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Profound impairment in social interaction is a core symptom of autism, a severe neurodevelopmental disorder. Deficits can include a lack of interest in social contact and low levels of approach and proximity to other children. In this study, a three-chambered choice task was used to evaluate sociability and social novelty preference in five lines of mice with mutations in genes implicated in autism spectrum disorders. Fmr1tm1Cgr/Y(Fmr1−/y) mice represent a model for fragile X, a mental retardation syndrome that is partially comorbid with autism. We tested Fmr1−/ymice on two genetic backgrounds, C57BL/6J and FVB/N-129/OlaHsd (FVB/129). Targeted disruption of Fmr1 resulted in low sociability on one measure, but only when the mutation was expressed on FVB/129. Autism has been associated with altered serotonin levels and polymorphisms in SLC6A4 (SERT), the serotonin transporter gene. Male mice with targeted disruption of Slc6a4 displayed significantly less sociability than wild-type controls. Mice with conditional overexpression of Igf-1 (insulin-like growth factor-1) offered a model for brain overgrowth associated with autism. Igf-1 transgenic mice engaged in levels of social approach similar to wild-type controls. Targeted disruption in other genes of interest, En2 (engrailed-2) and Dhcr7, was carried on genetic backgrounds that showed low levels of exploration in the choice task, precluding meaningful interpretations of social behavior scores. Overall, results show that loss of Fmr1 or Slc6a4 gene function can lead to deficits in sociability. Findings from the fragile X model suggest that the FVB/129 background confers enhanced susceptibility to consequences of Fmr1 mutation on social approach.
While mouse models cannot fully recapitulate diverse behavioral elements of complex neuropsychiatric disorders such as autism, engineered mutations in mouse lines provide a way to investigate the association between candidate endophenotypes and specific genes or signaling pathways implicated in the human disease (Hranilovic & Bucan 2001). The following studies used genetic mouse models to investigate social approach deficits as a possible endophenotype for the broad domain of abnormal social function in autism. Mutant lines were selected for alterations in genes linked to heritable, biochemical or neuropathological aspects of autism. Our hypothesis was that one or more of these mouse lines would show deficient social approach and thus provide a link between a single gene (Fmr1, Slc6a4, Igf-1, En2 or Dhcr7) and a quantifiable social endophenotype for more global social impairment.
In humans, fragile X syndrome is associated with mental retardation, physical abnormalities and autistic symptoms (Hagerman et al. 1986). The disease is caused by disrupted function of the FMR1 (Fragile X Mental Retardation 1) gene, which has been modeled in the Fmr1−/ymouse (Bakker et al. 1994). Numerous studies have provided evidence for significant parallels between alterations observed in children with fragile X and abnormal behavior in Fmr1−/ymice, including deficits in attention and learning (Bakker et al. 1994; Kooy et al. 1996; Moon et al. 2006; Paradee et al. 1999), changes in reactions to sensory stimuli (Chen & Toth 2001; Frankland et al. 2004) and abnormal social responses (McNaughton et al. 2008; Mineur et al. 2006; Spencer et al. 2005). One interesting feature of this mouse model is that the phenotype of Fmr1 loss of function may be dependent on the genetic background. For example, researchers have proposed that the C57BL/6J background confers resistance to effects of Fmr1 deficiency on spatial learning, while FVB/N-129/OlaHsd (FVB/129) leads to greater susceptibility (Dobkin et al. 2000, see also Paradee et al. 1999). The direction of neuroanatomical changes in Fmr1-null mice, such as labeling of mossy fiber terminals, can also be dependent on background strain (Ivanco & Greenough 2002; Mineur et al. 2002), suggesting that modifier genes play an important role in Fmr1-related phenotypes. The present report evaluates Fmr1−/ymice on both C57BL/6J and FVB/129 backgrounds for alterations in social approach.
Both cross-sectional and longitudinal studies have shown that a subset of autistic children show age-dependent brain overgrowth (Courchesne et al. 2001, 2003; Hazlett et al. 2005). Brain overgrowth at an early age can be modeled by the conditional overexpression of Igf-1 (insulin-like growth factor-1) in brain, which induces significant increases in brain volume during the embryonic and early postnatal period (Popken et al. 2004). In comparison to wild-type controls, nestin-Igf-1 transgenic mice exhibit an almost 30% greater brain size, without concomitant changes in overall body weight (Popken et al. 2004). Interestingly, Mills et al. (2007) reported both greater head circumference and higher levels of plasma IGF-1 in children with autism and autism spectrum disorder (ASD) compared with normal controls. The correlation between head size and IGF-1 levels was highly significant in the autism/ASD group but not in the control group. The present studies investigated whether conditional overexpression of Igf-1 was associated with autism-like social behavior in mice.
Mice deficient for engrailed-2 (En2), a gene crucial for normal development of the cerebellum, were also assessed. Several studies in human populations have reported that variants of En2 may confer risk for ASDs (Benayed et al. 2005; Brune et al. 2008; Gharani et al. 2004; Wang et al. 2008), although not all findings have been positive (Zhong et al. 2003). Researchers have observed parallels between neuroanatomical changes in brain of En2−/−mice and alterations in cerebellar structure reported in autistic children (Kuemerle et al. 2007; Murcia et al. 2005). Last, we examined a genetic mouse model for Smith–Lemli–Opitz syndrome (SLOS), a disease with a markedly high co-occurrence with autism (Bukelis et al. 2007; Sikora et al. 2006; Tierney et al. 2001). Smith–Lemli–Opitz syndrome arises from mutations in DHCR7 (7-dehydrocholesterol reductase), the last enzyme in the cholesterol biosynthesis pathway, with subsequent disruption of cholesterol synthesis. During the prenatal period, Dhcr7-null mice have overt increases in serotonin immunoreactivity (Waage-Baudet et al. 2003). Unfortunately, the loss of Dhcr7 function is lethal in mice (Fitzky et al. 2001). Our study evaluated heterozygous animals (Dhcr7tm1Gst/+ or Dhcr7+/−), which have a mild reduction in the Dhcr7 enzyme.
An important issue for interpreting results from social approach tests is that low preference for the social partner may be associated with changes in activity and/or anxiety-like behavior (Kalueff et al. 2007a; Moy et al. 2007; Spencer et al. 2005). In the present studies, information from one or more control measures, including motor co-ordination, activity levels in an open field and anxiety-like behavior in an elevated plus maze, was considered in the interpretation of results from the social approach assays.
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- Materials and methods
In addition to profound deficits in social interaction, the core symptoms of autism include aberrant repetitive behavior and restricted interests (American Psychiatric Association 2000). The impairments in social function may involve a different set of genes than symptoms related to the repetitive behavior domain (Ronald et al. 2006; see also Ronald et al. 2005). Complex neuropsychiatric disorders with this type of genetic heterogeneity and phenotypic diversity present difficulties for large-scale genome linkage and candidate gene association studies. Recently, investigators have focused on endophenotyping approaches for genetic analysis of clinical syndromes such as autism or schizophrenia, measuring social or neurocognitive traits (Duvall et al. 2007; Gur et al. 2007; Horan et al. 2008). The present studies used genetically engineered mouse lines to evaluate a quantifiable social trait as a heritable marker for impaired social function relevant to autism.
One challenge for the development of mouse models for autism is that the fundamental mechanisms underlying symptomatology are unknown. However, several of the candidate genes implicated in autism play a role in synaptic function, suggesting that disruption of synaptic mechanisms may be a common factor across ASDs (Abrahams & Geschwind 2008). In the present studies, the Fmr1- and Slc6a4-null mouse lines provided models of dysregulated synaptic function associated with specific candidate genes for autism. Fmr1 silencing can lead to abnormal synaptic plasticity, which has been linked to prolonged glutamatergic signaling (Hou et al. 2006; Huber et al. 2002; Nakamoto et al. 2007; Nosyreva & Huber 2006). Synaptic disruption in Fmr1-null mice includes aberrant dendritic morphology, characterized by longer, thinner spines and a higher spine density, comparable to abnormalities observed in fragile X syndrome (Comery et al. 1997; Irwin et al. 2002; McKinney et al. 2005). Similarly, activation of serotonergic pathways is regulated by the serotonin transporter. Loss of Slc6a4 results in prolonged signaling, which may have a profound impact on normal brain development and function (Murphy & Lesch 2008). Regionally specific alterations in dendritic morphology and increased spine density have been reported in Slc6a4-null mice (Wellman et al. 2007).
Our results show that deficits in social approach are found with the targeted disruption of either Fmr1 or Slc6a4. The mice null for Fmr1 on an FVB/129 background failed to show significant preference for spending time in the social partner side in the choice task in contrast to Fmr1−/ymice on a C57BL/6J background. The lack of preference could not be attributed to low exploration, low activity or higher levels of anxiety-like behavior in the mutant mice. In line with these findings, male Slc6a4−/−mice (on a C57BL/6J background) spent significantly less time than wild-type controls in the proximity of the unfamiliar social partner. As with the fragile X model, lack of social preference was not associated with low total number of entries during the test or with changes in anxiety-like behavior. A similar link between genetic changes leading to altered synaptic function and deficient social approach has been reported for Gabrb3 (GABAA receptor subunit β3)-null mice (DeLorey et al. 2008), Mecp2 (methyl-cytosine–phosphate–guanosine-binding protein-2)-mutant mice (Moretti et al. 2005) and Nlgn3 (neuroligin-3) R451C knockin mice (Tabuchi et al. 2007). It is notable that GABRB3, MECP2 and NLGN3, as well as FMR1 and SLC6A4, are found on chromosomal loci associated with susceptibility for ASDs (Abrahams & Geschwind 2008).
In addition to specific behavioral characteristics, age-dependent brain overgrowth has been observed in autism (Aylward et al. 2002; Courchesne et al. 2003; Hazlett et al. 2005) and fragile X syndrome (Chiu et al. 2007). Igf-1 transgenic mice were used to model this neuroanatomical abnormality. A previous study with Igf-1 null mice provided evidence that this gene is important for dendritic growth and synaptogenesis (Cheng et al. 2003). Interestingly, loss of Igf-1 led to significant decreases in dendritic spine length and density, which are opposite to the alterations observed with Fmr1 deficiency (Comery et al. 1997; Irwin et al. 2002; McKinney et al. 2005). These findings suggest that Igf-1 overexpression might induce abnormal growth of dendritic spines and therefore have detrimental effects on synapse function similar to targeted disruption of Fmr1. However, in contrast to the fragile X model mice, the Igf-1 transgenic mice did not show deficits in social approach or in any other behavioral measure. The unchanged phenotype of the Igf-1 mutants shows that even overt alterations in normal brain development do not necessarily lead to social endophenotypes.
En2-null mice served as a model of altered cerebellar morphology observed in autism (Kuemerle et al. 2007; Murcia et al. 2005). Cheh et al. (2006) found that En2−/− mice have higher levels of serotonin than wild-type controls in cerebellum, but not frontal cortex, hippocampus or striatum. Thus, the En2−/− mice could provide information on the behavioral effects of a regionally specific enhancement of serotonin signaling. The Dhcr7+/− mice were investigated as another interesting mutant with dysregulation of serotonin signaling. These mice reflect the disrupted cholesterol biosynthesis observed in SLOS (Fitzky et al. 2001). There is evidence that reductions in cholesterol lead to decreased activity of the serotonin transporter (Magnani et al. 2004; Nomura et al. 2008; Scanlon et al. 2001), which could underlie increases in hindbrain serotonin observed in Dhcr7-null mice during prenatal development (Waage-Baudet et al. 2003). Unfortunately, the behavioral phenotypes of the En2 and Dhcr7 lines, both wild type and mutant, included markedly low exploration. Other researchers have reported general hypoactivity in mutant mouse lines on a 129S2/SvPas (Gerlai et al. 1996) or a 129S6/SvEvTac (Holmes et al. 2003) background. Inbred strain distributions of anxiety-like behavior (Bouwknecht & Paylor 2002; Brooks et al. 2005; Rodgers et al. 2002; see also Cook et al. 2002) confirm low exploration in specific 129 substrains. In the present studies, the lack of exploration in the social approach task precluded the detection of social endophenotypes in the En2 and Dhcr7 mutant mice.
Our findings with the Fmr1-null mice illustrate the importance of background strain in determining the effects of genetic alteration. Recently, Fmr1-null mice on a C57BL/6J x FVB/NJ hybrid background were reported to have normal social preference in a three-chambered choice task (McNaughton et al. 2008). However, depending on the behavioral assay, Fmr1−/ymice on a C57BL/6J background can exhibit altered social responses. Fmr1-null C57BL/6J mice have been found to have deficits in social interaction with repeated presentations of an ovariectomized female during a habituation procedure (Mineur et al. 2006). Spencer et al. (2005) evaluated Fmr1-null C57BL/6J mice across several domains of social behavior. In a repeated partition test, the mutant mice had decreased social interest for the unfamiliar stranger mouse at the beginning of the 20-min procedure and increased social interest by the end of the testing period. We observed a similar lack of significant preference in the Fmr1−/yFVB/129 mice during the first 10-min assay (the sociability test), but not the following 10-min assay (the social novelty test). Spencer et al. (2005) have suggested that decreased social interest at the beginning of a test may reflect increased social anxiety in Fmr1 mutants. However, changes in anxiety-like behavior may be dependent on the particular assay. In line with previous reports (Mineur et al. 2002; Nielsen et al. 2002), our study on elevated plus maze performance did not indicate a general increase in anxiety-like behavior in Fmr1−/y mice on either background strain.
Other researchers have found changes in social behavior in Slc6a4−/−mice on a C57BL/6J background. Holmes et al. (2002) noted decreased aggression in male Slc6a4-null mice during a resident-intruder test, without any changes in investigatory social interest. The mutant mice were also hypoactive in the home cages and in an open field. Kalueff et al. (2007a) found that female Slc6a4−/−mice had less initiation of sniffing directed towards the social partner in a free interaction test. The Slc6a4−/−mice also had decreased exploration in an open-field test as well as reduced approaches in a novel object test. Therefore, results from these social interaction tests may have reflected hypoactivity and higher levels of neophobia in the Slc6a4-null mice rather than an intrinsic deficit in social interest. The issue of hypoactivity and low exploration is also problematic for the evaluation of depression-like behavior in Slc6a4-null mice (Kalueff et al. 2006). In the present study, reduced social approach was observed in Slc6a4−/−mice without decreases in approach towards a nonsocial novel object (the empty wire cage) or fewer entries during the test. The low percent time (ranging from 5% to 9%) spent in the open arms of the elevated plus maze by the Slc6a4 line may have prevented the detection of increases in anxiety-like behavior in the mutant mice.
The dependence of social preference on background strain in the Fmr1−/−mice suggests that modifier genes can attenuate or exacerbate the consequences of Fmr1 loss. One important conclusion from the findings in the Slc6a4 line is that the C57BL/6J background does not necessarily confer protection from the effects of genetic alteration on social approach in the three-chambered choice task. In the Slc6a4-null mice, modifier genes present in the C57BL/6J background did not prevent the changes induced by disrupted transporter function, which may indicate a stronger association of the serotonin signaling pathway, rather than Fmr1-mediated events, with fundamental alterations in social motivation. However, many other factors could have affected social behavior in the mutant lines, including altered learning ability, deficits in sustained attention, subtle olfactory dysfunction or other traits not assessed in these experiments.
The social approach test used in the present studies included an assay for social novelty preference to provide a secondary measure of social approach based on discrimination between two partners (stranger 1 and the more novel stranger 2). Previous work has shown that high sociability does not predict subsequent preference for social novelty in inbred mouse strains, suggesting that the two assays are measuring different components of social behavior (Moy et al. 2007, 2008). A similar dissociation between sociability and social novelty preference was evident in the mutant mouse lines of the present studies. In particular, neither the Slc6a4 nor the Igf-1 wild-type groups had significant preference for the stranger 2 mouse, even though both groups had significant sociability, and were on a background characterized by positive social novelty preference (C57BL/6J; Moy et al. 2007, 2008). The lack of social novelty preference suggests that, across multiple generations, the Slc6a4 and Igf-1 mouse lines have diverged from the original C57BL/6J background.
In the Slc6a4 groups, only the heterozygous mice showed a significant preference for social novelty. Previous work has shown that Slc6a4+/−mice retain about 50% of normal serotonin transporter binding (Bengel et al. 1998; Montanez et al. 2003). Behavior in Slc6a4+/−mice is usually not different from wild-type mice, or else parallels, to a lesser extent, changes observed in Slc6a4−/−mice, supporting a gene dose-dependent function for some behavioral domains (Holmes et al. 2002, 2003; Kalueff et al. 2007a). However, one study found that serotonin levels in the frontal cortex were significantly increased in Slc6a4+/− mice, but decreased in null mutant mice, in comparison to controls (Bengel et al. 1998). It is possible that a reduction, vs. a loss, of transporter function could lead to qualitatively different alterations in specific brain regions and to different profiles of social behavior.
Our findings, together with published reports in other mutant mouse lines (DeLorey et al. 2008; Moretti et al. 2005; Tabuchi et al. 2007), provide evidence that synaptic dysfunction through various mechanisms can lead to similar deficits in social approach. The results are in line with human genetic analyses that have identified disruption of synaptic function as a possible cellular mechanism underlying symptoms in ASDs (Abrahams & Geschwind 2008). In addition, recent work has shown that genetic alterations thought to restore normal synaptic function can reverse, either fully or partially, abnormalities in dendritic morphology, plasticity and behavior in Fmr1-null double transgenic mice (Dolen et al. 2007; Hayashi et al. 2007). Overall, these studies support the utility of mouse models to link specific genes and signaling pathways to heritable social endophenotypes and to examine possible underlying mechanisms relevant to autism.