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. Author manuscript; available in PMC: 2023 Jul 10.
Published in final edited form as: Brain Behav Immun. 2023 Mar 29;111:61–75. doi: 10.1016/j.bbi.2023.03.023

Fig. 6. ß-Estradiol Treatment rescued microglia structure and function in NLGN4−/− male mice.

Fig. 6.

(A) Schematic of experimental layout for 5 weeks of beta-estradiol (E2) treatment of NLGN4−/− male mice (NLGN4−/−-E2) and aged-matched untreated male WT and NLGN4−/− mice. (B) Representative photomicrographs from WT and NLGN4 treated and untreated male brain samples after laser lesion (microglia in green EGFP, scale bar 10 μm). Quantitative analysis of lesion-induced, microglial process movement expressed in arbitrary units (AU) in 20-week-old male WT, NLGN4−/− and NLGN4−/−-E2 mice. Impaired process extension in male NLGN4−/− mice are rescued by estradiol treatment (right graph, ordinary one-way ANOVA following Bonferroni’s multiple comparisons test: number of sections (and mice): male WT, 12 (3); male NLGN4−/−, 12 (3); male NLGN4−/−-E2, 18 (4)). We performed 3 independent experiments. (C) Average current density to voltage relationships of microglial ATP-induced metabotropic purinergic responses (P2YR12) in the hippocampus of 20-week-old WT, NLGN4−/− and NLGN4−/−-E2 mice (left graph). Summary of the outward conductance’s, which were calculated from ATP (10 μM)-evoked currents at potentials between + 20 mV and + 60 mV (right graph). Both datasets suggested that beta-estradiol treatment reversed the effect of loss of NLGN4 in male microglia (Ordinary one-way ANOVA following Bonferroni’s multiple comparisons test; number of cells (and mice): male WT, 20 (5); male NLGN4−/−, 18 (3); male NLGN4−/−-E2, 22 (4)). We performed 3 independent experiments. (D) The percentage of AnnexinV positive microglial cells is reduced to the level of WT after the treatment in NLGN4−/− male (2 independent experiments, ordinary one-way ANOVA following Bonferroni’s multiple comparisons test: male: n(WT) = 38, n(NLGN4−/−) = 14, n(E2- NLGN4−/−) = 20). (E) Representative photomicrographs of microglia morphology from WT, NLGN4−/− treated and untreated males (scale bar 5 μm, Iba-1 staining for microglia). Microglia morphology analysis shows that the total number of processes, the average process length and the number of branch points per cell in CA3 was significantly altered between male WT and NLGN4−/− microglia in 20 weeks old animals, and beta estradiol treatment normalized the morphology (2 independent experiments, ordinary one-way ANOVA following Bonferroni’s multiple comparisons: n(WT) = 5, n(NLGN4−/−) = 5, and n(E2- NLGN4−/−) = 6). Asterisk represents significant levels as *p < 0.05; **p < 0.01; ***p < 0.0001. All mice used for the study were on a C57BL/6J genetic background.