Juvenile Shank3b deficient mice present with behavioral phenotype relevant to autism spectrum disorder

Abstract

Autism spectrum disorder (ASD) is a pervasive, multifactorial neurodevelopmental disorder diagnosed according to deficits in three behavioral domains: communication, social interaction, and stereotyped/repetitive behaviors. Mutations in Shank genes account for ∼1% of clinical ASD cases with Shank3 being the most common gene variant. In addition to maintaining synapses and facilitating dendritic maturation, Shank genes encode master scaffolding proteins that build core complexes in the postsynaptic densities of glutamatergic synapses. Male mice with a deletion of the PDZ domain of Shank3 (Shank3B KO) were previously shown to display ASD-like behavioral phenotypes with reported self-injurious repetitive grooming and aberrant social interactions. Our goal was to extend these previous findings and use a comprehensive battery of highly detailed ASD-relevant behavioral assays including an assessment of mouse ultrasonic communication carried out on key developmental days and male and female Shank3B KO mice. We demonstrate that ASD-related behaviors, atypical reciprocal social interaction and indiscriminate repetitive grooming, are apparent in juvenile stages of development of Shank3B KO mice. Our findings underscore the importance of utilizing Shank mutant models to understand the impact of this gene in ASD etiology, whichmay enable future studies focusing on etiological gene-environment interactions in ASD.

Keywords: Autism; Autism spectrum disorder; Communication; Shank3; Social behavior.

Fig. 1.. Micro-behaviors of 3-Chamber, Social Proximity and Self-grooming tests.

Micro-behaviors were scored according to previously published criteria: 3-Chamber [57], Grooming [25], Social Proximity [26].

Fig. 2.. Ultrasonic Vocalization (USV).

The Ultrasonic Vocalization test was performed for Shank3B KO (KO) mice and their wild-type (WT) littermates, grouped by sex (A,C: females; B,D: males) at postnatal days (PND) 8, 10 and 12. The data are expressed as a combined call duration during the test period (A-B) and call frequency reflecting number of calls per minute (CD). The graphs are averages ± SEM, with n=10 for WT and KO males and WT females, and n=9 for KO females.

Fig. 3.. 3-Chamber.

The 3-Chamber test was performed for Shank3B KO (KO) mice and their wild-type (WT) littermates, grouped by sex (A,C,E: females; B,D,F: males). The duration spent in one of the three chambers (A-B) as well as the duration (C-D) and the frequency of 7 different micro-behaviors (E-F) were examined. The graphs are averages ± SEM, with n=10 for WT and KO males and WT females, and n=9 for KO females. Statistical significance: * P<0.05; ** P<0.01.

Fig. 4.. Social Proximity.

The Social Proximity (forced social interaction) test was performed for Shank3B KO (KO) mice and their wild-type (WT) littermates, grouped by sex (A,C: females; B,D: males). The duration (A-B) and the frequency (C-D) of eight different social interaction micro-behaviors were measured. The graphs are averages ± SEM, with n=10 for WT and KO males and WT females, and n=9 for KO females. Statistical significance: * P<0.05.

Fig. 5.. Self-Grooming.

The Self-grooming test assaying for restricted, repetitive behavior was performed for Shank3B KO (KO) mice and their wild-type (WT) littermates, grouped by sex (A,C: females; B,D: males). The duration (A-B) and the frequency (C-D) of six different self-grooming micro-behaviors, based on the body part that was groomed, were measured. The graphs are averages ± SEM, with n=10 for WT and KO males and WT females, and n=9 for KO females. Statistical significance: * P<0.05, ** P<0.01; *** P<0.001.

Fig. 6.. Elevated Mazes.

Elevated plus maze (EPM) and elevated zero maze (EZM) tests were performed to assess anxiety-like behaviors in Shank3B KO (KO) mice and their wild-type (WT) littermates. The duration of time spent within enclosed or open area were measured and the data are expressed as percentage of time spent in the open area. The graphs are averages ± SEM, with n=10 for WT and KO males and WT females, and n=9 for KO females. Statistical significance (two sample t-test): P values are shown within graph.