MOESM1 of Dopamine receptor D3 signalling in astrocytes promotes neuroinflammation
Creators: name: Montoya, Andro
name: Elgueta, Daniela
name: Campos, Javier
name: Chovar, Ornella
name: Falcón, Paulina
name: Matus, Soledad
name: Alfaro, Iván
name: Bono, María
name: Pacheco, Rodrigo
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. Analysis of astrocyte activation in different areas of the mouse brain upon LPS treatment. (A) Wild-type mice received an i.p. injection of PBS (left panel) or 5 mg/kg of LPS (right panel) and 3h later were sacrificed and immunofluorescence analysis was performed in brain slices. Specific DRD3 (green) and GFAP (red) immunostaining was performed as indicated in the legend of Fig. 2. Nuclei were stained with DAPI (blue). Representative images of brain sections showing DRD3-immunostaing (top panels) and GFAP-immunostaining (middle panels). Pseudocolored image of a brain section showing the intensity of GFAP-associated immunoreactivity (bottom panel). Different regions of interest (ROI) were selected in different brain areas from LPS-treated mice (shown in white-framed squares; bottom-right panel). (B) High resolution images of ROIs selected (A; bottom-right panel) showing GFAP-immunostaining in different brain areas including cortex (ROI 1 and 2), subhypotalamic zone (ROI 3), corpus callosum (ROI 4), cerebral nuclei striatum (ROI 5) and cerebral nuclei pallidum (ROI 6). Figure S2. Systemic inflammation induced by LPS triggers the increase of inflammatory cytokines in the brain. WT mice were treated with an i.p. injection of LPS (5 mg/kg) or PBS (Control). 4h or 24h later, the midbrain/striatum structures were isolated, disaggregated, and the RNA was extracted and analysed by quantitative RT-PCR. (A) The transcript for TNF-α was determined 24 h after LPS administration. ***, p&lt;0.001 by two-tailed unpaired Student’s t-test. (B) The transcript for IL-1β was quantified after 4 and 24 h after LPS administration.**, p&lt;0.01; ***, p&lt;0.0001 by one-way ANOVA followed by Tukey’s post-hoc test. (A and B) Gapdh transcript was used as a house keeping for normalization. Data from 4-8 mice per group is shown. Values are the mean ± SEM. Figure S3. Genetic deficiency or pharmacologic antagonism of DRD3-signalling reduces the M1-to-M2 ratio of microglial cells in the midbrain of mice undergoing systemic inflammation induced by LPS. Associated to Fig. 4. Wild-type (WT) or DRD3 knockout (DRD3KO) mice were pre-treated or not with an i.p. injection of a DRD3-selective antagonist (PG01037; 30 mg/kg) and 1h later received an i.p. injection of LPS (5 mg/kg) or PBS. 24h after LPS administration, the midbrain/striatum structures were isolated, disaggregated, and M1 (CD16/32+CD206- cells) and M2 (CD16/32+CD206+ cells) phenotypes were analysed in living (ZAq-) microglial cells (CD11b+ CD45+) by flow cytometry. Representative contour-plots are shown. Numbers in red and blue indicate the percentage of M1 (CD16/32+CD206- cells) and M2 (CD16/32+CD206+ cells) microglia in each sample. Figure S4. Similar density of expression of microglial markers in the midbrain of mice WT and DRD3-deficient mice undergoing systemic inflammation induced by LPS. Wild-type (WT) or DRD3 knockout (DRD3KO) mice were pre-treated or not with an i.p. injection of a DRD3-selective antagonist (PG01037; 30 mg/kg) and 1h later received an i.p. injection of LPS (5 mg/kg) or PBS. 24h after LPS administration, the midbrain/striatum structures were isolated, disaggregated, and different molecular markers were analysed in microglial cells by flow cytometry. The density of CD16/32, CD206, CD11b and CD45 was determined as the mean-fluorescence intensity (MFI) in the population of living (ZombieAqua-) microglial cells (CD11b+ CD45+). Top panels show data from 4-5 mice per group. Each symbol represents a WT (white) or a DRD3KO (black) animal. In each experimental group, the line and error bars represent the mean ± SEM respectively. *, p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001; ****, p&lt;0.0001 by one-way ANOVA followed by Tukey’s post-hoc test. Bottom panels show representative histograms. Figure S5. Similar behaviour of microglia and astrocytes in the midbrain of WT and DRD3KO mice at early time-points after the induction of systemic inflammation triggered by LPS. WT or DRD3KO mice were treated with an i.p. injection of LPS (5 mg/kg) or PBS (Control). 4h later, the midbrain/striatum structures were isolated, disaggregated, and the inflammatory and anti-inflammatory phenotypes of microglia (A) and astrocytes (B) were analysed by flow cytometry as described in Figs. 4 and 6 respectively. (A and B) Top panels show representative contour-plots indicating the percentage of pro-inflammatory glia (red numbers) and anti-inflammatory glia (blue numbers). Bottom panels show the quantification of the frequencies of inflammatory (left-bottom panels) and anti-inflammatory (middle-bottom panels) phenotypes and the inflammatory-to-anti-inflammatory ratio (right-bottom panels). Data from 4 mice per group is shown. Each symbol represents a WT (white) or a DRD3KO (black) animal. In each experimental group, the line and error bars represent the mean ± SEM, respectively. *, p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001 by one-way ANOVA followed by Tukey’s post-hoc test. Figure S6. Similar density of expression of astrocytic markers in the midbrain of wild-type and DRD3-deficient mice undergoing systemic inflammation induced by LPS. WT or DRD3KO mice were treated with an i.p. injection of LPS (5 mg/kg) or PBS (Control). 24h later, the midbrain/striatum structures were isolated, disaggregated, and astrocytic markers were analysed by flow cytometry. The density of iNOS (left panels), Arg1 (middle panels) and GFAP (right panels) was determined as the mean fluorescence intensity (MFI) in the population of living (ZombieAqua-) astrocytes (GFAP+ cells). Top panels show the quantification from 8 mice per group. Each symbol represents a WT (white) or a DRD3KO (black) animal. In each experimental group, the line and error bars represent the mean ± SEM respectively. No significant differences were found among the different experimental groups. Bottom panels show representative histograms.