Fig. 1: 2HIT stress susceptibility associated with sex differences in mice is underlain by increased cerebellar microglia.

a Experimental timeline for 2HIT mouse generation. We applied poly(I:C) to the pregnant mothers at embryonic day (E)12.5 as maternal immune activation (MIA). Repeated social defeat stress (RSDS) was exposed at 4 weeks old for 10 days. For 2HIT-microglia replacement (2HIT+rMG), a CSF1R inhibitor, Ki20227 was administered from 2 days before exposure to RSDS for seven days to deplete existing microglia. b The higher stress susceptibility in male 2HIT. The time difference in the zone with or without a stressor mouse during the social avoidance test (left plot) and the proportion of male and female mice susceptible (sus) and resilient (res) to RSDS (right pie charts) are shown. c A density increase of cerebellar Iba1(+)-microglia. Normalized density plots of coronal brain slices containing medial prefrontal cortex (mPFC), hippocampus (HPC), and cerebellum of male Control (n = 3) and 2HIT susceptible (n = 3) are shown. Cell density of Iba1(+) microglia. Bar graphs of the cerebellar cortex (CbCrx), cerebellar nuclei (CbN), HPC, ventral tegmental areas (VTA), and mPFC in the experimental C57BL/6N mouse groups (Control, RSDS susceptible (sus), RSDS resilient (res), MIA, 2HIT sus, 2HIT res, and 2HIT+rMG) across sex (male (d) and female (e)) and developmental periods (2-, 5-, and 9-weeks of age). The microglia density was restored after 1 week (at 5 weeks old) and four weeks (at 9 weeks old) after CSF1R inhibitor Ki20227 treatment. Data are shown as mean ± SEM in (d) and (e). *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA with multiple comparisons using the Tukey-Kramer method.