Microglial Gi-dependent dynamics regulate brain network hyperexcitability

Abstract

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that G_i-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating Mg^PTX mice to genetically inhibit G_i in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, G_i-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.

Extended Data Fig. 1. Microglia-specific PTX expression in Mg^PTX mice

a, Confocal microscopy of PTX (red), neurons (white), microglia (green), and astrocytes (cyan) in cortex of Mg^PTX mice (left); right, schematic of Mg^PTX genetic background. PTX expressed in 63.9 ± 1.3% (mean ± s.e.m.) of GFP-positive microglia and 0.69 ± 0.43% (mean ± s.e.m.) of NeuN-positive neurons. Scale bar, 75 μm. n = 5 mice, 3 brain sections/mouse. ND, not detected. Quantification in an independent mouse cohort of n = 3 mice with similar results is shown in Fig. 1a. PTX expression in microglia (white arrows) and rare occasion of NeuN-positive cell co-localizing with PTX (yellow arrow). b, Confocal microscopy of PTX (red, not detected), neurons (white), microglia (green), and astrocytes (cyan) in cortex of Mg^WT mice (left); right, schematic of Mg^WT genetic background. Scale bar, 75 μm. n = 5 Mg^WT mice, 3 brain sections/mouse. ND, not detected.

Extended Data Fig. 2. Characterization of microglia in Mg^PTX mice

a, Initial slope of microglial surveillance (% volume fill/min) in Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 6 mice per genotype. * P = 0.0174 by unpaired two-sample t-test. b, Confocal images (top) and filament reconstruction (bottom) of GFP-expressing microglia in Mg^PTX and Mg^WT mice and quantification of total process branch points and total process length per cell. Scale bar, 10 μm. Data are mean ± s.e.m. n = 4 mice per genotype. * P = 0.0142 and ** P = 0.0067 by unpaired two-tailed t-test. c, Confocal images of GAD65/67 (red) in Mg^PTX and Mg^WT cortex with microglia contacts (GFP, green) onto neuronal somata (NeuN, blue) surrounded by GAD65/67 puncta. Scale bar, 10 μm. Data are mean ± s.e.m. n = 5 mice per genotype. **** P < 0.0001 by unpaired two-tailed t-test. d, Relative expression of P2ry12 and Kcnk13 normalized to Gapdh in FACS-sorted microglia from cortex and hippocampus of Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 3 mice per genotype. ns, not significant by unpaired two-tailed t-test. e, Confocal images of THIK-1 (red) and microglial GFP (green) co-localization (yellow, arrows) in Mg^WT and Mg^PTX hippocampus. Scale bar, 10 μm. Data are mean ± s.e.m. n = 4 mice per genotype, 3 brain sections/brain region/mouse. **P = 0.0053, ns, not significant by two-way ANOVA. f, In vivo 2P images of microglia (green) and F-actin (red) in Mg^WT and Mg^PTX mice. Scale bar, 15 μm. Data are mean ± s.e.m. n = 3 mice per genotype. 5–8 microglia quantified/mouse. * P = 0.0128 by unpaired two-tailed t-test.

Extended Data Fig. 3. Dendritic spine density and synaptic markers in Mg^PTX mice

a, 2P in vivo images of cortical dendritic spines (YFP). Scale bar, 10 μm. Data are mean ± s.e.m. n = 5 mice per genotype. No significant difference by unpaired two-tailed t-test. b, 3D reconstruction of excitatory synapses (green spheres) in cortical volumes obtained by SBEM. Scale bar, 2 μm. Data are mean ± s.e.m. n = 5 mice per genotype. No significant difference by unpaired two-tailed t-test. c, Epifluorescent images of NeuN-positive neurons (red) in the cortex. Scale bar, 100 μm. Data are mean ± s.e.m. n = 5 mice per genotype. No significant difference by unpaired two-tailed t-test. d, Confocal images of cortical GAD65/67. Scale bar, 50 μm. Data are mean ± s.e.m. n = 5 mice per genotype. No significant difference by unpaired two-tailed t-test. e, Confocal images of cortical vGLUT1. Scale bar, 20 μm. Data are mean ± s.e.m. n = 5 mice per genotype. No significant difference by unpaired two-tailed t-test.

Extended Data Fig. 4. Effects of microglia-specific Gi inhibition on neural excitability.

a, Gamma oscillatory power for the different EEG stages of pilocarpine-induced network hyper-synchronization in Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 7 mice per genotype. N, Normal; HS1, 1^st hypersynchrony; D1, 1^st depression; HS2, 2^nd hypersynchrony; D2, 2^nd depression. No significant difference (n.s.) between genotypes for each stage by two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 1. b, Pre-pilocarpine in vivo baseline EEG recordings in Mg^WT and Mg^PTX mice. Data are mean ± s.e.m. n = 13 Mg^PTX and n = 10 Mg^WT mice. No significant difference by unpaired two-tailed t-test. c, Confocal images of GFP-positive microglia in Mg^PTX and Mg^WT cortex. Scale bar, 50 μm. Data are mean ± s.e.m. n = 4 Mg^PTX mice and n = 4 Mg^WT mice. *** P = 0.0004 by unpaired two-tailed t-test.

Extended Data Fig. 5. Microglia-specific PTX expression in Mg^PTX-ind mice

a, Schematic of Cx3cr1^CreER/+:Rosa^PTX/tdTomato mice (Mg^PTX-ind) with inducible Gi shutdown and microglia-specific tdTomato expression. b, Confocal images of PTX (blue), Iba1 (green), tdTomato (red), and tdTomato-expressing microglia (yellow, co-localization) in cortex of Mg^PTX-ind mice 1.5 months after tamoxifen administration. Scale bar, 20 μm. Representative images are shown from n = 3 Mg^PTX-ind mice. c, Confocal microscopy of PTX (green), neurons (white), microglia (tamoxifen-induced tdTomato expression, red), and astrocytes (cyan) in cortex of Mg^PTX-ind mice. Exclusive expression of PTX in microglia (white arrows). PTX expressed in 97.3 ± 0.76% (mean ± s.e.m.) of tdTomato-positive microglia. Expression in neurons and astrocytes not detected (ND). Scale bar, 75 μm. Data are mean ± s.e.m. n = 5 mice, 3 brain sections/mouse. d, Confocal microscopy of PTX (green), neurons (white), microglia (tamoxifen-induced tdTomato expression, red), and astrocytes (cyan) in cortex of Ctrl mice. PTX expression not detected (ND). Scale bar, 75 μm. n = 5 mice, 3 brain sections/mouse.

Extended Data Fig. 6. Tamoxifen-dependent microglial PTX expression in Mg^PTX-ind mice

a, Confocal images of PTX (green) and nuclei (DAPI, blue) in cortex of Mg^PTX-ind mice with or without tamoxifen. PTX expression in 97.2 ± 0.62% (mean ± s.e.m.) of tdTomato-expressing microglia after tamoxifen; 69.6 ± 3% (mean ± s.e.m.) tdTomato-positive cells after tamoxifen and 11.67 ± 1.32% (mean ± s.e.m.) tdTomato-positive cells prior to tamoxifen. Scale bar, 75 μm. Data are mean ± s.e.m. n = 3 Mg^PTX-ind mice per condition, 3 brain sections/mouse. *** P = 0.0001 by unpaired two-tailed t-test. ND, not detected. b, Confocal images of PTX (green) and nuclei (DAPI, blue) in cortex of Ctrl mice with or without tamoxifen (left). PTX expression was not detected; 74.7 ± 1.72% (mean ± s.e.m. tdTomato expression after tamoxifen and 7.65 ± 0.35% (mean ± s.e.m.) tdTomato-positive cells prior to tamoxifen. Scale bar, 75 μm. Data are mean ± s.e.m. n = 3 Ctrl mice per condition, 3 brain sections/mouse. *** P < 0.0001 by unpaired two-tailed t-test. ND, not detected.

Extended Data Fig. 7. Microglia dynamics and seizures in Mg^PTX-ind mice

a, In vivo 2P time-lapse imaging of cumulative microglial surveillance in Mg^PTX-ind and Ctrl mice after tamoxifen induction. Scale bar, 20 μm. Data are mean ± s.e.m. n = 5 Mg^PTX-ind and n = 4 Ctrl mice. Overall genotype effect P-value = 0.04 by two-sample t-test of mean AUC. * P < 0.05 for comparison at individual time points by unpaired two-sample t-test. b, In vivo 2P time-lapse imaging of microglial directed process motility toward laser ablation in Mg^PTX-ind and Ctrl mice after tamoxifen induction. Scale bar, 20 μm. Data are mean ± s.e.m. n = 5 Mg^PTX-ind and n = 4 Ctrl mice. Overall genotype effect ^# P = 0.005 by two-sample t-test of mean AUC. * P < 0.05, ** P < 0.01 for comparison at individual time points by two-sample t-test. c, Pilocarpine dose (left) and latency (right) to reach Stage 4 seizures. Data are mean ± s.e.m. n = 8 Mg^PTX-ind and n = 4 Ctrl mice. * P = 0.0138, ** P = 0.0061, respectively by unpaired two-tailed t-test. d, Percentage of THIK-1 co-localization with tdTomato-expressing microglia in Mg^PTX-ind and Ctrl mice. Data are mean ± s.e.m. n = 4 mice per genotype, 3 brain sections/brain region/mouse. *P = 0.0105. n.s., not significant by one-way ANOVA. e, Confocal images of cortical Tomato-positive microglia in Mg^PTX-ind and Ctrl mice 1–2 months after tamoxifen administration. Scale bar, 50 μm. Data are mean ± s.e.m. n = 3 Mg^PTX-ind mice and n = 4 Ctrl mice. Not significant by unpaired two-tailed t-test. sections/brain region/mouse. *P = 0.0105. n.s., not significant by one-way ANOVA. e, Confocal images of cortical Tomato-positive microglia in Mg^PTX-ind and Ctrl mice 1–2 months after tamoxifen administration. Scale bar, 50 μm. Data are mean ± s.e.m. n = 3 Mg^PTX-ind mice and n = 4 Ctrl mice. Not significant by unpaired two-tailed t-test.

Extended Data Fig. 8. Glutamate-induced microglia–neuron interactions

a, Location of microglia process contacts onto jRCaMP1b-expressing neurons in the whisker barrel cortex of Mg^WT mice (images shown in Fig. 2d). Each data point represents the average percentage of all microglia–neuron contacts quantified in an individual mouse. Data are mean ± s.e.m. n = 6 mice. AIS, axonal initial segment. b, Number of microglia in the FOV during in vivo 2P time-lapse imaging of microglial motilities and neuronal activity in awake Mg^WT and Mg^PTX mice. Data are mean ± s.e.m. n = 8 mice per genotype. Not significant, (P = 0.114) by two-tailed unpaired t-test. c, Correlation analysis of intraneuronal Ca²⁺ accumulation and number of microglia in awake Mg^WT and Mg^PTX mice. n = 8 mice per genotype. R² = 0.0059; deviation from zero = not significant (P = 0.856) by linear regression analysis (dotted red line). d, Gene expression of metabotropic (Grm) and ionotropic (Grin) receptors and glutamate transporters (Slc) in Mg^PTX microglia compared to Mg^WT microglia. Data from microglia from n = 3 mice/genotype. Dotted lines indicate 1.5-fold change threshold levels. *P = 0.0245; n.s., not significant by unpaired two-tailed t-test. e, In vivo 2P time-lapse imaging of focal (arrows) glutamate uncaging-induced neuronal Ca²⁺ transients (jRCaMP1b, red) and directed microglia motilities (green) in the cortex of awake Mg^WT mice. Scale bar, 25 μm. Representative images are shown for n = 3 mice.

Extended Data Fig. 9. Impaired microglia dynamics in Mg^PTX mice

a, In vivo 2P time-lapse imaging of individual microglial process extensions and retractions in awake Mg^WT and Mg^PTX mice during whisker stimulation (“Stim”) and non-stimulation (“Unstim”) conditions. Data are mean ± s.e.m. n = 6 mice per genotype. *P = 0.018 (Mg^WT Stimulated vs. all other conditions) and ***P = 0.0083 (Mg^WT vs. Mg^PTX mice) by two-way ANOVA. b, Velocity (blue vectors) of individual microglia process dynamics (white) in Mg^WT and Mg^PTX mice. Scale bar, 25 μm. Data are mean ± s.e.m. n = 6 mice per genotype. No significant difference by two-way ANOVA with post hoc Tukey’s multiple comparison test. c, Ca²⁺ peak amplitudes of whisker stimulation-induced neuronal Ca²⁺ transients in Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 14 Mg^WT and n = 9 Mg^PTX mice. No significant difference by-two-sample t-test of mean AUC and Permutation test.

Extended Data Fig. 10. Rho GTPase effects on microglia and neuronal activity in Mg^PTX mice

a, In vivo 2P images of microglial process extensions at baseline and after Rho/Rac/Cdc42 Activator I or vehicle (ACSF) in Mg^PTX mice. Scale bar, 25 μm. Data are mean ± s.e.m. n = 7 mice per condition, 5–10 microglia/mouse/condition. ** P = 0.0054 by paired two-tailed t test. b, Correlation between microglia process fill (MPF) and cumulative whisker stimulus-induced neuronal Ca²⁺ levels 5 h after Rho/Rac/Cdc42 Activator I administration. Data are mean ± s.e.m. n = 7 Mg^PTX mice. Deviation from zero = significant (P = 0.0023); R = 0.553 (linear regression analysis [red line]); 95% confidence intervals (dotted black lines). c, Cumulative Ca²⁺ levels in neurons surrounded by microglia with MPF increase at t₃₅ compared to t₀ of the whisker stimulation (MPF_high microglia) 5 h after administration of Rho/Rac/Cdc42 Activator I. Data are mean ± s.e.m. n = 7 Mg^PTX mice. *** P = 0.0007 for overall treatment effect by unpaired two-tailed t test. d, Total Ca²⁺ signal in neurons surrounded by either MPF_low or MPF_high microglia in the FOVs described in (b) and (c). Data are mean ± s.e.m. n = 7 Mg^PTX mice. * P = 0.01073 and ** P = 0.00672 at individual time points (multiple unpaired t tests, Holm-Sidak). e, Intraneuronal Ca²⁺ peak FWHM of neurons surrounded by MPF_low microglia at t₀ and by MPF_high microglia at t₃₅. Data are mean ± s.e.m. n = 7 Mg^PTX mice. ** P = 0.0057 for overall treatment effect (paired two-tailed t test).

Fig. 1:. Effects of Gi inhibition on microglia dynamics and seizures.

a, Generation of Mg^PTX mice (left); confocal microscopy in cortex of PTX (red) and microglia (green) in Mg^PTX and Mg^WT mice. Arrows indicate PTX-positive microglia. Scale bar, 50 μm. 60.5 ± 2.7% (mean ± s.e.m.) of cortical GFP-positive cells express PTX. n = 3 mice. b, In vivo 2P time-lapse imaging of cumulative microglial surveillance. Data are mean ± s.e.m. n = 6 mice per genotype. Overall genotype effect ^# P = 0.024 by unpaired two-sample t-test of mean AUC. * P < 0.05, ** P < 0.01 at individual time points by permutation test. c, In vivo 2P time-lapse imaging of microglial directed process motility towards laser ablation. Scale bar, 20 μm. Data are mean ± s.e.m. n = 4 mice per genotype. Overall genotype effect *** P = 0.0004 by unpaired two-sample t-test of mean AUC. Dotted line indicates * P < 0.05, ** P < 0.01, ^# P < 0.001 at individual time points by unpaired two-sample t-test. d, Survival curves of Mg^PTX and Mg^WT mice. n = 13 mice per genotype. ** P = 0.0056 by Log-rank test. e, Pilocarpine dose (left) and latency (right) to reach Stage 4 seizures for Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 6 mice per genotype. * P = 0.014 and 0.017, respectively, by-unpaired two-tailed t-test. f, Spectrograms (top) and gamma oscillatory power traces (bottom) from EEG recordings during pilocarpine induced-seizures. EEG stages: N, normal; HS1, 1^st hypersynchrony; D1, 1^st depression; HS2, 2^nd hypersynchrony; D2, 2^nd depression; M, Mortality. Representative data from n = 7 mice per genotype with similar results. g, Cumulative time to each pilocarpine-induced seizure stage in Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 7 mice per genotype. * P = 0.032 for genotype effect by two-way repeated-measures ANOVA. h, Survival curves for pilocarpine-induced time to death (left) and from 1^st epileptiform spike to death (right). n = 7 mice per genotype. * P = 0.0127 (time to death) and 0.0333 (time from 1^st spike to death) by Gehan-Breslow-Wilcoxon test.

Fig. 2:. Microglial Gi-dependent dynamics and evoked neuronal activity in awake mice.

a, In vivo 2P imaging of microglia (GFP, green) and neuronal Ca²⁺ (jRCaMP1b, red) transients in the barrel cortex during whisker stimulation in awake Cx3cr1^GFP/+ mice. Scale bar, 50 μm. Data are mean ± s.e.m. n = 12 mice. **** P < 0.0001 by two-way ANOVA. b, In vivo 2P images of cumulative microglial surveillance during whisker stimulation. Scale bar, 20 μm. Data are mean ± s.e.m. n = 12 mice per condition. Overall condition effect ^# P = 0.021 by unpaired two-sample t-test of mean AUC. Dotted line indicates * P < 0.05 at individual time points by permutation test. c, SBEM and volumetric image analysis of microglial cell contacts with surrounding neurons. (i) Transmitted light microscopy of immunoperoxidase-labeled P2Y₁₂R cell bodies (arrows) and processes (arrowheads). Scale bar, 20 μm. 2D electron micrographs of microglia (yellow) contact sites on nearest neighbor neuron somata (ii, iii; deep and light blue), a dendrite (iv, light blue), a distal neuron soma (v, green). Scale bars, 1 μm. (vi) 3D reconstruction of a microglia (yellow) contacting two nearest neurons (deep and light blues) and a distal neuron (green). SBEM volume dimensions (inset). Representative 3D reconstruction from 4 microglia from n = 3 Mg^WT mice. d, Evoked and non-evoked neuronal somata upon whisker stimulation in contact with microglia in Mg^WT and Mg^PTX mice. Data are mean ± s.e.m. n = 5 mice per genotype. *** P = 0.0003 and **** P < 0.0001 by two-way ANOVA and Tukey’s multiple comparisons test; Right, in vivo 2P image (top) and 3D reconstruction (bottom) of microglia (green) contacting a jRCaMP1b-expressing neuronal soma (red) in Mg^WT barrel cortex during whisker stimulation. Scale bar, 20 μm. e, In vivo 2P time-lapse of microglial directed motility in response to glutamate uncaging in somatosensory cortex (left); right, microglia area fill around uncaging site (yellow dot). Scale bar, 20 μm. Data are mean ± s.e.m. n = 5 Mg^PTX and n = 4 Mg^WT mice. Overall genotype effect ^# P = 0.015 by unpaired two-sample t-test of mean AUC. * P < 0.05, ** P < 0.01 at individual time points by unpaired two-sample t-test.

Fig. 3.. Hypersynchronized neuronal activity after microglia-specific G_i inhibition.

a, In vivo 2P imaging of cumulative microglia surveillance (green) and neuronal activity (red) in the barrel cortex upon whisker stimulation in awake Mg^WT and Mg^PTX mice. Scale bar, 20 μm. Data are mean ± s.e.m. n = 7 mice (unstimulated) and n = 8 mice (whisker stimulation). Overall stimulation effect ^# P = 0.042 by unpaired two-sample t-test of mean AUC. * P < 0.05 at individual time points by permutation test. n.s., no significant difference for overall effect by-unpaired two-sample t-test of mean AUC. b, Heatmaps of cumulative evoked neuronal Ca²⁺ and quantification of neuronal activity and peak full width at half maximum (FWHM) during whisker stimulation in awake Mg^PTX and Mg^WT mice. Data are mean ± s.e.m. n = 9 Mg^PTX and n = 14 Mg^WT mice. Neuronal activity, overall genotype effect ^# P = 0.016 by unpaired two-sample t-test of mean AUC. * P < 0.05, ** P < 0.01, *** P < 0.001 at individual time points by permutation test. FWHM, overall genotype effect ^# P = 0.00009 by unpaired two-sample t-test of mean AUC and * P < 0.05, ** P < 0.01, *** P < 0.001 for individual time points by permutation test. c, Clustered correlation matrix of synchronized neuronal network firing (top) and raster plots of synchronized neuronal network burst activity (bottom) during whisker stimulation in Mg^WT and Mg^PTX mice. Red arrowheads indicate whisker stimulus. Data are mean ± s.e.m. n = 5 Mg^PTX and n = 6 Mg^WT mice. * P = 0.0139, ** P = 0.0092 by unpaired two-tailed t-test.