Abstract
Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi 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, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.
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The open-source machine-learning algorithm for large-scale EM data, CDeep3M (National Center for Microscopy and Imaging Research; UCSD, School of Medicine) is freely available at https://github.com/CRBS/cdeep3m2. Matlab and R code written for data analysis of this study is available in Supplementary Software 1.
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Acknowledgements
We thank D. J. Julius for the anti-P2Y12R antibody, J. Heller Brown and L. Mucke for discussions, G. Maki for graphics, K. Claiborn and F. Chanut for editorial assistance, and the Gladstone Histology and Microscopy Core for Imaris. The EM studies were in part supported by grants for shared infrastructure from NIGMS P41 GM10341, R24GM137200, S10OD021784 (to M.H.E.), and the Gladstone animal facility by NCRR RR18928. This research was supported by the Berkelhammer Award for Excellence in Neuroscience (to V.A.R. and A.S.M.), the UCSF Molecular and Cellular Immunology NIAID T32 AI007334 (to V.A.R., A.S.M., E.G.S.), AHA Postdoctoral Fellowship, and NINDS F32 NS096920 (to V.A.R.), NIA K99 AG062776 (to K.M.), NMSS Postdoctoral Fellowships FG-1708-28925 and FG-1944-A-1 (to A.S.M. and S.B.), NINDS K02 NS110973 (to M.A.P.), CTSI grant 5TL1TR001871 (to E.G.S.), NIA RF1 AG064926 (to K.A., J.J.P. and M.H.E.), NIA AG047313 and AG062234 (to J.J.P.), and the Simon Family Trust, the Dagmar Dolby Family Fund, Edward and Pearl Fein, the Conrad N. Hilton Foundation (17348), and NINDS R35 NS097976 (to K.A.).
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Authors and Affiliations
Contributions
M. Merlini and V.A.R. co-designed the study, performed imaging, analyzed and interpreted data and co-wrote the manuscript. K.M. and J.J.P. designed and performed the electrophysiology experiments and analyzed data. K.-Y.K. and E.A.B. designed and performed the EM experiments and analyzed data. P.E.R.C., T.D., Z.Y., M.G.H., M. Madany, D.N.S. and R. Tognatta quantified and analyzed data. A.S.M., Z.Y. and S.B. performed the FACS and gene expression experiments. E.G.S. performed imaging. M.A.P. prepared brains for EM analysis. R. Thomas designed statistical analysis. R.M.A. and B.C. performed histology and mouse genotyping. J.K.R. performed immunohistochemistry and the pilocarpine experiments. S.R.C. designed experiments, and M.H.E. designed experiments, supervised the EM experiments and analyzed data. K.A. conceived the study, designed experiments, interpreted data and co-wrote the manuscript with input from all authors.
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Peer review information Nature Neuroscience thanks Ryuta Koyama, Valentin Nagerl, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Microglia-specific PTX expression in MgPTX mice.
a, Confocal microscopy of PTX (red), neurons (white), microglia (green), and astrocytes (cyan) in cortex of MgPTX mice (left); right, schematic of MgPTX 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 MgWT mice (left); right, schematic of MgWT genetic background. Scale bar, 75 μm. n = 5 MgWT mice, 3 brain sections/mouse. ND, not detected.
Extended Data Fig. 2 Characterization of microglia in MgPTX mice.
a, Initial slope of microglial surveillance (% volume fill/min) in MgPTX and MgWT 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 MgPTX and MgWT 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 MgPTX and MgWT 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 MgPTX and MgWT 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 MgWT and MgPTX 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 2 P images of microglia (green) and F-actin (red) in MgWT and MgPTX 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 MgPTX mice.
a, 2 P 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 MgPTX and MgWT mice. Data are mean ± s.e.m. n = 7 mice per genotype. N, Normal; HS1, 1st hypersynchrony; D1, 1st depression; HS2, 2nd hypersynchrony; D2, 2nd 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 MgWT and MgPTX mice. Data are mean ± s.e.m. n = 13 MgPTX and n = 10 MgWT mice. No significant difference by unpaired two-tailed t-test. c, Confocal images of GFP-positive microglia in MgPTX and MgWT cortex. Scale bar, 50 µm. Data are mean ± s.e.m. n = 4 MgPTX mice and n = 4 MgWT mice. *** P = 0.0004 by unpaired two-tailed t-test.
Extended Data Fig. 5 Microglia-specific PTX expression in MgPTX-ind mice.
a, Schematic of Cx3cr1CreER/+:RosaPTX/tdTomato mice (MgPTX-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 MgPTX-ind mice 1.5 months after tamoxifen administration. Scale bar, 20 μm. Representative images are shown from n = 3 MgPTX-ind mice. c, Confocal microscopy of PTX (green), neurons (white), microglia (tamoxifen-induced tdTomato expression, red), and astrocytes (cyan) in cortex of MgPTX-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 MgPTX-ind mice.
a, Confocal images of PTX (green) and nuclei (DAPI, blue) in cortex of MgPTX-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 MgPTX-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-positive cells 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 MgPTX-ind mice.
a, In vivo 2 P time-lapse imaging of cumulative microglial surveillance in MgPTX-ind and Ctrl mice after tamoxifen induction. Scale bar, 20 µm. Data are mean ± s.e.m. n = 5 MgPTX-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 2 P time-lapse imaging of microglial directed process motility toward laser ablation in MgPTX-ind and Ctrl mice after tamoxifen induction. Scale bar, 20 μm. Data are mean ± s.e.m. n = 5 MgPTX-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 MgPTX-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 MgPTX-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 MgPTX-ind and Ctrl mice 1–2 months after tamoxifen administration. Scale bar, 50 µm. Data are mean ± s.e.m. n = 3 MgPTX-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 MgWT 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 MgWT and MgPTX 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 MgWT and MgPTX 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 MgPTX microglia compared to MgWT 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 MgWT mice. Scale bar, 25 µm. Representative images are shown for n = 3 mice.
Extended Data Fig. 9 Impaired microglia dynamics in MgPTX mice.
a, In vivo 2 P time-lapse imaging of individual microglial process extensions and retractions in awake MgWT and MgPTX mice during whisker stimulation (‘Stim’) and non-stimulation (‘Unstim’) conditions. Data are mean ± s.e.m. n = 6 mice per genotype. *P = 0.018 (MgWT Stimulated vs. all other conditions) and ***P = 0.0083 (MgWT vs. MgPTX mice) by two-way ANOVA. b, Velocity (blue vectors) of individual microglia process dynamics (white) in MgWT and MgPTX 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 MgPTX and MgWT mice. Data are mean ± s.e.m. n = 14 MgWT and n = 9 MgPTX mice. No significant difference bytwo-sample t-test of mean AUC and Permutation test.
Extended Data Fig. 10 Rho GTPase effects on microglia and neuronal activity in MgPTX mice.
a, In vivo 2 P images of microglial process extensions at baseline and after Rho/Rac/Cdc42 Activator I or vehicle (ACSF) in MgPTX 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 MgPTX mice. Deviation from zero = significant (P = 0.0023); R2 = 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 (MPFhigh microglia) 5 h after administration of Rho/Rac/Cdc42 Activator I. Data are mean ± s.e.m. n = 7 MgPTX mice. *** P = 0.0007 for overall treatment effect by unpaired two-tailed t test. d, Total Ca2+ signal in neurons surrounded by either MPFlow or MPFhigh microglia in the FOVs described in (b) and (c). Data are mean ± s.e.m. n = 7 MgPTX 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 MPFlow microglia at t0 and by MPFhigh microglia at t35. Data are mean ± s.e.m. n = 7 MgPTX mice. ** P = 0.0057 for overall treatment effect (paired two-tailed t test).
Supplementary information
Supplementary Video 1
Time-lapse in vivo 2P imaging of cumulative volume sampling of microglia surveillance in MgPTX and MgWT mice. A 20-μm z-stack was acquired in the cortex every 2 min for 1 h. Each frame is an overlay of the maximum-intensity projection acquired at ti and all previous frames. Scale bar, 20 μm.
Supplementary Video 2
Time-lapse in vivo 2P imaging of microglia-directed process motility toward a tissue laser ablation in MgPTX and MgWT mice. Maximum-intensity projection of a 40-μm z-stack acquired every 4 min for 116 min. Scale bar, 20 μm.
Supplementary Video 3
Animation of 3D reconstruction of cortical microglia in MgPTX and MgWT mice. Scale bar, 20 μm.
Supplementary Video 4
Spontaneous seizure in a MgPTX mouse. A spontaneous seizure was observed in a MgPTX mouse during routine cage inspection.
Supplementary Video 5
Time-lapse in vivo 2P imaging of cumulative volume sampling of microglia surveillance in MgPTX-ind and control mice. A 20-μm z-stack was acquired every 3 min for 57 min. Each frame is an overlay of the maximum-intensity z-projection acquired at t0 and all previous frames. Scale bar, 20 μm.
Supplementary Video 6
Time-lapse in vivo 2P imaging of microglia directed process motility toward a tissue laser ablation in MgPTX-ind and control mice. Maximum-intensity projection of a 40-μm z-stack acquired every 4 min for 1 h. Scale bar, 20 μm.
Supplementary Video 7
Simultaneous imaging of microglia and neuronal Ca2+ transients in awake mice during whisker stimulation. Ca2+ transients were detected through expression of jRCaMP1b in neurons (red) and microglia were visualized with the Cx3cr1GFP/+ reporter (green) in the whisker barrel cortex. Single-plane 2P imaging at 3.03 Hz for 55 s. Scale bar, 20 μm.
Supplementary Video 8
Time-lapse imaging of cumulative volume sampling by microglia in awake Cx3cr1GFP/+ mice during whisker stimulation. A 50-μm z-stack was acquired in the whisker barrel cortex every 5 min for 35 min. Each frame is an overlay of the maximum-intensity projection acquired at ti and all previous frames. Scale bar, 20 μm.
Supplementary Video 9
3D reconstruction of microglia contacts with neurons by SBEM and CDeep3M machine-deep-learning volumetric image analysis. A representative microglia (yellow) immunolabeled for P2Y12R with processes contacting two neighboring neurons (deep and light blues) and extending processes contacting the soma of a distal neuron (green).
Supplementary Video 10
Time-lapse imaging of microglia-directed process motility toward uncaged glutamate in MgPTX and MgWT mice in vivo. In vivo 2P time-lapse imaging of cortical microglia after bathing of the craniotomy/dura mater with caged l-glutamate or vehicle control (ACSF) with or without laser-induced uncaging. White dot indicates the uncaging location. Maximum-intensity projection of a 70-μm z-stack acquired every 3 min for 30 min. Scale bar, 20 μm.
Supplementary Video 11
Time-lapse imaging of cumulative volume sampling by microglia in awake MgPTX and MgWT mice during whisker stimulation. A 50-μm z-stack was acquired every 5 min for 35 min in the whisker barrel cortex. Each frame is an overlay of the maximum-intensity projection acquired at ti and all previous frames. Scale bar, 20 μm.
Supplementary Video 12
Time-lapse imaging of microglial surveillance in the whisker barrel cortex of MgPTX mice during cranial bathing with Rho/Rac/Cdc42 Activator I. In vivo 2P time-lapse imaging of microglia process motilities in the whisker barrel cortex during bathing of the craniotomy/dura mater with vehicle (ACSF) or Rho/Rac/Cdc42 Activator I. Maximum-intensity projection of a 70-μm z-stack acquired every 20 min for 5 h.
Supplementary Software 1
Merlini_Supplementary Software_R_code.txt (Code written in R used for statistical analyses) and Merlini_Supplementary Software_MATLAB_code.txt (Code written in Matlab used for neuronal activity analyses).
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Merlini, M., Rafalski, V.A., Ma, K. et al. Microglial Gi-dependent dynamics regulate brain network hyperexcitability. Nat Neurosci 24, 19–23 (2021). https://doi.org/10.1038/s41593-020-00756-7
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DOI: https://doi.org/10.1038/s41593-020-00756-7
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