Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by impairments in social interaction and communication, and repetitive behaviors. Its prevalence is high (1% globally), and while genetic factors are implicated (*SHANK3* being a key gene), environmental influences significantly impact severity. Currently, pharmacological treatments are limited in addressing core ASD features, while Applied Behavior Analysis (ABA) interventions, although effective for some, are expensive and time-consuming. This study explores the impact of the social environment on both WT and Shank3 KO mice, which exhibit autism-like traits, aiming to identify potential avenues for improving social skills and reducing repetitive behaviors.

Existing literature highlights the complex interplay of genetics and environment in ASD. While numerous genes are implicated, the variability in ASD severity between individuals suggests strong environmental influences. Pharmacological interventions have shown limited efficacy, often addressing co-occurring symptoms rather than core features. ABA-based therapies have demonstrated some success but face challenges in accessibility, cost, and limited generalizability. The existing literature shows conflicting results regarding the effects of social isolation on sociability in rodents, underscoring the need for further research, especially with regard to ASD models and the early social environment.

The study utilized Shank3 KO and WT mice, bred on a mixed C57BL/6J and 129S2 background. Mice were subjected to different housing conditions: group housing (2-4 mice) and chronic social isolation (1 or 4 weeks). Behavioral tests included reciprocal social interaction, three-chambered social interaction, spatial Y-maze (cognitive flexibility), motor stereotypy, and open field (locomotion and anxiety). Olfactory function was assessed using a two-choice preference test. Molecular analyses included quantitative PCR (qPCR) for Oxtr and related genes in olfactory tissues (MOE, VNO, OB), plasma and urine oxytocin concentration measurement using Enzyme ImmunoAssay, RNAscope in situ hybridization for Oxtr mRNA, and calcium imaging of vomeronasal sensory neurons (VSNs). Statistical analyses were performed using R and GraphPad Prism, with appropriate tests selected based on data distribution.

Contrary to expectations, 4 weeks of chronic social isolation in WT mice led to increased social interaction (nose contacts) in the reciprocal test, without affecting social preference or huddling. Shank3 KO mice, exhibiting impaired social novelty preference, showed enhanced social interaction and social novelty preference after 4 weeks of isolation, along with increased huddling. Social isolation normalized self-grooming in Shank3 KO mice, suggesting that this behavior is socially acquired. A novel phenomenon of 'synchronized grooming' was observed in group-housed Shank3 KO mice. Live Mouse Tracker analysis revealed that WT mice in enriched social environments (4 mice/cage) showed sustained social motivation across trials, unlike mice in less enriched environments. Shank3 KO mice displayed altered responses to different social contexts: increased social interaction with other KO mice but reduced interaction in enriched environments compared to WT. Chronic social isolation increased Oxtr mRNA levels in the VNO of both WT and Shank3 KO mice. Calcium imaging showed that VSNs from Shank3 KO mice had a reduced response to urine and oxytocin, which was restored by social isolation. Olfactory preference tests indicated that WT mice and, to a lesser extent, Shank3 KO mice showed a preference for oxytocin over saline. WT mice showed a preference for the urine of isolated mice over group-housed mice.

The findings challenge the assumption that social isolation always impairs sociability. The increased social interaction in isolated WT mice might reflect a compensatory mechanism, potentially involving the oxytocin-dopamine pathway. The normalization of social deficits and stereotypies in socially isolated Shank3 KO mice indicates a potential for environmental manipulation to ameliorate ASD-like phenotypes. The synchronized grooming observed in group-housed Shank3 KO mice highlights the influence of conspecifics on repetitive behaviors. The differential effects of social environment on WT and Shank3 KO mice underscore the importance of considering individual differences when designing behavioral interventions. The findings suggest that olfactory dysfunction contributes to the social deficits observed in Shank3 KO mice and that social isolation can restore this function via VNO modulation.

This study demonstrates that the social environment significantly impacts social behavior and olfactory function in both WT and Shank3 KO mice. Chronic social isolation can enhance social interaction and restore some ASD-like deficits in Shank3 KO mice. These findings support the development of targeted behavioral interventions and adaptive strategies for children with ASD, such as optimizing classroom size and social interaction dynamics within inclusive education.

The study focused on a specific mouse model of ASD and its findings may not be directly generalizable to all forms of ASD or other neurodevelopmental disorders. The sample size, particularly in certain subgroups, was modest. Further research with larger samples and different mouse models is required to strengthen the conclusions. The observed effects of social isolation may be specific to the duration and nature of the isolation implemented in this study.