Extended Data Figure 8. Microglia suppress neuronal activation via an ATP/AMP/ADO/A1R- dependent feedback mechanism.

a, Scheme for generation of mice with microglia-specific CD39 depletion by breeding Cd39^fl/fl mice to Cd39^fl/fl;Cx3cr1^{CreErt2/+(Jung)} mice followed by tamoxifen-mediated Cre induction at 4-6 weeks of age. b, Dot plots show relative expression of Entpd1, Il34, and Csf1 mRNA in the striatum of Cd39^fl/fl;Cx3cr1^CreErt2/+ mice and littermate controls normalized to Gapdh (n=5 and 6 mice, Entpd1: P=0.0012, Il34: P=0.38, Csf1: P=0.22, unpaired two-tailed t-test). c, left, Representative images of striatal sections from Cd39^fl/fl and Cd39^fl/fl;Cx3cr1^CreErt2/+ mice stained for IBA1 (microglia, green) and DAPI (nuclei, blue) (scale bar:100μm); right, dot plots show the average number of microglia per mm² per mouse in the striatum of Cd39^fl/fl and Cd39^fl/fl;Cx3cr1^CreErt2/+ mice (n=4 mice, P=0.33, unpaired two-tailed t-test with Welch’s correction for variance). d, Microglia-specific CD39 ablation leads to increased levels of neuronal PKA activity in the striatum as measured by phosphorylation levels of GLUR1 at Ser845 in striatal protein lysate from Cd39^fl/fl;Cx3cr1^CreErt2/+ and littermate controls, pGLUR1 levels have been normalized to total GLUR1 in each sample, (n=8 and 6 mice, P=0.029, two-tailed Mann-Whitney Test). e, f, Increased seizure response in Cd39^fl/fl;Cx3cr1^CreErt2/+: e, Dot plot shows number of stage IV-V seizures recorded within one hour in response to D1 agonist (SKF81297, 5mg/kg) (n=11 mice each, P=0.0004; unpaired two-tailed t-test). f, Bar graph showing percentage of mice (left) and dot plot showing number (right) of stage IV-V seizures in response to kainic acid (15mg/kg) in Cd39^fl/fl;Cx3cr1^CreErt2/+ mice as compared to littermate controls (n=5 and 8 mice; left, P=0.17, Fisher’s exact test with Yates correction, right, P=0.032, unpaired two-tailed t-test). g, left, Scheme for the generation of mice with a D1 neuron-specific Adora1 depletion by breeding Adora1^fl/fl mice to Drd1a^Cre/+ mice; right, dot plots show relative expression of Adora1 mRNA in the striatum of Adora1^fl/fl;Drd1a^Cre/+ mice and littermate controls normalized to Gapdh (n=5 and 4 mice, P=0.002, unpaired two-tailed t-test). h, Co-administration of A₁R agonist (CPA, 0.1mg/kg) and D1 agonist (SKF81297, 5mg/kg) does not prevent the increased seizure susceptibility in Adora1^fl/fl;Drd1a^Cre/+ mice (n=12 and 6 mice, P=0.009, Fisher’s exact test with Yates correction). i, Bar graph shows percentage of microglia deficient mice with seizures in response to D1 agonist alone (SKF81297, 5mg/kg, i.p.) or co-administered with an A_2AR agonist (CGS21680, 0.1mg/kg, i.p.) or an A₁R agonist (CPA, 0.1m/kg, i.p.) (n=9-10 mice, P=0.005, Chi-squared test with Bonferroni post hoc adjustment). j, A₁R agonist administration (CPA, 0.1mg/kg) normalizes increased PKA activity in Il34^fl/fl;Drd1a^Cre/+ mice but does not affect PKA activity in control Il34^fl/fl mice as measure by phosphorylation levels of GLUR1 at Ser845 in striatal protein lysate, pGLUR1 levels have been normalized to total GLUR1 expression in each sample (Il34^fl/fl mice, n=5 mice, P= 0.62, Il34^fl/fl;Drd1a^Cre/+ mice, n=5 mice, P=0.06, unpaired two-tailed t-test). All statistical tests are two-tailed; Data shown as mean± s.e.m.