMSO.

Techniques: In Vivo, Knockdown, In Vitro, Comparison, Expressing, Isolation, Transfection

Journal: Journal of Neuroinflammation

Article Title: mGlu3 receptor regulates microglial cell reactivity in neonatal rats

doi: 10.1186/s12974-020-02049-z

Figure Lengend Snippet: Effects of pharmacological mGlu3 receptor modulation and Grm3 knock-down on microglial Ccl9 expression in vitro. a , c mRNA expression of Ccl9 under the pro-inflammatory condition (IL-1β + IFNγ) in the presence of LY 379268 (1 μM) + Ro 64-5229 (25 μM) in CTRL and LPD/IL-1β cultured microglia at P4 ( a ) and at P7 ( c ). Data (mean ± SEM) are relative to the gene expression under basal CTRL conditions. Two-way ANOVA followed by the Newman-Keuls multiple comparison; * p < 0.05, *** p < 0.001 effect of LY 379268 + Ro 64-5229; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 effect of IL-1β + IFNγ; # p < 0.05, ### p < 0.001 effect of LPD/IL-1β. b , d mRNA expression of Ccl9 in microglial cells sorted from P4 ( b ) and P7 ( d ) rat pups cultured under basal or pro-inflammatory (IL-1β + INFγ) conditions ± LY 2389575 (5 μM). The data (mean ± SEM) are relative to the gene expression under basal CTRL conditions. One-way ANOVA followed by the Newman-Keuls multiple comparison test; ** p < 0.01, *** p < 0.001, **** p < 0.0001 effect of LY 2389575; $ p < 0.05 effect of IL-1β + INFγ. e Ccl9 mRNA expression in response to SCR and Grm3 siRNA (50 nM). Data (mean ± SEM) are relative to the mRNA expression for the respective SCR. Unpaired t test; **** p < 0.0001. f Pearson’s correlation between Grm3 expression and Ccl9 48 h after transfection with siGrm3 (50 nM)

Article Snippet: LY 2389575 hydrochloride, a selective negative allosteric modulator of mGlu3 receptors (Tocris), and Ro 64-5229, a selective, non-competitive mGlu2 receptor antagonist (Tocris), were dissolved in DMSO.

Techniques: Knockdown, Expressing, In Vitro, Cell Culture, Gene Expression, Comparison, Transfection