Fig. 10: Amelioration of 2HIT phenotypes with cerebellum-specific microglia replacement.

a Schematic of stereotaxic injection of PBS or clodronate disodium salt (CDS) into the male cerebellum. PBS or CDS was administered at four sites in 4-week-old male mice. b Representative images of Iba1(+) microglia in the cerebellar cortex at 1-, 5-, and 30-days post-injection (dpi) of PBS or CDS. c Percent changes in Iba1(+) microglia count across brain regions at 5 dpi of PBS or CDS. Bar graphs show data from the cerebellar cortex (CbCrx), cerebellar nuclei (CbN), hippocampus (HPC), ventral tegmental areas (VTA), periaqueductal gray (PAG), and medial prefrontal cortex (mPFC). *p < 0.05, two-tailed unpaired Student’s t-test. d Experimental timeline for Control+PBS, 2HIT + PBS, and 2HIT + CDS groups. e Iba1(+) microglia cell density in 9-week-old Control+PBS, 2HIT + PBS, and 2HIT + CDS animals (five weeks post-injection of PBS or CDS); see also Fig. 1d. f Density of Iba1-, MHC class II-, TREM2-triple-positive cells from CbCrx and CbN for Control+PBS, 2HIT + PBS, and 2HIT + CDS; see also Fig. 5i. g Bar graphs showing the density of cerebellar Purkinje cells in Control+PBS, 2HIT + PBS, and 2HIT + CDS groups. Data from lobules II-IV, VIa- VIb, and IX-X are presented (mean ± SEM). *p < 0.05, one-way ANOVA with multiple comparisons using the Tukey-Kramer method (e–g). h Representative action potential traces of Purkinje cells from Control+PBS and 2HIT + CDS mice evoked by 400 pA and 500  ms step-pulse current. i Firing frequency of Purkinje cells from Control+PBS, 2HIT + PBS, and 2HIT + CDS groups in response to gradually increasing depolarization pulses from 0 to 550 pA. Two-way ANOVA group × injected current, interaction: F(20, 770) = 0.78, p = 0.7412; main effect of group: F(2, 770) = 37.64, p < 0.0001. *p < 0.001, multiple comparisons using the Bonferroni method. Purkinje cell action potential properties, including input resistance (j), basement potential of action potential (k), peak voltage of action potential (l), action potential threshold (m), full width of half maximum (FWHM) (n), 10–90% rise time (o), and rheobase (p) is shown. There are significant differences between Control+PBS and 2HIT + PBS or between 2HIT + PBS and 2HIT + CDS in (j), (k), (m), (n), (o), and (p). q-z Behavioral tests in Control+PBS, 2HIT + PBS, and 2HIT + CDS mice: social avoidance time in response to a stressor in the social avoidance test (q); open field test measures of travel distance (r), mean speed (s), and center entries (t); time spent in open arms of the elevated plus maze (u); preference index in the 3-chamber test (v); marble burying score (w); motor coordination assessed by latency to fall on a rotating rod at 10, 20, 30, and 40 rpm (x) [Two-way ANOVA group × rotation frequency, interaction: F(6, 148) = 6.2535, p < 0.0001]; and hind paw stride length (y) and stride time (z) in footprint analysis. *p < 0.05, one-way ANOVA with multiple comparisons using the Tukey-Kramer method (q-w,y,z). *p < 0.001, two-way ANOVA with multiple comparisons using the Bonferroni method (x). All data are represented as mean ± SEM.