Fig 2: 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-1ß + IFN?) in the presence of LY 379268 (1 µM) + Ro 64-5229 (25 µM) in CTRL and LPD/IL-1ß 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-1ß + IFN?; #p < 0.05, ###p < 0.001 effect of LPD/IL-1ß. 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-1ß + INF?) 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-1ß + INF?. 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)

Fig 3: Transcriptomic analysis of Grm3 deregulation associated with neonatal neuroinflammation in rats. a Functional network enrichment in microglial cells magnetically sorted from P4 animals under basal conditions (CTRL) and following LPD/IL-1ß challenge inducing neonatal neuroinflammation. The 20 most enriched molecular functions are reported in the graph, sorted by increasing enrichment score. The ontologies associated with glutamate receptor activity are underlined in red. b Description of the number of genes mapped and their respective FDR values for each molecular function. In red are reported the gene ontology pathways that were associated with glutamate receptor activity. c Grm gene expression in microglia cells sorted from CTRL and LPD/IL-1ß animals at P4 according to a Gene Set Enrichment Analysis against the Gene Ontology Database. Data (mean ± SEM) are relative to Grm1 expression in CTRL animals. Multiple t test; #p < 0.05. d Grm3 gene expression in microglial cells just sorted from CTRL and LPD/IL-1ß animals at P4 analyzed by quantitative RT-PCR. Data (mean ± SEM) are relative to Grm3 expression in CTRL animals. Unpaired t test; ##p < 0.01. e Grm3 gene expression assessed in primary microglia from CTRL and LPD/IL-1ß animals at P4 after 48 h cell culture. Data (mean ± SEM) are relative to Grm3 expression in CTRL animals. Unpaired t test; ####p < 0.0001. f Minimal Grm3 network showing the centrality of Grm3 gene deregulation following LPD/IL-1ß exposure, selecting deregulated genes that are connected to more than 10 genes. Betweenness centrality of hub genes is quantified by the circle size, and the number of connections among genes by the color code. The figure shows that Grm3 has a central position in the network. g The same Grm3 minimal network as in f showing the expression of Grm3 and Grm3-associated genes. The blue color vs the red color intensity is associated with down- and upregulation, respectively

Fig 4: 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-1ß injections. Representative photomicrographs at × 10 magnification are shown in a (scale bar = 50 µm). Data (mean ± SEM) are expressed as number of Iba1+/mm2 in b. Two-way ANOVA followed by the Newman-Keuls multiple comparison; $p < 0.05, $$p < 0.01 effect of IL-1ß + INF?; #p < 0.05, ##p < 0.01 effect of Grm3-/-. c, d Iba1+ cells within Cingular white matter of P4 WT and Grm3-/- mice under basal conditions and after IL-1ß injections. Representative photomicrographs at × 40 magnification are shown in c (scale bar = 25 µm). The cell area, cell perimeter, and cell circularity were assessed in d. Data (mean ± SEM). Two-way ANOVA followed by the Newman-Keuls multiple comparison; $$p < 0.01, $$$p < 0.001 effect of IL-1ß + INF?; #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-1ß 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-1ß + INF?; #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

Fig 5: mGlu3 receptor activation and microglial reactivity in CTRL and LPD/IL-1ß cultured microglia at P4. a, b Microglial cells were stained with Iba1 (green) and DAPI (blue) under basal and challenged conditions (IL-1ß + IFN?) ± 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-1ß + IFN?; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 effect of LPD/IL-1ß. 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-1ß; $p < 0.05, $$$p < 0.001; $$$$p < 0.0001 effect of IL-1ß + IFN?