4

Figure S1. Consequences of early postnatal microglia depletion for cortical connectivity, related to Figure 1 (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 pyramidalneurons) 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.

Figure S2. Microglia-PV synapse interactions during development, related to Figure 2 (A) Masks showing the criteria for contact identification in the in vivo imaging experiments. Percentages indicate the fraction of bouton area contacted by microglia.

(B) Proportion of PV boutons contacted by one microglia during the in vivo imaging experiments in deep layer 3 (n = 15 cells contacting 68 boutons) or layer 4 (n = 36 cells contacting 154 boutons). Data are the same as in (C) and (I) where they are shown as pooled dataset. ns p > 0.05, Mann-Whitney test. Data are mean ± SEM, each data point represents one cell.

(C) Distribution of Cx3cr1GFP/+microglia contacting the indicated percentages of PV boutons in a 20 μmradius around the microglia cell body over 20 minutes (n = 51 cells from 6 mice) during the in vivo imaging experiments. PV boutons are labeled with PVe-Syp-tdTomato. The distribution is shown as density histogram and kernel density estimation. The first peak corresponds to 11.7% of PV boutons contacted by microglia and the second peak to 60.9%. The local minimum of the bimodal distribution is 26.4%. These data were pooled with data in (I) to generate the graph in Figure 3C.

(D) Distribution of microglia-PV boutons interactions for the indicated duration during the in vivo imaging experiments. The distribution is shown as density histogram and kernel density estimation of the proportion of Syp-tdTomato+ PV synaptic boutons contacted by microglia for the indicated duration (n = 222 PV boutons from 6 mice).

(E) Duration of contacts between microglia interacting with a minority (n = 11 cells) or the majority (n = 25 cells) of local PV boutons during the in vivo imaging experiments. *p < 0.01, Mann-Whitney test. These data represent the subset of data shown in (D) for which it was possible to visualize the microglia cell body and they were pooled with data in (K) to generate the graph shown in Figure 3D. Data were split based on the local minimum of the kernel density estimation in (C). Data are mean ± SEM, each data point represents one cell.

(F) Time-lapse images from Video S2 showing a Syp-tdTomato+ PV bouton being enwrapped by a microglia process (Cx3cr1GFP/+) and contacted by a second process over 10 minutes. Scale bar equal 10 μm.

(G) Time-lapse images from Video S3 showing a Cx3cr1GFP/+ microglial process that terminates into what resembles a phagocytic cup engulfing a Syp-tdTomato+ PV bouton over 20 minutes. Scale bar equal 10 μm.

(H) Time-lapse image from in vivo imaging of Syp-Gamillus+ PV boutons and microglia genetically labeled using Tmem119CreER/+;Ai14 mice. Scale bar equal 10 μm.

(I) Distribution of Tmem119CreER/+;Ai14 microglia contacting the indicated percentages of PV boutons (labeled with PVe-Syp-Gamillus) in a 20 μm radius around the cell body over 20 minutes (n = 37 cells from 6 mice). The distribution is shown as density histogram and kernel density estimation. The first peak corresponds to 18.1% of PV boutons contacted by microglia and the second peak to 57.4%. The local minimum of the bimodal distribution is 34.4%. These data were pooled with data in (C) to generate the graph in Figure 3C.

(J) Distribution of microglia-PV boutons interactions for the indicated duration. The distribution is shown as density histogram and kernel density estimation of the proportion of Syp-Gamillus+ PV synaptic boutons contacted by microglia for the indicated duration (n = 115 PV boutons from 6 mice).

(K) Duration of contacts between microglia interacting with a minority (n = 13 cells) or the majority (n = 23 cells) of local PV boutons. *p < 0.01, Student’s t test. These data represent the subset of data shown in (J) for which it was possible to visualize the microglia cell body and they were pooled with data in E to generate the graph shown in Figure 3D. Data are mean ± SEM, each data point represents one cell.

(L) Representative images of the same 3D Imaris space in two different orientations. Note that, in the visualization of a 3D volume, boutons can appear closer than they are depending on the orientation. In contrast, “real contacts” appear close regardless of the orientation. Scale bar equal 5 μm.

(M) Representative 3D reconstruction of a microglia process and surrounding PV boutons. Contacted PV boutons are reconstructed as red spots whereas PV boutons not contacted by microglia are reconstructed as light blue spots. Full and empty arrowheads indicate positive and negative (i.e., boutons that did not meet the contact criteria) examples, respectively. The yellow empty arrowhead indicates a bouton that is far from the process. In all relevant figures, only contacted boutons (see methods for criteria) have been highlighted as “reconstructed spots.” Scale bar equal 5 μm.

Figure S3. Expression of GABAb receptors in microglia, related to Figure 2 (A) Schematic of analysis and heatmaps showing ligands-receptor pairs that are leading candidates for mediating interneuron-microglia communication. The heatmap on the left is showing the expression (at the indicated developmental time points) of putative ligands enriched in interneurons as compared to pyramidal cells. For P5 and P10 interneurons, the corresponding values in SST and PV cells were averaged. The list is ranked according to specificity and developmental upregulation. For GABA, the expression of the GABA-synthetizing enzyme Gad1 is shown. The heatmap on the right is showing the expression (at the indicated developmental time points) of receptors expressed in microglia that are experimentally validated interactors of the ligands shown on the left. For the P3-P9 time point, values at P3 and P9 were averaged.

(B) Venn diagram depicting the fraction of microglia expressing Gabbr1+ mRNA, Gabbr2+ mRNA as well as double positive for both Gabbr1+ and Gabbr2+ mRNAs at P15 (smFISH: single molecule fluorescent in situ hybridization). The diagram is a visualization of the data shown in Figure 3F.

(C) Validation of the specificity of GABAB1R antibody. Representative images from P15 controls and VGlut1Cre;Gabbr1 cKO mice, lacking GABAB1R in cortical excitatory neurons. Note the absence of GABAB1R signal within NeuN+ excitatory neurons in VGlut1Cre cKO mice. Scale bar equal 10 μm.

(D) Left: quantitation of the percentage of microglia (Iba1+) expressing GABAB1R protein at P0, P10 and P15 in control and Cx3cr1Cre GABAB1R cKO mice (n = 3–6 each). ns p > 0.05, **p < 0.01, ***p < 0.001, One-Way ANOVA followed by Sidak’s multiple comparisons test. Note the higher expression of GABAB1R in controls at P15 compared to P0 or P10. Right: quantitation of the percentage of microglia (Iba1+) expressing GABAB1R protein at P15 in control and Tmem119CreER GABAB1R cKO mice (n = 4 each). *p < 0.05, Student’s t test. Data are mean ± SEM, each data point represents one experimental animal.(E) Representative images and quantitation of microglia (Iba1+) expressing both GABAb-|R and GABAB2R proteins in P15 control mice (n = 3) within layer 4 of S1. Scale bar equal 5 μm. See STAR Methods for details about noise in the IHC experiment versus smFISH. Arrowheads indicate colocalization. Data are mean ± SEM, each data point represents one experimental animal.(F) Gabbr1 and Gabbr2 expression in adult microglia/macrophages from different brain regions. Data and visualization are from Saunders et al. (2018). FC: frontal cortex, TH: thalamus, STR: striatum, PC: posterior cortex, CB: cerebellum, SN: substantia nigra, HC: hippocampus, GP: globus pallidus. The reported confidence intervals reflect statistical sampling noise.(G) Fraction of GABA-receptive microglia (defined as expressing both Gabbr1 and Gabbr2 mRNAs by single molecule fluorescent in situ hybridization) in layer 4 of S1 and the CA1 region of the hippocampus of Cx3cr1GFP/+ mice at P15. S1 data are the same as shown in Figure 3F. Data are mean ± SEM, each data point represents one experimental animal.(H) 3D reconstruction and fraction of PV boutons (PVe-Syp-tdTom) encased by Gabbr2+ and Gabbr2-microglia (Cx3cr1GFP/+) at P15 (n = 58 Gabbr2+ and 59 Gabbr2-cells from 4 mice). ***p < 0.001, Mann-Whitney test. Scale bar equal 1 μm. Data are mean ± SEM, each data point represents one cell.(I) 3D reconstruction and fraction ofVGlut2+ boutons encased by Gabbr2+ and Gabbr2-microglia (Cx3cr1GFP/+) at P15 (n = 23 Gabbr2+ and 36 Gabbr2-cells from 4 mice). *p < 0.05, Student’s t test. Scale bar equal 1 mm. Data are mean ± SEM, each data point represents one cell.

Figure S4. Synaptic phenotype of GABAB1R cKO mice, related to Figure 3 (A) Left: quantitation of PV boutons contacted by microglia at P15 in wild-type control mice (n = 7, Cx3cr1+/+;GABAB1Rfl/fl), GABAB1R cKO mice (n = 5, Cx3cr1Cre/+; GABAB1Rfl/fl) and Cx3cr1−/+ heterozygous control mice (n = 8, Cx3cr1Cre/+; GABAB1R+/+) mice. One-Way ANOVA followed by Tukey’s multiple comparisons test. Wild-type control and cKO data are the same as shown in Figure 4A. Right: quantitation of PV boutons contacted by microglia at P15 in control (n = 4, Tmem119+/+;GABAB1Rfl/fl) and cKO (n = 6, Tmem119CreER/+;GABAB1Rfl/fl) mice. Student’s t test. *p < 0.05, **p < 0.01.(B) Left: quantitation of VGlut2 boutons contacted by microglia at P15 in wild-type control mice (n = 7, Cx3cr1+/+;GABAB1Rfl/fl), GABAB1R cKO mice (n = 5, Cx3cr1Cre/+; GABAB1Rfl/fl) and Cx3cr1−/+ heterozygous control mice (n = 6, Cx3cr1Cre/+; GABAB1R+/+) mice. One-Way ANOVA followed by Tukey’s multiple comparisons test. Wild-type control and cKO data are the same as shown in Figure 4B. Right: quantitation of VGlut2 boutons contacted by microglia at P15 in control (n = 8, Tmem119+/+;GABAB1Rfl/fl) and cKO (n = 8, Tmem119CreER/+;GABAB1Rfl/fl) mice. Student’s t test. ns p > 0.05.(C) Distribution of microglia-PV boutons interactions for the indicated duration during the in vivo imaging experiments in controls (n = 181 PV boutons from 6 mice) and cKOs (Cx3cr1GFP/+;Tmem119CreER/+;GABAB1Rfl/fl, n = 99 PV boutons from 3 mice) cKO mice. The distribution is shown as kernel density estimation of the proportion of PV boutons contacted by microglia for the indicated duration. Control data are the same as in Figure S2D but limited to a total duration of 20 minutes to allow comparison with cKO data (see STAR Methods). **p < 0.01, Kolmogorov–Smirnov test. Note the complex effect of GABAB1R removal on the contact duration, the cKO distribution is bimodal with some contacts having a shorter duration and others being abnormally long.(D) Left: quantitation of Syt2+Gephyrin+ synapses made by PV cells onto excitatory neurons at P15 in wild-type control mice (n = 8, Cx3cr1+/+;GABAB1Rfl/fl), GABAB1R cKO mice (n = 6, Cx3cr1Cre/+; GABAB1Rfl/fl) and Cx3cr1−/+heterozygous control mice (n = 6, Cx3cr1Cre/+; GABAB1R+/+) mice. One-Way ANOVA followed by Holm-Sidak’s multiple comparisons test. Wild-type control and cKO data are the same as shown in Figure 4H. Right: Syt2+Gephyrin+ synapses made by PV cells onto excitatory neurons at P15 in control (n = 8, Tmem119+/+;GABAB1Rfl/fl) and cKO (n = 6, Tmem119CreER/+;GABAB1Rfl/fl) mice. Student’s t test. *p < 0.05, **p < 0.01.(E) Image, relative frequency histogram and overlaid kernel density estimation of Syt2+Gephyrin+ synapses imaged using STED super-resolution microscopy andmade by PV cells onto excitatory neurons at P15 in wild-type controls (n = 58 cells from 3 Cx3cr1+/+;GABAB1Rfl/fl mice) and GABAB1R cKO mice (n = 66 cells from 3 Cx3cr1Cre/+; GABAB1Rfl/fl mice). ***p < 0.001, Kolmogorov–Smirnov test. The dotted line indicates the soma outline.(F) Left: quantitation of VGlut2+Homer1+ synapses made onto excitatory neurons at P15 in wild-type control mice (n = 6, Cx3cr1+/+;GABAB1Rfl/fl), GABAB1R cKO mice (n = 7, Cx3cr1Cre/+; GABAB1Rfl/fl) and Cx3cr1−/+ heterozygous control mice (n = 9, Cx3cr1Cre/+; GABAB1R+/+) mice. One-Way ANOVA followed by Holm-Sidak’s multiple comparisons test. Wild-type control and cKO data are the same as shown in Figure 4I. Right: VGlut2+Homer1+ synapses made onto excitatory neurons at P15 in control (n = 9, Tmem119+/+;GABAB1Rfl/fl) and cKO (n = 8, Tmem119CreER/+;GABAB1Rfl/fl) mice. Student’s t test. ns p > 0.05.(G) Quantitation of VGlut2+Homer1+ synapses onto L4 PV neurons in P15 control (n = 6, Cx3cr1+/+;GABAB1Rfl/fl) and cKO (n = 5, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. ns p > 0.05, Student’s t test.(H) Quantitation of VGlut1+Homer1+ synapses onto L4 excitatory cells (left) or pyramidal cell dendrites (right) in control (n = 7–9, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 6–7, Cx3cr1Cre/+; GABAB1Rfl/fl) mice at P15. ns p > 0.05, Mann-Whitney test for somatic analysis and Student’s t test for dendritic analysis. (I) and (J) Traces, frequency and amplitude of mIPSCs (n = 26 cells from 3 Tmem119+/+;GABAB1Rfl/fl control mice and n = 28 cells from 4 Tmem119CreER/+; GABAB1Rfl/fl cKO mice) and mEPSCs (n = 26 cells from 3 Tmem119+/+; GABAB1Rfl/fl control mice and n = 31 cells from 4 Tmem119CreER/+;GABAB1Rfl/fl cKO mice) recorded from layer 4 excitatory neurons at P15. ***p < 0.001, ns p > 0.05, Student’s t test except for mEPSC amplitude where Mann-Whitney test was used.(K) mEPSC/mIPSC frequency ratio (n = 26 cells from 3 Tmem119+/+; GABAB1Rfl/fl control mice and n = 28 cells from 4 Tmem119CreER/+;GABAB1Rfl/fl cKO mice). ***p < 0.001, Mann-Whitney test.(L) Left: quantitation of Syt2+Gephyrin+ synapses made by PV cells onto S1 L4 excitatory neurons in control (n = 7, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 7, Cx3cr1Cre/+; GABAB1Rfl/fl) mice at P30. Right: quantitation of Syt2+Gephyrin+ synapses made by PV cells onto S1 L4 excitatory neurons in control (n = 6, Tmem119+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 7, Tmem119CreER/+;GABAB1Rfl/fl) mice at P30. **p < 0.01, Student’s t test.(M) Left: quantitation of Syt2+Gephyrin+ synapses made by PV cells onto V1 L4 excitatory neurons at P30 in control (n = 6, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 7, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. Right: quantitation of PV+ Gephyrin+ synapses made onto NeuN+ cells in the dorsolateral striatum of P30 control (n = 5, Cx3cr1+/+;GABAB1Rfl/fl) and cKO (n = 6, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. *p < 0.05, **p < 0.01, Student’s t test.(N) Images, 3D reconstruction and quantitation of SST synapses (GAD65+Gephyrin+ synapses with GAD65 presynaptic boutons completely encased within genetically labeled SST axons) in layer 1 of S1 at P15 in control (n = 5; SSTFlp/+; RCE:FRT; Cx3cr1Cre/+; GABAB1Rfl/fl) and GABAB1R cKO (n = 4; SSTFlp/+; RCE:FRT; Cx3cr1+/+; GABAB1Rfl/fl) mice. *p < 0.05, Student’s t test.All data are mean ± SEM, each data point represents one experimental animal except in I-K where they represent cells. Scale bars equal 1 mm. Arrowheads indicate colocalization.

Figure S5. The synaptic phenotype of GABAB1R cKO mice is not due to off-target effects, related to Figure 3 (A) Representative images and quantitation of the density of microglia in control (n = 7, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 5, Cx3cr1Cre/+; GABAB1Rfl/fl) mice at P15. ns p > 0.05, Student’s t test. Scale bar equal 100 μm.(B) Representative images and quantitation of the density of PV cells in control (n = 6, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 5, Cx3cr1Cre/+; GABAB1Rfl/fl) mice at P15. ns p > 0.05, Student’s t test. Scale bar equal 100 μm.(C) Representative images and quantitation of the density of SST cells (GFP+) in control (n = 8, SSTFlp/+; RCE:FRT; Cx3cr1Cre/+; GABAB1Rfl/fl) and GABAB1R cKO (n = 7, SSTFlp/+; RCE:FRT; Cx3cr1+/+; GABAB1Rfl/fl) mice at P15. ns p > 0.05, Student’s t test. Scale bar equal 100 μm.(D) Quantitation of the density of PV and SST cells in control (n = 6, Lhx6GFP/+; Cx3cr1+/+; GABAB1Rfl/fl) and GABAB1R cKO (n = 6, Lhx6GFP/+; Cx3cr1Cre/+; GABAB1Rfl/fl) mice at P10. PV cells were identified as GFP+SST− cells. ns p > 0.05, Student’s t test.(E) Quantitation of Syt2+Gephyrin+ synapses made by PV cells onto L4 excitatory neurons at P10 in control (n = 3, Cx3cr1+/+;GABAB1Rfl/fl) and cKO (n = 6, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. ns p > 0.05, Student’s t test.(F) Representative images and quantitation of the fraction of L4 PV cells surrounded by perineuronal nets (WFA) in control (n = 4, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 5, Cx3cr1Cre/+; GABAB1Rfl/fl) mice at P15 and P30. ns p > 0.05, One-Way ANOVA and Holm-Sidak multiple comparisons test. Scale bar equal 20 μm.(G) Quantitation of neurons (labeled with NeuN) expressing Gabbr1 mRNA in control (n = 4, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 3, Cx3cr1Cre/+; GABAB1R+/+) mice. Gabbr1 mRNA was detected by single-molecule in situ hybridization specifically for the exons deleted in the cKOs (7 and 8). ns p > 0.05, Student’s t test.(H) Time course of mean Racine scores of seizures induced by intraperitoneal injection of kainic acid (20 mg/kg) into control (n = 7, Cx3cr1+/+;GABAB1Rfl/fl) and GABAB1R cKO (n = 7, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. Seizures were scored as described in the STAR Methods. Two-Way ANOVA for repeated-measures with genotype (p > 0.05) and time (p < 0.001) as factors. Interaction between time and genotype p > 0.05.(I) Maximum and cumulative seizure scores over 90 minutes from the time of kainic acid injection. ns p > 0.05, Mann-Whitney test.

(J) Latency to reach the status epilepticus and/or death. ns p > 0.05, Mann-Whitney test.

(K) and (L) Traces, frequency and amplitude of mIPSCs (n = 16 cells from 3 control and n = 22 cells from 3 cKO mice) and mEPSCs (n = 18 cells from 3 control andn = 25 cells from 3 cKO mice) recorded from layer 4 excitatory neurons at P60. **p < 0.01, ns p > 0.05, Mann-Whitney test for mIPSCs and Student’s t test for mEPSCs.(M) Images and quantitation of Syt2+Gephyrin+ synapses made by PV cells onto excitatory neurons in P60 control (Ctl, n = 5, Cx3cr1+/+;GABAB1Rfl/fl) and cKO (n = 8, Cx3cr1Cre/+; GABAB1Rfl/fl) mice as well as cKO (n = 5) and control (n = 3) mice that underwent microglia depletion from P30 to P60. ns p > 0.05, *p < 0.05, One-Way ANOVA followed by Sidak’s multiple comparisons test. Scale bar equal 2 μm.(N) Quantitation of VGlut2+Homer1+ synapses made onto excitatory neurons in P60 control (Ctl, n = 5, Cx3cr1+/+;GABAB1Rfl/fl) and cKO (n = 8, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. ns p > 0.05, Student’s t test.(O) Quantitation of PV+ Gephyrin+ synapses made onto NeuN+ cells in the dorsolateral striatum of P60 control (Ctl, n = 7, Cx3cr1+/+;GABAB1Rfl/fl) and cKO (n = 8, Cx3cr1Cre/+; GABAB1Rfl/fl) mice. *p < 0.05, Student’s t test.All data are mean ± SEM, each data point represents one experimental animal except in K and L where they represent cells. Arrowheads indicate colocalization.

Figure S6. Loss of GABAB1Rs does not fundamentally alter the range of microglial states but causes a downregulation of pruning-related genes, related to Figure 5 (A) UMAP plots showing the integration of P15 wild-type microglia with embryonic, early postnatal and P30 datasets from Hammond et al. (2019).(B) UMAP plot of Cre-Het control (Cx3cr1Cre/+) microglia showing 5 clusters and representative genes enriched in each cluster.(C) UMAP plot of Cre-Het control microglia from cluster 1 further subjected to subclustering. Representative enriched genes are shown.

(D) UMAP plot of GABAB1R cKO microglia from P15 S1 cortex showing 7 main clusters and representative genes enriched in each cluster.(E) Pie charts indicating the contribution of each wild-type or cKO cluster to the clusters of the integrated dataset. The overall filled area is proportional to the total number of cells within each cluster.

(F) UMAP plots of Cre-Het control and cKO integrated scRNaseq dataset. See methods for the different integration of this dataset as compared to WT control andcKO data.(G) Same as (F), showing 10 mixed clusters and representative genes enriched in each cluster.

(H) Mixed cluster contributions to total differentially expressed genes (DEGs) between Cre-Het control and cKO microglia.

(I) Violin plots of normalized log-expression values for representative genes significantly downregulated in cKO microglia from Cre-Het-cKO cluster 3. See methods for the different integration of Cre-Het control and cKO data as compared to WT control and cKO data.(J) Violin plots of normalized log-expression values for representative genes significantly downregulated in cKO microglia from Cre-Het-cKO cluster 5.

(K) Heatmap showing the output of an elastic net regularized classifier trained to learn which genes are predictive of control wild-type and GABAB1R cKO microglia using all genes as features and wild-type or cKO as labels. Genes are ordered by increasing mean expression. The top 5 genes are predictive of wild-type microglia (downregulated in cKO) and the remaining 3 genes are predictive of cKO microglia (upregulated in cKO).(L) UMAP plots of control and cKO integrated scRNaseq dataset after denoising expression data with a weighted affinity kernel and self-supervision method(DEWÄ KSS, see STAR Methods).(M) Same as (L) followed by unsupervised clustering and showing 8 mixed clusters. See also Table S2 for genes enriched in each cluster.(N) Volcano plot showing differentially expressed genes between control and cKO microglia from cluster 4 after denoising. The negative log10-transformed p values are plotted against the log2 fold change. Differentially expressed genes with an absolute log2 fold change higher than 0.5 and -log10 (p value) higher than or equal to 20 are depicted in orange. Top downregulated genes are highlighted. A full list is shown in Table S2.

Figure S7. MoSeq reveals a biphasic behavioral phenotype in GABAB1R cKO mice. Related to Figure 7 (A) Expression probability of syllable usage in P30 wild-type control (n = 11), cKO (n = 15) and Cre-Het control (n = 5) female mice. Syllables are ordered by differential usage (left: cKO-enriched syllables; right: cKO-downregulated syllables). Here and in the rest of the figure, only relevant or significantly differentially used syllables are shown. *p < 0.05, z-test on bootstrapped syllable usage distribution corrected for false discovery rate. Data are mean ± SEM. Here and in the rest of the figure, syllable labels were assigned by a human observer.(B) Expression probability of syllable usage in P30 wild-type control (n = 6), cKO (n = 14) and Cre-Het control (n = 7) male mice. Syllables are ordered by differential usage. *p < 0.05, z-test on bootstrapped syllable usage distribution corrected for false discovery rate. Data are mean ± SEM.

(C) Expression probability of syllables enriched in P60 control (n = 19, both sexes) and cKO (n = 14, both sexes) mice. Syllables are ordered by differential usage(left: cKO-enriched syllables; right: cKO-downregulated syllables). *p < 0.05, z-test on bootstrapped syllable usage distribution corrected for false discovery rate. Data are mean ± SEM.

(D) Heatmap depicting the position of P60 control (n = 10 males and 9 females) and cKO (n = 5 males and 9 females) mice during MoSeq.(E) Graph showing the mean syllable speed in control (n = 19) and GABAB1R cKO (n = 14) mice. Syllables are ordered by differential speed (left: syllables having a higher speed in cKOs and right: syllables with a lower speed in the cKOs). *p < 0.05, t test on bootstrapped syllable speed distribution corrected for false discovery rate. Data are mean ± SEM.(F) Expression probability of syllables enriched in P60 Cx3cr1Cre cKOs (n = 9 females and 10 males) and Tmem119CreER cKOs (n = 3 females and 3 males) mice. Only significantly differentially used syllables are shown. In females, almost all syllables were similarly used. Males from both groups used high velocity syllables although Tmem119CreER cKOs appeared even more active than Cx3cr1Cre cKOs. *p < 0.05, z-test on bootstrapped syllable usage distribution corrected for false discovery rate. Data are mean ± SEM.(G) Summary of the scalar information for P60 control (n = 19) and GABAB1R cKO (n = 14) mice. Velocity is measured in mm/s; height, width and length are measured in mm. ***p < 0.001, ns p > 0.05, Student’s t test. The inset shows the probability density function (PDF) of the two-dimensional velocity, p < 0.001, Two-sample Kolmogorov–Smirnov test.