Extended Data Fig. 1: DREADD-based mouse models to study microglia responses to neuronal activation and inhibition reveals distinct microglia responses.
From: Negative feedback control of neuronal activity by microglia
a, b, Neuron-specific activation (a) and inhibition (b) has been achieved by the expression of the Gq-coupled (activating) hM3Dq or Gi-coupled (inhibiting) hM4Di in CaMKII+ forebrain neurons. The CaMKII-tTa mice were bred to either tetO-CHRM3 or tetO-CHRM4 mice to generate CaMKII-tTa; tetO-CHRM3 or CaMKII-tTa; tetO-CHRM4 mice. hM3Dq or hM4Di were activated by i.p. injection of clozapine-N-oxide (CNO) to activate (0.25 mg kg–1) or inhibit (1 mg kg–1) CaMKII+ neuronal activity, respectively. c-e, Validation of CNO-mediated neuronal activation and inhibition: c, Heatmap (left) and violin plot (right) show RNA expression levels of 18 immediate early genes in total striatum 2 h after CNO-mediated neuronal inhibition (orange) or neuronal activation (blue) as compared with controls (n = 2 CaMKII-tTa; tetO-CHRM4, n = 5 control, and n = 3 CaMKII-tTa; tetO-CHRM3 mice) (right, P = 0.0001, One-way ANOVA (Kruskal–Wallis test) with Dunn’s multiple comparison test). d, Dot plot showing quantification of the average number of cFOS+ cells in the dorsal striatum of CaMKII-tTa; tetO-CHRM4 (orange, n = 4 mice), control (black, n = 6 mice), and CaMKII-tTa; tetO-CHRM3 (blue, n = 4 mice) mice one hour after treatment with CNO (P = 0.0004, One-way ANOVA with Tukey’s post hoc test). e, Representative images showing cFOS+ cells (green) in the striatum of CaMKII-tTa; tetO-CHRM4 (top), control (middle), and CaMKII-tTa; tetO-CHRM3 (bottom) mice in response to CNO, DAPI (blue) (image are representative of two independent cohorts of mice). f, To allow for the microglia-specific analysis of changes in ribosome-associated RNA levels following neuron inhibition, the CaMKII-tTa; tetO-CHRM4 mice were bred to Cx3cr1CreErt2/+(Litt); Eef1a1LSL.eGFPL10a/+ mice followed by tamoxifen-induced Cre-mediated L10a-eGFP expression in microglia. g, Changes in ribosome-bound mRNA levels in striatal microglia were determined using the TRAP-sequencing approach. The heatmap shows the variation in the expression levels of 135 upregulated and 220 downregulated genes (z-scored log2(RPKM) at 2 h following CNO-mediated neuronal inhibition. h, Selected gene ontology (using GO) annotations for upregulated genes (using DESeq2) in striatal microglia in response to neuronal inhibition, GO analysis was performed using ENRICHR analysis69,70 (dotted line, P = 0.05). i, Venn diagrams comparing microglial genes up- and downregulated following CaMKII+ neuronal activation and inhibition reveals highly differential microglia response. j, qPCR confirmation of increased mRNA expression (lower ΔCT, normalized to Gapdh) in microglia upon neuronal activation (Ccl3, left, n = 3 mice, P = 0.059, unpaired two-tailed t-test) and neuronal inhibition (Cd74, right, n = 2 mice). k, Dot plots show lack of expression changes in selected genes in the striatum of wild type mice 2 h after saline, 0.25 mg kg–1 CNO injection, or 1 mg kg–1 CNO injection (n = 3, 3, and 4 mice; Kdm6b: P = 0.70 Adrb1: P = 0.22, Ccl24: P = 0.54, Ccl3: P = 0.43, Kcnk13: P = 0.37, Ikbkb, P = 0.62, One-way ANOVA with Tukey’s post hoc test). RPKM: reads per kilobase of transcript per million mapped reads, TRAP: translating ribosome affinity purification; Data shown as mean ± s.e.m.
