A Novel Shank3-Deficient Rat Model to Understand the Neural Basis of Autism

 

Background: Shank3 is a scaffolding protein that forms a key structural part of the postsynaptic density (PSD) of excitatory synapses, where it recruits glutamate receptors and binds cytoskeletal elements regulating glutamate signaling. Haploinsufficiency of SHANK3 causes a monogenic form of autism spectrum disorders (ASD). Characterization of mouse models with Shank3mutations provided evidence for impaired glutamatergic synaptic function in hippocampal and striatal brain slices and deficits in several behavioral measures related to ASD. Yet, our knowledge about the affected molecular pathways and the alterations in brain circuitries and their relation to the behavioral deficits observed in ASD remains incomplete.

Objectives: The aim of our study is to analyze the effect of Shank3 deficiency on ASD associated behaviors and to uncover the alterations in synaptic plasticity in brain regions and circuits associated with the impaired behaviors. At the molecular level, we aim to identify the affected pathways that underlie the behavioral and synaptic deficits. To achieve our aims, we have developed a genetically modified rat model with a targeted disruption of Shank3and completed a battery of behavioral, biochemical, electrophysiological and genome wide expression analyses.

Methods: Behavior: To study social behavior, we applied a battery of social behaviors tests including long and short-term social recognition memory (SRM). To evaluate attentional function and behavioral flexibility, we used the 5-choise serial reaction time  (5-CSRT) task and a strategy-shifting paradigm, respectively. Electrophysiological recording: To analyze synaptic function and plasticity we used in vitro recording from acute hippocampal brain slices. To study the long-range synaptic connectivity in the Hippocampal-medial prefrontal cortex (mPFC) circuitry, we stimulated the hippocampus and recorded the synaptic responses in the mPFC in anesthetized rats. Genome wide expression and pathway analysis: We used proteomic approaches and RNA sequencing technology to compare gene expression signatures between the Shank3-deficient rats and their WT littermate and applied gene ontology (GO) analysis to reveal the affected molecular pathways.

Results: Behaviorally, we found that Shank-deficient rats are impaired in long-term SRM, attention, and reversal learning (an essential component of behavioral flexibility), all of which are prevalent in subjects with ASD. These behaviors are highly dependent on the intact function of brain regions previously associated with ASD, i.e., the hippocampus and the PFC. Our in vitro recordings demonstrated impairment in long-term potentiation (LTP) and depression (LTD) in the hippocampus while in vivo recordings showed impaired mPFC LTP in response to hippocampal stimulation. Proteomics analysis revealed decreased levels of postsynaptic components, including Homer and PSD-95, while genome wide expression and GO analysis revealed a list of differentially expressed genes in Shank3-deficient rats highly enriched in pathways and cellular components involved in cell communication, extracellular matrix (ECM) receptor interaction and focal adhesion.

Conclusions: Shank3 deficiency has an effect on behaviors relevant to ASD and modulates synaptic plasticity in brain regions and circuits underlying them. These deficits could be in part due to the disruptive effect of Shank3 deficiency on ECM-associated factors that are known to play a critical role in synaptic plasticity and in forming neuronal connections during development.