Unusual Adult Reciprocal Social Interactions, Ultrasonic Vocalizations, Self-Grooming, Seizure Activity and EEG Gamma-Power in Shank3B Knockout Mice: Replications and New Discoveries

Background: Translocation and breakpoint mutations in SHANK3 have been implicated in autism spectrum disorder (ASD).  Shank3 is a synaptic scaffolding protein, localized in the postsynaptic density.  Mutant mouse models have been generated to evaluate the biological and behavioral consequences of Shank3 gene mutations, including targeted mutations in the ankyrin (Shank3A), PDZ (Shank3B) or Homer binding domains within the Shank3 gene (Bozdagi et al., 2010; Peca et al., 2011; Bangash et al., 2011; Wang et al., 2011; Kouser et al., 2013). 

Objectives: The present experiments in Shank3B mice were designed to (a) evaluate replicability of published phenotypes; (b) evaluate novel behavioral and physiological phenotypes of high relevance to ASD. The longer-term objective is to select mouse models with robust ASD-relevant phenotypes for discovery of pharmacological interventions for the symptoms of ASD. Our investigations of Shank3B knockout mice are a component of the Autism Speaks Preclinical Autism Consortium on Therapeutics (PACT). 

Methods: Social and repetitive behavioral assays were conducted as previously described (Silverman et al., 2010, 2012).  An analysis of male–female reciprocal social interactions and ultrasonic vocalizations was conducted in male subjects paired with freely moving unfamiliar estrus C57BL/6J females, within a sound attenuating chamber.   Videos were scored by investigators uninformed of genotypes, using Noldus Observer software, as previously described (Yang et al., 2012).  A sensitive ultrasonic Avisoft Bioacoustics microphone collected audiofiles, which were later quantified with SAS-LAB software. Physiological parameters of EEG and core body temperature were recorded using wireless telemetry transmitters, implanted into 7-week-old male Shank3B null and wildtype subject mice.  Data were collected for 8 days in a 12 hour light/12 hour dark cycle followed by 8 days of a 24 hour dark cycle. Animals were then administered pentylenetetrazole (PTZ; 40 mg/kg, i.p.).  EEG seizures, gamma oscillations and circadian rhythms were analyzed using automated algorithms.

Results: Adult male-female social interactions were lower in Shank3B null mutants than wildtype littermates on several key social parameters.  Fewer ultrasonic vocalizations were emitted during the session.  Self-grooming was higher in the null mutants than in their wildtype littermates, in both males and females, in our standard assay, replicating and extending the original report (Peca et al., 2012).  Shank3B null mutant males exhibited more frequent and longer spontaneous seizures during the dark cycle and increased gamma activity (30-80 Hz) in the light phase. Upon PTZ-treatment, the Shank3B null mutants showed a significantly reduced number of myoclonic jerks.

Conclusions: We confirmed the robustness of elevated self-grooming in Shank3B null mutant mice. We discovered reduced ultrasonic vocalizations and reciprocal social interactions by adult male Shank3B null mutant mice.  We also show that Shank3B null mutant males have increased spontaneous seizures during night, and protection against PTZ-induced seizures during day, which may be related to increased gamma EEG power.  These results suggest that Shank3B null mutants have a reliable behavioral phenotype that is reflective of deficits in core symptom domains of ASD. Moreover, our neurophysiological analyses point to EEG abnormalities that may represent quantitative, translational biological markers of pathophysiology associated with mutations in the Shank3 gene.