Sensitive Periods for Cerebellar-Mediated Autistic-like Behaviors

Abstract

Despite a prevalence exceeding 1%, mechanisms underlying autism spectrum disorders (ASDs) are poorly understood, and targeted therapies and guiding parameters are urgently needed. We recently demonstrated that cerebellar dysfunction is sufficient to generate autistic-like behaviors in a mouse model of tuberous sclerosis complex (TSC). Here, using the mechanistic target of rapamycin (mTOR)-specific inhibitor rapamycin, we define distinct sensitive periods for treatment of autistic-like behaviors with sensitive periods extending into adulthood for social behaviors. We identify cellular and electrophysiological parameters that may contribute to behavioral rescue, with rescue of Purkinje cell survival and excitability corresponding to social behavioral rescue. In addition, using anatomic and diffusion-based MRI, we identify structural changes in cerebellar domains implicated in ASD that correlate with sensitive periods of specific autism-like behaviors. These findings thus not only define treatment parameters into adulthood, but also support a mechanistic basis for the targeted rescue of autism-related behaviors.

Keywords: Purkinje cell; autism; cerebellum; sensitive periods; treatment; tuberous sclerosis.

Figure 1.. Sensitive Period Remains Open at 6 Weeks for Treatment of Social and Motor Behaviors

(A) Schema for treatment paradigm. (B) Social testing in three-chambered apparatus. (C–F) Rapamycin initiated at 6 weeks rescues (C and D) social behaviors in three chambered apparatus, (E) social olfaction, and (F) motor function on rotarod. (G and H) No rescue is achieved for (G) repetitive grooming nor (H) reversal learning on water T maze. Two-way ANOVA, Bonferroni post hoc analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. FA, familiar animal; NA, novel animal; NO, novel object; Ns, not significant. Rapa, rapamycin; RevDay, reversal day; VEH, vehicle. Data are reported as mean ± SEM.

Figure 2.. Treatment Initiated at 6 Weeks Rescues PC Survival and Impaired Spontaneous Firing and Excitability in PC Tsc1 Mice

Recordings were performed at 11 weeks of age (5 weeks after treatment initiation at 6 weeks of age). (A) PC loss is rescued with rapamycin therapy (quantification of PCs on left). Shown are midline vermis sagittal sections stained with calbindin (to identify PCs) and phosphoS6 (to evaluate mTOR activity) antibodies. (B and C) Rapamycin initiated at 6 weeks rescues (B) intrinsic firing properties and (C) excitability deficits in mutant mice. Two-way ANOVA, Bonferroni post hoc analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. Ns, not significant; Rapa, rapamycin; VEH, vehicle. Data are reported as mean ± SEM.

Figure 3.. Brain Alterations in PC Tsc1-Mutant Mice Are Partially Rescued with Rapamycin Therapy

(A) Voxelwise differences in relative volume between control and mutant mice are partially rescued with rapamycin therapy. (B–F) Bar plots summarizing data showing partial rescue of (B) CrusI white matter (WM), (C) CrusII WM, (D) lateral CN, (E) interposed CN, and (F) medial CN. (G and H) Bar plots also summarize data demonstrating areas altered in PC Tsc1-mutants that are not rescued with rapamycin treatment, including anterior (Ant) lobules 4 and 5 (G) gray and (H) WM. *FDR < 0.05; **FDR < 0.01; and ns, FDR > 0.1. CON, control; MUT, PC Tsc1 mutant; Rapa, rapamycin; VEH, vehicle. Data are reported as mean ± SEM.

Figure 4.. At 10 Weeks, Sensitive Period Is Open for Social Behaviors in the Presence of PC Survival

(A–C) Rapamycin initiated at 10 weeks is able to rescue social behaviors in (A) social approach, (B) social novelty, and (C) social olfaction behavioral paradigms only in heterozygous (het), not homozygous (mutant), PC Tsc1 mutants. (D and E) No rescue is seen for either genotype in (D) reversal learning in water T maze or (E) with repetitive grooming. (F) Despite these findings, persistent rescue of motor learning on the rotarod was observed in homozygous PC Tsc1-mutant mice. (G) PC viability is not rescued in homozygous PC Tsc1 mutants with rapamycin initiated at 10 weeks of age. Two-way ANOVA, Bonferroni post hoc analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. FA: familiar animal; NA, novel animal; NO, novel object; Ns, not significant; Rapa, rapamycin; Rev, reversal; VEH, vehicle. Data are reported as mean ± SEM.

Figure 5.. Electrophysiology Phenotypes with 10 Week Treatment Paradigm

Recordings were performed at 15 weeks of age (5 weeks after treatment initiation at 10 weeks of age). (A and B) Intrinsic PC firing properties are rescued in mutants with rapamycin therapy (A), while PC excitability is not rescued in mutant mice even with rapamycin therapy (B). Two-way ANOVA, Bonferroni post hoc analysis. **p < 0.01 and ***p < 0.001. Ns, not significant; Rapa, rapamycin; VEH, vehicle. Data are reported as mean ± SEM.