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• W3178113810 abstract "•GABA-receptive microglia interact with inhibitory synapses during development•Removing GABABRs from microglia alters inhibitory but not excitatory connectivity•Synapse pruning genes are altered in GABA-receptive microglia lacking GABABRs•Mice lacking microglial GABAB1Rs exhibit behavioral abnormalities Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types. Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types. Brain function relies on interactions among diverse cell types. Microglia are the primary brain macrophages and play diverse roles in tissue defense during infection and injury (Ransohoff and Perry, 2009Ransohoff R.M. Perry V.H. Microglial physiology: unique stimuli, specialized responses.Annu. Rev. Immunol. 2009; 27: 119-145Crossref PubMed Scopus (1307) Google Scholar). In the healthy developing brain, microglia regulate a plethora of processes that impact the organization of neural circuits, including synapse pruning (Thion et al., 2018Thion M.S. Ginhoux F. Garel S. Microglia and early brain development: An intimate journey.Science. 2018; 362: 185-189Crossref PubMed Scopus (125) Google Scholar; Bohlen et al., 2019Bohlen C.J. Friedman B.A. Dejanovic B. Sheng M. Microglia in Brain Development, Homeostasis, and Neurodegeneration.Annu. Rev. Genet. 2019; 53: 263-288Crossref PubMed Scopus (44) Google Scholar; Neniskyte and Gross, 2017Neniskyte U. Gross C.T. Errant gardeners: glial-cell-dependent synaptic pruning and neurodevelopmental disorders.Nat. Rev. Neurosci. 2017; 18: 658-670Crossref PubMed Scopus (129) Google Scholar; Wilton et al., 2019Wilton D.K. Dissing-Olesen L. Stevens B. Neuron-Glia Signaling in Synapse Elimination.Annu. Rev. Neurosci. 2019; 42: 107-127Crossref PubMed Scopus (99) Google Scholar). Synapses exhibit a striking molecular and functional heterogeneity, the best example of which is the dichotomy between excitatory and inhibitory synapses that possess distinct molecular components and properties (Favuzzi and Rico, 2018Favuzzi E. Rico B. Molecular diversity underlying cortical excitatory and inhibitory synapse development.Curr. Opin. Neurobiol. 2018; 53: 8-15Crossref PubMed Scopus (15) Google Scholar; Vogels and Abbott, 2009Vogels T.P. Abbott L.F. Gating multiple signals through detailed balance of excitation and inhibition in spiking networks.Nat. Neurosci. 2009; 12: 483-491Crossref PubMed Scopus (226) Google Scholar). These fundamental differences have profound implications for circuit function (Sohal and Rubenstein, 2019Sohal V.S. Rubenstein J.L.R. Excitation-inhibition balance as a framework for investigating mechanisms in neuropsychiatric disorders.Mol. Psychiatry. 2019; 24: 1248-1257Crossref PubMed Scopus (203) Google Scholar). However, whether microglia are generic effectors of synapse pruning or specialized microglia are able to discriminate between distinct synapse types is unknown. Our understanding of microglia diversity in both development and disease has been greatly enhanced by the examination of their transcriptomic differences at the single cell level (Hammond et al., 2019Hammond T.R. Dufort C. Dissing-Olesen L. Giera S. Young A. Wysoker A. Walker A.J. Gergits F. Segel M. Nemesh J. et al.Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.Immunity. 2019; 50: 253-271.e6Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar; Keren-Shaul et al., 2017Keren-Shaul H. Spinrad A. Weiner A. Matcovitch-Natan O. Dvir-Szternfeld R. Ulland T.K. David E. Baruch K. Lara-Astaiso D. Toth B. et al.A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease.Cell. 2017; 169: 1276-1290.e17Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar; Krasemann et al., 2017Krasemann S. Madore C. Cialic R. Baufeld C. Calcagno N. El Fatimy R. Beckers L. O’Loughlin E. Xu Y. Fanek Z. et al.The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases.Immunity. 2017; 47: 566-581.e9Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar; Li et al., 2019Li Q. Cheng Z. Zhou L. Darmanis S. Neff N.F. Okamoto J. Gulati G. Bennett M.L. Sun L.O. Clarke L.E. et al.Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing.Neuron. 2019; 101: 207-223.e10Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar; Masuda et al., 2019Masuda T. Sankowski R. Staszewski O. Böttcher C. Amann L. Sagar Scheiwe C. Nessler S. Kunz P. van Loo G. et al.Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.Nature. 2019; 566: 388-392Crossref PubMed Scopus (398) Google Scholar; Matcovitch-Natan et al., 2016Matcovitch-Natan O. Winter D.R. Giladi A. Vargas Aguilar S. Spinrad A. Sarrazin S. Ben-Yehuda H. David E. Zelada González F. Perrin P. et al.Microglia development follows a stepwise program to regulate brain homeostasis.Science. 2016; 353: aad8670Crossref PubMed Scopus (585) Google Scholar). Such analysis led to the discovery of disease-associated microglia (DAM), which act as universal immune sensors of neurodegeneration (Deczkowska et al., 2018Deczkowska A. Keren-Shaul H. Weiner A. Colonna M. Schwartz M. Amit I. Disease-Associated Microglia: A Universal Immune Sensor of Neurodegeneration.Cell. 2018; 173: 1073-1081Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). However, whether variations in microglial transcriptomes map onto differences in function in the healthy developing brain remains poorly understood. We explored the hypothesis that functional microglia diversity has evolved to ensure the selective pruning of excitatory versus inhibitory synapses. Thus far, the examination of microglia-mediated synaptic pruning has focused on excitatory synapses (Paolicelli et al., 2011Paolicelli R.C. Bolasco G. Pagani F. Maggi L. Scianni M. Panzanelli P. Giustetto M. Ferreira T.A. Guiducci E. Dumas L. et al.Synaptic pruning by microglia is necessary for normal brain development.Science. 2011; 333: 1456-1458Crossref PubMed Scopus (2133) Google Scholar; Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1991) Google Scholar). An association between microglia and inhibitory synapses has been suggested in the adult and under pathological conditions (Chen et al., 2014Chen Z. Jalabi W. Hu W. Park H.-J. Gale J.T. Kidd G.J. Bernatowicz R. Gossman Z.C. Chen J.T. Dutta R. Trapp B.D. Microglial displacement of inhibitory synapses provides neuroprotection in the adult brain.Nat. Commun. 2014; 5: 4486Crossref PubMed Scopus (168) Google Scholar; Lui et al., 2016Lui H. Zhang J. Makinson S.R. Cahill M.K. Kelley K.W. Huang H.-Y. Shang Y. Oldham M.C. Martens L.H. Gao F. et al.Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation.Cell. 2016; 165: 921-935Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, Liu et al., 2021Liu Y.-J. Spangenberg E. Tang B. Holmes T.C. Green K.N. Xu X. Microglia elimination increases neural circuit connectivity and activity in adult mouse cortex.J. Neurosci. 2021; 41: 1274-1287Crossref PubMed Scopus (21) Google Scholar), the latter of which are often characterized by an aberrant reactivation of developmental programs (Wilton et al., 2019Wilton D.K. Dissing-Olesen L. Stevens B. Neuron-Glia Signaling in Synapse Elimination.Annu. Rev. Neurosci. 2019; 42: 107-127Crossref PubMed Scopus (99) Google Scholar). However, support for this hypothesis has been to date limited to studies in the embryonic brain, where prenatal immune challenges regulate the laminar positioning and connectivity of neocortical parvalbumin (PV) interneurons (Squarzoni et al., 2014Squarzoni P. Oller G. Hoeffel G. Pont-Lezica L. Rostaing P. Low D. Bessis A. Ginhoux F. Garel S. Microglia modulate wiring of the embryonic forebrain.Cell Rep. 2014; 8: 1271-1279Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar; Thion et al., 2019Thion M.S. Mosser C.-A. Férézou I. Grisel P. Baptista S. Low D. Ginhoux F. Garel S. Audinat E. Biphasic Impact of Prenatal Inflammation and Macrophage Depletion on the Wiring of Neocortical Inhibitory Circuits.Cell Rep. 2019; 28: 1119-1126.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Here, we demonstrate that GABA-receptive microglia remodel inhibitory but not excitatory synapses during mouse postnatal cortical development. The selectivity revealed by this process identifies specialized microglia dedicated to remodeling distinct synapse types. To investigate whether microglia are required for inhibitory synapse development, we examined inhibitory connectivity after depleting myeloid cells—including microglia—for the first two postnatal weeks. Daily injections of the colony-stimulating factor 1 receptor inhibitor PLX5622 efficiently depleted microglia beginning at postnatal day 4 (P4) (Figures 1A and 1B ). We focused on the barrels in the mouse somatosensory (S1) cortex and its most abundant interneuron subtype, PV cells. At P15, PV inhibitory synapses onto excitatory neurons were increased in microglia-depleted mice compared to controls (Figure 1C). Of note, this phenotype was not due to changes in the distribution or number of PV interneurons (Figure S1A). Upon microglia depletion, significant changes in PV synapses were detected only after their initial assembly (P12) (Figures S1B and S1C), suggesting a role for microglia in the maturation or refinement rather than formation of these connections. Conversely, the depletion of microglia during the third and fourth postnatal weeks did not alter the density of PV synapses (Figures S1C and S1D). Synapse development in different neocortical areas follows common principles but is asynchronous (Pinto et al., 2013Pinto J.G.A. Jones D.G. Murphy K.M. Comparing development of synaptic proteins in rat visual, somatosensory, and frontal cortex.Front. Neural Circuits. 2013; 7: 97Crossref PubMed Scopus (29) Google Scholar). Consistent with this, the increase in PV synapses upon microglia depletion was also observed in the visual cortex (V1), although the exact time window was shifted in accordance with its later development (Figure S1E). Notably, inhibitory synapses made by dendrite-targeting somatostatin (SST) interneurons were also increased in P15 microglia-depleted mice (Figure S1F). As for PV cells, the density of SST interneurons was unaltered (Figure S1G).Figure S1Consequences of early postnatal microglia depletion for cortical connectivity, related to Figure 1Show full caption(A) Representative images and quantitation of the density and layer distribution of PV cells in control (n = 4) and microglia-depleted (n = 4) mice at P15 after P1-P15 microglia depletion. ns p > 0.05, Student’s t test (for density) and One-Way ANOVA followed by Sidak’s multiple comparisons test (for layer distribution). Scale bar equal 100 μm.(B) Quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons in P10 control (n = 4) or depleted (n = 4) mice after P1-P10 microglia depletion and in P12 control (n = 3) or depleted (n = 5) mice after P1-P12 microglia depletion. ns p > 0.05, One-Way ANOVA followed by Tukey’s multiple comparisons test.(C) Representative images and quantitation of Iba1+ microglia density in control (n = 4) and microglia-depleted (n = 4) mice at P30 after P15-P30 microglia depletion. ∗∗∗p < 0.001, Student’s t test. Scale bar equal 100 μm.(D) Quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons in P30 control (n = 4) and depleted (n = 3) mice after P15-P30 microglia depletion. One brain with incomplete depletion was excluded from the analysis. ns p > 0.05, Student’s t test.(E) Representative images and quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons in the primary visual cortex (V1) of P15 control (n = 6) and depleted (n = 9) mice after P1-P15 microglia depletion and of P25 control (n = 12) and depleted (n = 10) mice after P1-P25 microglia depletion. ns p > 0.05 and ∗p < 0.05, One-Way ANOVA followed by Tukey’s multiple comparisons test. Scale bar equal 2 μm.(F) Representative images and quantitation of synapses made in L1 of S1 by SST+ cells (SSTCre;Ai34 Synaptophysin-tdTomato labeled SST+ presynaptic terminals colocalizing with Gephyrin) in P15 control (n = 8) and depleted (n = 6) mice after P1-P15 microglia depletion. ∗p < 0.05, Student’s t test. Scale bar equal 2 μm.(G) Quantitation of the density of SST cells in P15 control (n = 6) and microglia-depleted (n = 5) mice after P1-P15 microglia depletion. ns p > 0.05, Mann-Whitney test.(H) Traces, frequency and amplitude of sIPSCs (n = 14 cells from 3 control and n = 17 cells from 5 depleted mice). ∗∗∗p < 0.001, ns p > 0.05, Student’s t test.(I) Traces, frequency and amplitude of sEPSCs (n = 17 cells from 4 control and n = 18 cells from 5 depleted mice). ∗∗∗p < 0.001, ns p > 0.05, Student’s t test.(J) sEPSC/sIPSC ratio (n = 14 cells from 3 control and n = 15 cells from 3 depleted mice). ns p > 0.05, Mann-Whitney test.(K) Schematic of synapses analyzed in L and M.(L) Representative images and quantitation of VGlut2+Homer1+ synapses onto excitatory cell dendrites (TdTomato+, labeled by virus injection of pyramidal neurons) in P15 control (n = 6) and depleted (n = 4) mice. ∗∗p < 0.01, Student’s t test. Scale bar equal 2 μm.(M) Quantitation of VGlut1+Homer1+ synapses made onto L4 excitatory cells (NeuN) in P15 control (n = 6) and depleted (n = 4) mice (left), onto PV cells in P15 control (n = 9) or depleted (n = 10) mice (middle) and onto excitatory cell dendrites (TdTomato+, labeled by virus injection of pyramidal neurons) in P15 control (n = 7) or depleted (n = 4) mice. ns p > 0.05, Mann-Whitney test for excitatory neurons and Student’s t test for PV cells and dendrites.(N) Representative images of Iba1+ microglia in control and P1-P15 microglia-depleted brains at the indicated stages of repopulation and quantitation of microglia density in control and microglia-repopulated brains at P17, P19, P21, P25 and P30 (n = 3-4 mice per condition). ∗∗∗p < 0.001, ns p > 0.05, One-Way ANOVA followed by Sidak’s multiple comparisons test. Scale bar equal 100 μm.(O) Quantitation of synapses made in L1 of S1 by SST+ cells (SSTCre;Ai34 labeled presynaptic terminals colocalizing with Gephyrin) in P30 control (n = 4) and microglia-repopulated (n = 5) mice after P1-P15 microglia depletion. ∗p < 0.05, Student’s t test.(P) Quantitation of Syt2+Gephyrin+ synapses (left), SSTCre;Ai34+Gephyrin+ synapses (middle) and VGlut2+Homer1+ synapses (right) made by PV, SST and thalamic cells in P60 control (n = 6-8) and microglia-repopulated (n = 4-7) mice after P1-P15 microglia depletion. ExCs: excitatory cell soma. ns p > 0.05, Student’s t test for SST+ and thalamic synapses and Mann-Whitney test for PV synapses.All data are mean ± SEM, each data point represents one experimental animal except in F, I and J where it represents one cell. Arrowheads indicate colocalization.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Representative images and quantitation of the density and layer distribution of PV cells in control (n = 4) and microglia-depleted (n = 4) mice at P15 after P1-P15 microglia depletion. ns p > 0.05, Student’s t test (for density) and One-Way ANOVA followed by Sidak’s multiple comparisons test (for layer distribution). Scale bar equal 100 μm. (B) Quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons in P10 control (n = 4) or depleted (n = 4) mice after P1-P10 microglia depletion and in P12 control (n = 3) or depleted (n = 5) mice after P1-P12 microglia depletion. ns p > 0.05, One-Way ANOVA followed by Tukey’s multiple comparisons test. (C) Representative images and quantitation of Iba1+ microglia density in control (n = 4) and microglia-depleted (n = 4) mice at P30 after P15-P30 microglia depletion. ∗∗∗p < 0.001, Student’s t test. Scale bar equal 100 μm. (D) Quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons in P30 control (n = 4) and depleted (n = 3) mice after P15-P30 microglia depletion. One brain with incomplete depletion was excluded from the analysis. ns p > 0.05, Student’s t test. (E) Representative images and quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons in the primary visual cortex (V1) of P15 control (n = 6) and depleted (n = 9) mice after P1-P15 microglia depletion and of P25 control (n = 12) and depleted (n = 10) mice after P1-P25 microglia depletion. ns p > 0.05 and ∗p < 0.05, One-Way ANOVA followed by Tukey’s multiple comparisons test. Scale bar equal 2 μm. (F) Representative images and quantitation of synapses made in L1 of S1 by SST+ cells (SSTCre;Ai34 Synaptophysin-tdTomato labeled SST+ presynaptic terminals colocalizing with Gephyrin) in P15 control (n = 8) and depleted (n = 6) mice after P1-P15 microglia depletion. ∗p < 0.05, Student’s t test. Scale bar equal 2 μm. (G) Quantitation of the density of SST cells in P15 control (n = 6) and microglia-depleted (n = 5) mice after P1-P15 microglia depletion. ns p > 0.05, Mann-Whitney test. (H) Traces, frequency and amplitude of sIPSCs (n = 14 cells from 3 control and n = 17 cells from 5 depleted mice). ∗∗∗p < 0.001, ns p > 0.05, Student’s t test. (I) Traces, frequency and amplitude of sEPSCs (n = 17 cells from 4 control and n = 18 cells from 5 depleted mice). ∗∗∗p < 0.001, ns p > 0.05, Student’s t test. (J) sEPSC/sIPSC ratio (n = 14 cells from 3 control and n = 15 cells from 3 depleted mice). ns p > 0.05, Mann-Whitney test. (K) Schematic of synapses analyzed in L and M. (L) Representative images and quantitation of VGlut2+Homer1+ synapses onto excitatory cell dendrites (TdTomato+, labeled by virus injection of pyramidal neurons) in P15 control (n = 6) and depleted (n = 4) mice. ∗∗p < 0.01, Student’s t test. Scale bar equal 2 μm. (M) Quantitation of VGlut1+Homer1+ synapses made onto L4 excitatory cells (NeuN) in P15 control (n = 6) and depleted (n = 4) mice (left), onto PV cells in P15 control (n = 9) or depleted (n = 10) mice (middle) and onto excitatory cell dendrites (TdTomato+, labeled by virus injection of pyramidal neurons) in P15 control (n = 7) or depleted (n = 4) mice. ns p > 0.05, Mann-Whitney test for excitatory neurons and Student’s t test for PV cells and dendrites. (N) Representative images of Iba1+ microglia in control and P1-P15 microglia-depleted brains at the indicated stages of repopulation and quantitation of microglia density in control and microglia-repopulated brains at P17, P19, P21, P25 and P30 (n = 3-4 mice per condition). ∗∗∗p < 0.001, ns p > 0.05, One-Way ANOVA followed by Sidak’s multiple comparisons test. Scale bar equal 100 μm. (O) Quantitation of synapses made in L1 of S1 by SST+ cells (SSTCre;Ai34 labeled presynaptic terminals colocalizing with Gephyrin) in P30 control (n = 4) and microglia-repopulated (n = 5) mice after P1-P15 microglia depletion. ∗p < 0.05, Student’s t test. (P) Quantitation of Syt2+Gephyrin+ synapses (left), SSTCre;Ai34+Gephyrin+ synapses (middle) and VGlut2+Homer1+ synapses (right) made by PV, SST and thalamic cells in P60 control (n = 6-8) and microglia-repopulated (n = 4-7) mice after P1-P15 microglia depletion. ExCs: excitatory cell soma. ns p > 0.05, Student’s t test for SST+ and thalamic synapses and Mann-Whitney test for PV synapses. All data are mean ± SEM, each data point represents one experimental animal except in F, I and J where it represents one cell. Arrowheads indicate colocalization. We next asked whether the structural increase in PV synapses was paralleled by a functional increase in PV inhibition. While recording from excitatory neurons, we stimulated PV cells expressing Channelrhodopsin-2 (PVCre/+;Ai32) and found that the amplitude of optogenetically evoked inhibitory postsynaptic currents (IPSCs) was significantly increased in microglia-depleted animals compared to controls (Figure 1D). The increased inhibitory connectivity onto excitatory neurons in microglia-depleted mice was also confirmed by a higher frequency of miniature and spontaneous inhibitory synaptic currents (mIPSCs and sIPSCs) (Figures 1E and S1H). Together, these results demonstrate the existence of sequential and temporally restricted waves during which the maturation of cortical inhibitory circuits is regulated by microglia. For comparison, we examined the impact of microglia depletion on glutamatergic connectivity. The frequency of miniature and spontaneous excitatory synaptic currents (mEPSCs and sEPSCs) was higher in P15 microglia-depleted mice compared to controls (Figures 1F and S1I). Importantly, developmental microglia depletion did not significantly alter miniature or spontaneous EPSC/IPSC frequency ratios (Figures 1G and S1J). Consistently, structural synapse analyses showed that both PV and excitatory neurons received more thalamocortical synapses in microglia-depleted mice (Figures 1H and S1K–S1M). Taken together, these experiments suggest that microglia regulate the development of both excitatory and inhibitory synapses. We next asked whether the exuberant connectivity recovers once microglia are allowed to repopulate the brain. Within 48 h after cessation of the P1–P15 depletion treatment, microglia sequentially repopulated subcortical and cortical regions. Cortical repopulation was complete by P21 (Figures 1I and S1N). At P30, the supernumerary inhibitory and excitatory synapses persisted (Figures 1J, 1K, and S1O). However, in the adult (P60), synapse density returned to control levels (Figure S1P). This indicates that depleting microglia during development causes long-lasting, albeit not permanent, defects in inhibitory and excitatory connectivity. The previous experiments are consistent with microglia refining inhibitory synapses during development. Microglia-mediated synapse remodeling has been posited to depend on distinct and sequential processes: chemotaxis, target recognition, and phagocytosis (Neniskyte and Gross, 2017Neniskyte U. Gross C.T. Errant gardeners: glial-cell-dependent synaptic pruning and neurodevelopmental disorders.Nat. Rev. Neurosci. 2017; 18: 658-670Crossref PubMed Scopus (129) Google Scholar; Wilton et al., 2019Wilton D.K. Dissing-Olesen L. Stevens B. Neuron-Glia Signaling in Synapse Elimination.Annu. Rev. Neurosci. 2019; 42: 107-127Crossref PubMed Scopus (99) Google Scholar). In response to various chemotactic signals, microglia are attracted to and interact with neurons and synapses (Badimon et al., 2020Badimon A. Strasburger H.J. Ayata P. Chen X. Nair A. Ikegami A. Hwang P. Chan A.T. Graves S.M. Uweru J.O. et al.Negative feedback control of neuronal activity by microglia.Nature. 2020; 586: 417-423Crossref PubMed Scopus (175) Google Scholar; Cserép et al., 2020Cserép C. Pósfai B. Lénárt N. Fekete R. László Z.I. Lele Z. Orsolits B. Molnár G. Heindl S. Schwarcz A.D. et al.Microglia monitor and protect neuronal function through specialized somatic purinergic junctions.Science. 2020; 367: 528-537Crossref PubMed Scopus (160) Google Scholar; Madry and Attwell, 2015Madry C. Attwell D. Receptors, ion channels, and signaling mechanisms underlying microglial dynamics.J. Biol. Chem. 2015; 290: 12443-12450Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To test whether microglia directly interact with inhibitory synapses during development, we used confocal and stimulated emission depletion (STED) super-resolution microscopy (Figure 2A). We generated mice expressing fluorescent reporters in both microglia and PV synaptic terminals. To this end, we injected Cx3cr1GFP/+ mice with adeno-associated viruses (AAVs) expressing synaptophysin-tdTomato under the control of a PV-specific enhancer (Vormstein-Schneider et al., 2020Vormstein-Schneider D. Lin J.D. Pelkey K.A. Chittajallu R. Guo B. Arias-Garcia M.A. Allaway K. Sakopoulos S. Schneider G. Stevenson O. et al.Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans.Nat. Neurosci. 2020; 23: 1629-1636Crossref PubMed Scopus (40) Google Scholar). At P15, microglia contacted 10% of PV boutons and their processes ensheathed these presynaptic terminals. Moreover, the fraction of PV boutons contacted by microglia increased between P12 and P15, peaked at P15–P17, and decreased by P30 (Figure 2B). The developmental interactions of microglia with excitatory synapses involve their phagocytic engulfment and elimination (Neniskyte and Gross, 2017Neniskyte U. Gross C.T. Errant gardeners: glial-cell-dependent synaptic pruning and neurodevelopmental disorders.Nat. Rev. Neurosci. 2017; 18: 658-670Crossref PubMed Scopus (129) Google Scholar; Wilton et al., 2019Wilton D.K. Dissing-Olesen L. Stevens B. Neuron-Glia Signaling in Synapse Elimination.Annu. Rev. Neurosci. 2019; 42: 107-127Crossref PubMed Scopus (99) Google Scholar). We therefore examined whether similar processes occur at inhibitory synapses. Because fluorescence quenching and protein degradation by lysosomal proteases may affect the detection of fluorescent proteins inside microglial lysosomes, we used the acid-tolerant monomeric green fluorescent protein Gamillus (Katayama et al., 2008Katayama H. Yamamoto A. Mizushima N. Yoshimori T. Miyawaki A. GFP-like proteins stably accumulate in lysosomes.Cell Struct. Funct. 2008; 33: 1-12Crossref PubMed Scopus (143) Google Scholar; Shinoda et al., 2018aShinoda H. Ma Y. Nakashima R. Sakurai K. Matsuda T. Nagai T. Acid-Tolerant Monomeric GFP from Olindias formosa.Cell Chem. Biol. 2018; 25: 330-338.e7Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, Shinoda et al., 2018bShinoda H. Shannon M. Nagai T. Fluorescent Proteins for Investigating Biological Events in Acidic Environments.Int. J. Mol. Sci. 2018; 19: 1548Crossref Scopus (44) Google Scholar). We injected AAVs expressing synaptophysin-Gamillus to label PV synaptic terminals in mice with genetically labeled microglia (Tmem119CreER/+;Ai14) (Figure 2A). At P15, a subset of these boutons was encapsulated within microglia and colocalized with microglial lysosomes (Figures 2C and 2D). Classical complement proteins tag subsets of excitatory synapses for elimination by microglia (Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1991) Google Scholar; Stevens et al., 2007Stevens B. Allen N.J. Vazquez L.E. Howell G.R. Christopherson K.S. Nouri N. Micheva K.D. Mehalow A.K. Huberman A.D. Stafford B. et al.The classical complement cascade mediates CNS synapse elimination.Cell. 2007; 131: 1164-1178Abstract Full Text Full Text PDF PubMed Scopus (1806) Google Scholar). We examined complement C1q accumulation on PV boutons and found that C1q was deposited on 10% of PV synaptic terminals at P15 (Figure 2E). Next, we compared inhibitory and excitatory connectivity in controls versus C1qa knockout mice (C1q−/−). At P15, PV inhibitory synapses onto excitatory neurons were increased in C1q−/− mice (Figure 2F). The frequency of both mIPSCs and mEPSCs was also increased (Figures 2G and 2H). As with microglia depletion, there was no significant change in the ratio of mEPSCs/mIPSC frequency received by each cell (Figure 2I). Thus, C1q deficiency mimicked the defects observed in microglia-depleted mice, demonstrating that C1q is involved in regulating inhibitory connectivity. To visualize real-time interactions, we performed in vivo two-photon imaging of microglia and PV boutons during the peak contact period (P15–P17) in S1 (Figures 3A–3D and S2). The imaging experiments revealed a bimodal distribution in microglia-PV synapse dynamics. Microglia either contacted few PV puncta (14%) or engaged" @default.
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• W3178113810 date "2021-07-01" @default.
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• W3178113810 title "GABA-receptive microglia selectively sculpt developing inhibitory circuits" @default.
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