Microglia regulate chandelier cell axo-axonic synaptogenesis

Abstract

SignificanceChandelier cells (ChCs) are a unique type of GABAergic interneuron that form axo-axonic synapses exclusively on the axon initial segment (AIS) of neocortical pyramidal neurons (PyNs), allowing them to exert powerful yet precise control over PyN firing and population output. The importance of proper ChC function is further underscored by the association of ChC connectivity defects with various neurological conditions. Despite this, the cellular mechanisms governing ChC axo-axonic synapse formation remain poorly understood. Here, we identify microglia as key regulators of ChC axonal morphogenesis and AIS synaptogenesis, and show that disease-induced aberrant microglial activation perturbs proper ChC synaptic development/connectivity in the neocortex. In doing so, such findings highlight the therapeutic potential of manipulating microglia to ensure proper brain wiring.

Keywords: axon initial segment; chandelier cells; inflammation; microglia; synaptogenesis.

Fig. 1.

AIS cytoskeleton-dependent enrichment of AXIS microglia in the neocortex between P12 and P28. (A) Immunofluorescent labeling of Iba1⁺ microglia and AnkG⁺ AISs in L2/3 of the SSC in wild-type mice at P14. (Scale bars, 10 μm.) Arrows in A and F depict AXIS microglia–PyN AIS contact. (B) Schematic depicting the two key morphological features of AXIS microglia: 1) at least 50% microglial coverage of AIS length and 2) <1 μm distance between microglia and AIS surface. (C) Quantification of the percentage of PyN AISs contacted by AXIS microglia at P14. (D) Quantification of the percentage of AISs contacted by AXIS microglia (black line) and percentage of AISs innervated by single, labeled ChCs (dotted blue line) at time points spanning P8 to adulthood in L2/3 of the SSC. Dashed gray line and red arrow denote spike in the percentage of AISs with AXIS microglia and AISs innervated by ChCs starting at P12. (E) Schematic depicting cytoskeletal components and GABA_ARs at PyN AISs. (F) Representative images of GFP⁺ (green) and neighboring GFP⁻ PyNs and AnkG⁺ AISs (blue) with or without Iba1⁺ AXIS microglia (white) contact in L2/3 of the SSC from P14 mice electroporated at E15.5 with vectors coexpressing EGFP and short-hairpin RNAs targeting AnkG, βIV-spectrin, PSD-93, or control firefly luciferase (Ctrl). (Scale bars, 10 μm.) (G) Quantification of the percentage of GFP⁺ and neighboring GFP⁻ PyN AISs contacted by AXIS microglia. Data are mean ± SEM, *P < 0.05, ***P < 0.001 (Student’s t tests); n values and statistical information are listed in SI Appendix, Table S1.

Fig. 2.

Microglia–PyN AIS–ChC cartridge tripartite interactions are associated with increased ChC cartridge length and bouton number during postnatal development. (A) Immunofluorescent labeling of Iba1⁺ microglia, AnkG⁺ AISs, and RFP⁺ ChCs in L2/3 of the SSC in Nkx2.1-CreER;Ai9 mice at indicated time points. (Scale bars, 20 μm.) (B and C) Representative images of Iba1⁺ AXIS microglia–AnkG⁺ PyN AIS-RFP⁺ ChC cartridge tripartite interactions at P14 (B) and P18 (C). (Scale bars, 5 μm.) (D) Quantification of the percentage of RFP⁺ cartridges from a single ChC contacted by microglia at the AIS in L2/3 of the SSC. (E) Schematic of AXIS microglia–PyN AIS–ChC interactions with arrows denoting individual boutons of a single ChC cartridge and bracket denoting ChC cartridge length. (F) Representative image of RFP⁺ ChC cartridge–PyN AIS innervation with AXIS microglia contact in L2/3 of the SSC at P14. (Scale bar, 5 μm.) Three-dimensional representation of dashed boxed region is included (Right). (G and H) Quantification of the average number of boutons per ChC cartridge (G) and the average length of ChC cartridges (H) innervating AISs with or without AXIS microglia contact at indicated time points. Data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with post hoc Tukey’s multiple comparison test in D; Student’s t tests in G and H); n values and statistical information are listed in SI Appendix, Table S1.

Fig. 3.

Early postnatal depletion of microglia impairs neocortical ChC cartridge/bouton development and AIS synaptogenesis. (A and G) Representative images of single RFP⁺ ChCs, neighboring AnkG⁺ AISs, and Iba1⁺ microglia in L2/3 of the SSC from P14 (A) or P18 (G) Nkx2.1-CreER;Ai9 mice administered PLX or vehicle once daily from P7 to P14 (A) or P18 (G). (Scale bars, 20 μm.) (B and H) Representative images of RFP⁺ ChC cartridge–PyN AIS innervation with or without AXIS microglia contact from P14 (B) or P18 (H) Nkx2.1-CreER;Ai9 mice under PLX or vehicle conditions. (Scale bars, 3 μm.) (C_i, D_i, I_i, and J_i) Quantification of the average number of boutons per ChC cartridge (C_i and I_i) and the average length of ChC cartridges (D_i and J_i) innervating PyN AISs with or without AXIS microglia contact at P14 (C_i and D_i) or P18 (I_i and J_i) in the SSC of Nkx2.1-CreER;Ai9 mice under PLX or vehicle conditions. (C_ii, D_ii, I_ii, and J_ii) Frequency distribution histograms showing the percentage of ChC cartridges with bouton number per cartridge ranging from 2 to 17 boutons (C_ii and I_ii) and cartridge length ranging from 0 to ≥26 μm (D_ii and J_ii) with or without AXIS microglia contact at P14 (C_ii and D_ii) or P18 (I_ii and J_ii) under PLX or vehicle conditions. (E and K) Representative images of PyN AISs with or without AXIS microglia contact in L2/3 of the SSC from P14 and P18 mice treated with PLX or vehicle from P7 to P14 (E) or P18 (K). GABAergic synapses are visualized by immunostaining for gephyrin (Geph). (Scale bars, 3 μm.) (F and L) Quantification of average gephyrin puncta density at AISs with or without AXIS microglia contact at P14 (F) or P18 (L). For C_i, D_i, I_i, J_i, F, and L, data are mean ± SEM. For C_ii, D_ii, I_ii, and J_ii, error bars indicate SEM for a multinomial distribution. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t tests in C_i, D_i, I_i, J_i, F, and L; two-tailed Mann–Whitney U tests in C_ii, D_ii, I_ii, and J_ii); n values and statistical information are listed in SI Appendix, Table S1.

Fig. 4.

Microglia are important for the maintenance of neocortical ChC cartridges/boutons. (A) Representative images of single RFP⁺ ChCs, neighboring AnkG⁺ AISs, and Iba1⁺ microglia in L2/3 of the SSC from P58 Nkx2.1-CreER;Ai9 mice administered PLX or vehicle once daily from P48 to P58. (Scale bars, 20 μm.) (B) Representative images of RFP⁺ ChC cartridge–PyN AIS innervation with or without AXIS microglia contact from P58 Nkx2.1-CreER;Ai9 mice under PLX or vehicle conditions. (Scale bars, 3 μm.) (C_i and D_i) Quantification of the average number of boutons per ChC cartridge (C_i) and average length of ChC cartridges (D_i) innervating PyN AISs with or without AXIS microglia contact at P58 in the SSC of Nkx2.1-CreER;Ai9 mice under PLX or vehicle conditions. (C_ii and D_ii) Frequency distribution histograms showing the percentage of ChC cartridges with bouton number per cartridge ranging from 2 to 13 boutons (C_ii) and cartridge length ranging from 0 to ≥26 μm (D_ii) with or without AXIS microglia contact at P58 under PLX or vehicle conditions. (E) Representative images of PyN AISs with or without AXIS microglia contact in L2/3 of the SSC from P58 mice treated with PLX or vehicle from P48 to P58. GABAergic synapses are visualized by immunostaining for gephyrin (Geph). (Scale bars, 3 μm.) (F) Quantification of average gephyrin puncta density at AISs with or without AXIS microglia contact at P58. For C_i, D_i, and F, data are mean ± SEM. For C_ii and D_ii, error bars indicate SEM for a multinomial distribution. *P < 0.05, **P < 0.01, ****P < 0.0001 (Student’s t tests in C_i, D_i, and F; two-tailed Mann–Whitney U tests in C_ii and D_ii); n values and statistical information are listed in SI Appendix, Table S1.

Fig. 5.

Removal of GABA_B1Rs from microglia impacts AXIS microglia numbers and AIS GABAergic synaptogenesis. (A) Representative images of Iba1⁺ microglia and AnkG⁺ AISs in L2/3 of the SSC from P18 Cx3cr1-Cre;GABA_B1R^fl/fl (microglia GABA_B1R cKO) and wild-type littermates. Arrows denote AXIS microglia-PyN AIS contact. (Scale bar, 15 μm.) (B) Quantification of the percentage of PyN AISs contacted by AXIS microglia in P18 microglia GABA_B1R cKO and wild-type mice. (C) Representative images of PyN AISs with or without AXIS microglia contact in L2/3 of the SSC from P18 microglia GABA_B1R cKO and wild-type mice. GABAergic synapses are visualized by immunostaining for gephyrin (Geph). (Scale bars, 2 μm.) (D and E) Quantification of average AIS gephyrin puncta density (D) and average microglia number per 200 μm × 200 μm field-of-view (FOV) (E) in P18 microglia GABA_B1R cKO and wild-type mice. For B, D, and E, data are mean ± SEM, **P < 0.01, ****P < 0.0001, ns (not significant) indicates P ≥ 0.05 (Student’s t tests); n values and statistical information are listed in SI Appendix, Table S1.

Fig. 6.

Microglial activation perturbs neocortical ChC cartridge/bouton development and integrity. (A_i and B_i) Representative images of single RFP⁺ ChCs, neighboring AnkG⁺ AISs, and Iba1⁺ microglia in L2/3 of the SSC from P16 Nkx2.1-CreER;Ai9 mice administered LPS (B_i) or vehicle (A_i) once daily from P12 to P15. (Scale bars, 20 μm.) (A_ii and B_ii) Representative images of RFP⁺ ChC cartridge–PyN AIS innervation with or without AXIS microglia contact in P16 Nkx2.1-CreER;Ai9 mice under LPS (B_ii) or vehicle (A_ii) conditions. (Scale bars, 3 μm.) (C) Quantification of the percentage of AISs contacted by AXIS microglia in L2/3 of the SSC from P16 Nkx2.1-CreER;Ai9 mice under LPS or vehicle conditions. (D_i and E_i) Quantification of the average number of boutons per ChC cartridge (D_i) and average length of ChC cartridges (E_i) innervating PyN AISs with AXIS microglia contact at P16 in the SSC of Nkx2.1-CreER;Ai9 mice under LPS or vehicle conditions. (D_ii and E_ii) Frequency distribution histograms showing the percentage of ChC cartridges with bouton number per cartridge ranging from 2 to 13 boutons (D_ii) and cartridge length ranging from 0 to ≥26 μm (E_ii) with AXIS microglia contact at P16 under LPS or vehicle conditions. (F) Representative images of PyN AISs with AXIS microglia contact in L2/3 of the SSC from mice treated with LPS or vehicle from P12 to P15 and killed at P16. GABAergic synapses are visualized by immunostaining for gephyrin (Geph). (Scale bars, 3 μm.) (G) Quantification of average gephyrin puncta density at AISs with AXIS microglia contact at P16. (H_i) Representative images of L2/3 of the SSC from 14 mo. AβPP/PS1 and WT littermates immunostained for Aβ plaques (red), Iba1⁺ microglia (white), and AnkG⁺ AISs (blue). (Scale bars, 20 μm.) (H_ii) Magnified boxed Insets from H_i highlighting the close interactions between microglia, neurotoxic Aβ plaques, and AISs in AβPP/PS1, but not wild-type, animals. (Scale bars, 10 μm.) (I and J) Quantification of average microglia number per 200 μm × 200 μm FOV (I) and the percentage of AISs contacted by AXIS microglia (J) in 14 mo. AβPP/PS1 and wild-type littermates. (K) Representative images of PyN AISs with AXIS microglia contact in L2/3 of the SSC from 14 mo. AβPP/PS1 and wild-type littermates. GABAergic synapses are visualized by immunostaining for gephyrin (Geph). (Scale bars, 3 μm.) (L) Quantification of average gephyrin puncta density at AISs with or without AXIS microglia contact in 14 mo. AβPP/PS1 and WT littermates. For C, D_i, E_i, G, I, J, and L, data are mean ± SEM. For D_ii and E_ii, error bars indicate SEM for a multinomial distribution. **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t tests in C, D_i, E_i, G, I, and J; two-tailed Mann–Whitney U tests in D_ii and E_ii, one-way ANOVA with post hoc Tukey’s multiple comparison test in L); n values and statistical information are listed in SI Appendix, Table S1.