Environmental enrichment modulates affiliative and aggressive social behaviour in the neuroligin-3 R451C mouse model of autism spectrum disorder
Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disorder in which individuals display core deficits in social interaction and show repetitive and/or stereotyped behaviours (American Psychiatric Association and DSM-5 Task Force, 2013). The disorder is usually diagnosed in infancy and persists throughout adulthood and has an estimated prevalence of 1.5% in the general population (Christensen et al., 2018). Accordingly, the economic costs and social burden for individuals, families, and the community are significant (Kogan et al., 2008). Although some therapies such as Applied Behavioural Analysis (ABA) (Dillenburger and Keenan, 2009) can be an effective form of ameliorating core and associated symptoms, including aggression (Dawson, 2008; Howard et al., 2005), these interventions must be applied early to be most effective (Harris and Handleman, 2000). Limited therapeutic options exist beyond ABA as the aetiology of the disorder has yet to be fully understood.
ASD is thought to be predominantly caused by genetic variants, however complex interactions between genes and the environment are acknowledged to contribute (Yoo, 2015). The strong genetic basis of ASD is evident based on twin studies which have reported concordance rates of 70–80% between monozygotic twins (Bailey et al., 1995; Rosenberg et al., 2009), although there is no single genetic explanation for the cause of ASD. Common and rare variants have been identified as genetic risk factors through a variety of studies such as genetic linkage, candidate gene screening and genome-wide association studies (GWAS) (Bourgeron, 2015; Ramaswami and Geschwind, 2018). Genes linked to autism through genome-wide studies can be grouped into functional categories based on their established roles in the brain (Chakrabarti et al., 2009). Of these, mutations affecting synaptic proteins remain the most consistently reported and well-characterised (Boccuto et al., 2013; Durand et al., 2007; Jamain et al., 2003; Yanagi et al., 2012). Synaptic cell adhesion molecule (CAM) pathways implicated in ASD include proteins such as the neurexins, neuroligins and their downstream binding partner SHANK3 (Betancur et al., 2009; Geschwind and Levitt, 2007; Jamain et al., 2003; Zoghbi, 2003).
Mice expressing the autism-associated R451C mutation in neuroligin-3 (NL3) recapitulate core traits of ASD (Burrows et al., 2015; Burrows et al., 2017; Cao et al., 2018; Etherton et al., 2011; Hosie et al., 2018; Jaramillo et al., 2014; Kumar et al., 2014; Rothwell et al., 2014; Tabuchi et al., 2007) and are useful for understanding underlying neurobiology. NL3R451C mice have been assessed for social interaction impairments in a number of studies using the three-chamber social interaction arena assay, free reciprocal interaction and resident-intruder assay. While impairments in social behaviour have been reported (Burrows et al., 2015; Burrows et al., 2017; Cao et al., 2018; Etherton et al., 2011; Hosie et al., 2018; Jaramillo et al., 2014; Kumar et al., 2014; Tabuchi et al., 2007), a number of studies have shown no difference in social interaction phenotypes between NL3R451C mice and control littermates (Burrows et al., 2015; Chadman et al., 2008). These inconsistencies may be due to differences in genetic background strain and methodology (e.g. protocol details, lighting conditions and previous behavioural testing experience) however whether this phenotype is modulated by environmental enrichment has not been formally tested.
Environmental factors can strongly influence experimental results in behavioural tests and vary widely between different laboratories. While direct comparisons of these factors have been attempted in cases where identical genotypes are examined, assessment of their impact on outcomes is difficult and it is likely that they contribute to the large number of inconsistent results reported by different behavioural laboratories (Crabbe et al., 1999; Wahlsten, 2001; Wahlsten et al., 2006). Along these lines, it is not rare that the same mutation is reported to produce distinct phenotypes depending on the testing environment or tests applied (Crestani et al., 2000). In this study, we aimed to test whether environmental enrichment, a paradigm designed to boost somatosensory, cognitive and motor stimulation, could modulate the NL3R451C mouse social phenotype. Enrichment has been shown to boost social interaction (McQuaid et al., 2018; Mesa-Gresa et al., 2013), modulate social aggressive behaviour (Giles et al., 2018; Haemisch and Gärtner, 1997; Haemisch et al., 1994; Lima and Spinelli de Oliveira, 2014; Lockworth et al., 2015; Marashi et al., 2003; McQuaid et al., 2018; Pietropaolo et al., 2004) and reduce anxiety (McQuaid et al., 2013a, McQuaid et al., 2013b) in mice. To date, NL3R451C mice have not been housed in environmentally enriched conditions and we hypothesised that social and aggressive behaviour in NL3R451C mice is modulated by enriched housing.
Section snippets
Animals and housing
B6;129-Nlgn3tm1Sud/J mice were obtained from Jackson Laboratories (008475; Bar Harbor, Maine USA) and maintained on a Sv129/C57BL/6 background. NL3R451C and wild-type (WT) animals were derived by mating heterozygous females with NL3R451C males, which produced 50:50 WT and NL3R451C male offspring (Y/+ and Y/R451C) that were genotyped by real-time PCR (Transnetyx, Cordova, TN, USA). Mice were weaned at 3 weeks and housed 4 per cage, randomly assigned to one of two conditions with food and water
Environmental enrichment reduces body weight in mice
Mice housed with environmental enrichment (EE) exhibited lower weights, regardless of genotype, than standard-housed (SH) mice (Fig. 1; effect of environment, F(1, 34) = 8.071, p = 0.007; time ∗ environment, F(4,136) = 5.776, p = 0.003, pairwise SH vs EE p < 0.05 at 5,6,7 weeks of age). Overall, wild-type (WT) mice were heavier than NL3R451C mice (Fig. 1; effect of time ∗ genotype, F(4,136) = 3.520, p = 0.028, pairwise: NS trend).
Enrichment increased direct interactions with the mouse in the sociability trial however no differences were seen between NL3 and WT mice
All mice were highly sociable, with all four groups showing a
Discussion
This study aimed to investigate environmental modulation of social behaviour in Neuroligin-3 R451C mice. We found that environmental enrichment housing shifted both affiliative and aggressive social behaviour in mice, but did not show a specific effect in mice expressing the R451C gene mutation. Enrichment rearing did however lead to genotype-specific effects including reduced activity levels in NL3R451C mice and reduced anxiety-like behaviour in WT mice.
In conflict with previous findings, no
Authors' contributions and competing interests
ELB, ELH, and AJH conceived of the study and participated in its design and coordination. ELB supervised all behavioural tests, data collation/scoring and statistical analysis and carried out the locomotor test. LK carried out the social, anxiety and resident intruder tests and data analysis and revised the manuscript. CM performed the statistical analysis for aggression data. ELB, LK, CM, ELH and AJH drafted and edited the manuscript. All authors read and approved the final manuscript. The
Acknowledgements
We thank past and present laboratory members for useful discussions. Thank you to Leah Catalano for managing colony breeding and for dialogue regarding observations behind the barrier that led to us scrutinising aggression in this mouse model. Thank you also to Maria Bastias for diligent and continuous support in the animal house and to Brett Purcell and Craig Thomson for their assistance in facilitating behavioural testing.
Funding
ELB is supported by a National Health and Medical Research Council-Australian Research Council (NHMRC-ARC) Dementia Research Development Fellowship (1111552). EH-Y is supported by an ARC Future Fellowship (ARC FT 160100126) and an RMIT Vice Chancellor's Senior Research Fellowship. AJH has been supported by an Australian Research Council (ARC) FT3 Future Fellowship (FT100100835) and is currently supported by an NHMRC Principal Research Fellowship and NHMRC Project Grants.
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