Fig 2: Microglial processes contact the cell bodies, neurites, and synapses of DCX+ developing neurons(A) Confocal laser scanning microscopy (CLSM) image shows the distribution of IBA1+ microglia (green) in E15 pallium. Postmitotic neurons are labeled for doublecortin (DCX, red), NeuN is shown in blue, and white arrows point to the enrichment of microglia in the SVZ and VZ.(B) IBA1+ microglia are enriched in the subgranular zone of the DG in P90 mice. Staining is as in (A), and white arrows point to some microglia. The area within the white box in the top panel is enlarged in the bottom panel.(C and D) High-resolution CLSM images show some examples of microglia contacts in the E15 VZ (C) and the DG in P90 mice (D); white arrows point to contact sites.(E) CLSM image shows the lack of synapses in an E15 cortical plate. Samples are labeled for IBA1 (green), DCX (red), and VGLUT1 (cyan).(F) Staining is the same as in (E); the image shows the abundant presence of glutamatergic synapses outside of the granule cell layer.(G) IBA1+ microglial processes (yellow) contact developing glutamatergic synapses, as identified by the appositions of VGLUT1 (magenta) and Homer1 (green) on a DCX+ dendrite (red) in the stratum moleculare of a P90 DG. White arrows point to contacts.(H) Staining is the same as in (G); microglial processes contact synapses of mature neurons in the stratum moleculare of a P90 DG.(I–M) CLSM images show some examples of microglia-neuron somatic contact sites during both developmental and adult neurogenesis. Nuclei are visualized by DAPI (blue), microglia are stained for IBA1 (green) and postmitotic neurons for DCX (red), and white arrows point to contact sites. (I), E15; (J), P1; (K), P8; (L), P15; (M), P90.(N) Division of a DCX+ neuron’s cell body into “soma” and “proximal tuft” compartments.(O) Ratio of DCX+ neurons contacted by microglial processes on their somata, proximal tufts, or both. Percentage values next to the diagrams represent the ratio of cells that were contacted either way.(P) Somatic contact prevalence on DCX+ cell bodies (individual values represent different animals). Images and measurements are from the cortical plate in E15 mice, neocortex from P1–P15 mice, and hippocampal dentate gyrus from P90 mice; n = 3 mice in each age group.Scale bars represent 200 µm in (A); 100 µm in (B) and (F); 5 µm in (C); 10 µm in (D), (J), and (L); 50 µm in (E), 1 µm in (G) and (H); 15 µm in (I), (K), and (M); and 2 µm in (N). See also Figure S1 and Table S1.

Fig 3: Chaetocin induces proliferation and reduces differentiation of cultured adult hippocampal progenitors. (A) Immunostaining of H3K9me3 (red), DCX (green) and GFAP (white) and NucB (blue) in AHP differentiated for 24 h in the presence or absence of 1.25 and 2.5 nM chaetocin. Scale bar: 20 µm. (B,C) Quantification of the percentage of NucB-positive cells positive for DCX (b) or GFAP (c). (D) AHP were treated for 24 h with or without 1.25 and 2.5 nM chaetocin and were incubated with 10 µM BrdU for the last 2 h of treatment. Images show immunostaining of H3K9me3, BrdU and the nuclear staining NucB. Merged images show H3K9me3 (red), BrdU (green) and the progenitor cell marker nestin (blue). Scale bar: 20 µm. (E) Quantification of the percentage of NucB-positive cells that incorporated BrdU. Data are presented as mean ± SEM; N = 4 (b, c) or N = 5 (e) independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA followed by Bonferroni posthoc test.

Fig 4: PPAR? activation mediates effects of GRb1-treated microglia in vitro. (A) Representative micrographs after immunostaining against Iba1 display microglial activation phenotype. Fluorescence intensity reflects PPAR? expression. Scale bars: 20 µm. (B) Quantification of expression of PPAR? in mRNA levels. (C) Quantitation of PPAR? fluorescence intensity. (D) Representative western blot of of PPAR? and p-PPAR? protein expression. (E) Quantification of PPAR? and p-PPAR? protein (n = 5). (F) The unbiased stereological quantification of microglial cell area, and (G) total length of processes. (H) Experimental protocol to test the effects of conditioned medium from microglial cultures on NPCs. (I) NPC cultures were maintained in native medium (NM) and directly stimulated by GRb1, LPS, and GW9662; or they were maintained in conditioned medium (CM) of activated microglia. After 2 h or 10 days, the proliferation and differentiation of NPCs were tested, respectively, by immunolabeling BrdU (upper two rows) or DCX and GFAP (lower two rows). Scale bars: 15 µm. (J, K) The ratio of BrdU+-DAPI+ to DAPI+ cells in NPC cultures maintained in native medium or exposed to conditioned medium from microglial cultures. (L, M) The ratio of DCX+-DAPI+ to DAPI+ cells in NPC cultures maintained in native medium or exposed to conditioned medium. (N, O) Relative mRNA levels of DCX in treated groups (n = 6). The statistical results are shown in Table S8. *P < 0.05, ** P < 0.01, *** P < 0.001

Fig 5: Microglia at rest promote adult hippocampal neurogenesis. (A-F) Adult mice were given either normal or PLX-5622-supplemented chow for 3 weeks to deplete microglia. (A) PLX chow caused robust depletion of microglia as measured by Iba-1 IHC. (B) Quantification of Iba-1 staining in hippocampus found ~96% depletion of microglia. ****p < 0.0001, t-test vs. control. Scale bar: 500µm (C) Representative images of DCX staining in hippocampal dentate gyrus showing loss of neurogenesis with microglial depletion. Scale bar 100 µm. (D) Quantification found a ~30% reduction in DCX immunoreactivity. *p < 0.05, t-test vs. control. (E) Representative images of ki67 immunostaining after EtOH in dentate regions of the hippocampus in control and microglia depleted (PLX) mice. (F) Ethanol caused a 24% reduction in ki67. *p < 0.05, t-test. (G, H) OBSC were treated with PLX-3397 (1µM) for 10 days to deplete microglia and determine its effect on neurogenesis. (G) Representative images of DCX in hippocampal region of OBSC showing loss of neurogenesis with microglial depletion. Scale bar: 100µm. (H) Quantification found a 56% reduction in DCX staining with microglial depletion. **p < 0.01, t-test vs. control. (I, J) Microglial inhibition reduces neurogenesis. OBSC were treated with either AAV9.CD68.hM4di or AAV9.EGFP control for 24 hours prior to treatment with CNO (1µM or 5µM) or vehicle for 4 days, followed by IHC for DCX. (I) Quantification of DCX found that neither viral transfection alone nor CNO alone significantly altered neurogenesis. Addition of CNO to microglial hM4di-transfected slices caused a 77% reduction in DCX with 5µM CNO. 1-Way ANOVA, F5,61 = 7.148, p< 0.0001. ***p < 0.001, Sidak’s post-hoc test vs. AAV9.CD68.hM4di alone (J) Representative images of DCX +/- CNO and AAV9 transfection. Scale bar: 100µm.