Patients with ASD manifest behavioral abnormalities in three core areas: social, communication, and stereotypic behaviors. In the present study we have shown that the MALTT male mice display consistent deficits in all three domains – reduced social interest and interactions, altered USV communication during separation and during social interactions, and stereotypic circling. The MALTT line displays additional abnormalities consistent with variable features of ASD, including increased sensitivity to audiogenic seizures, impaired sensorimotor gating, enhanced tactile sensitivity, and hyperactivity. The MALTT mice have a transgene integrated on the X chromosome, which continues to be implicated in ASD based on a disproportionate (approximately 4:1) occurrence in males versus females and also evidence from linkage studies [57
]. However, some studies suggest the skewed ratio does not necessarily mean X chromosome insults are directly responsible for all cases of ASD, and certainly non-X chromosomal ASD-related mutations have been identified [13
]. The site of transgene integration also lies specifically within a region of mouse X chromosome syntenic to regions of human X chromosome implicated in autism susceptibility loci studies [58
]. Together our findings suggest a non-coding region insult on the X chromosome results in robust autism-relevant behaviors that fall within each of the primary ASD diagnosing criterion and within some of the variably associated symptom groups.
Clearly defining a mouse line as a model for ASD possesses multiple challenges because of the complex nature of the social, communicative, and perseverative abnormalities present in individuals with ASD. A number of researchers have identified various behavioral assessments for studying traits in mice that parallel aspects of the three core features of ASD [26
]. In the present study we used a number of these approaches to examine and characterize the MALTT line.
Pup separation-induced USVs have been used to assess early communication abilities in mice. There are no consistent reports of altered vocalizations in infants although automated tools for early-age vocal analysis in humans are being developed [62
]. Thus, this measure is not intended to directly parallel the human infant state. However, pup USVs are a well-defined measure for potentially identifying early neurodevelopmental and communicative abnormalities in mice. MALTT males and females respectively showed a four- and two-day extension of the normal WT USV pattern. For example, MALTT males did not persistently decrease their vocalizations from peak levels until PND 11 compared to PND 7 in WTs. Our results are consistent with several other studies of mouse models of neurodevelopmental disorders associated with ASD including the Tcs62 down syndrome model, a Rett syndrome mouse model, a chromosome 15q11–13 duplication model, and in the BTBR inbred strain autism mouse model [24
] indicating increased levels of vocalization for affected pups in multiple ASD mouse models. It is important to note that decreased vocalizations have been observed in other ASD mouse models [66
], suggesting that impaired separation-induced USV in ASD models could be reflected as either overall decreased number of calls or, as in the current study, a change in the normal developmental USV call pattern.
To further characterize USV communication in the MALTT mice, we studied the emission of USVs from older mice during the social interaction test. For each of the social interaction tests, all mice were juveniles so the social interactions were less influenced by reproductive motivations [69
], and this young age allowed us to avoid the potentially confounding stereotypies that begin in the MALTT line during adulthood. In the three-chamber test, WT mice clearly preferred to spend more time on the side with the social stimulus, a stranger mouse, and more time at the actual partition with the stranger mouse compared to the side with the object. In contrast, the MALTT mice did not show a preference for the stranger relative to a novel object for either measure. It is interesting that while the MALTT mice show a social interest deficit as measured by both time in side and sniffing at the partition, several other ASD mouse model lines exhibit a deficit in social preference when time in a social-paired chamber is considered but exhibit a normal preference when time directly (i.e. sniffing) investigating a social target is considered [19
]. Even the MALTT line trends toward a preference for stranger partition, but not stranger side. It is not clear what a disassociation between time on the side of the social stimulus mouse and actual time sniffing/investigating indicates, but it could reflect a spectrum of social preference in mice with a deficit in direct sniffing being the most sensitive indicator of disinterest in social cues.
Direct social interactions were then analyzed for a pair of mice allowed to freely interact. In our first experiment, a WT or MALTT mouse was paired with a novel standard partner FVB/NJ. This method ensured that the partner mouse for either genotype would have a similar baseline level of social behavior, allowing us to more clearly identify differences in WT and MALTT responses to a standardized social stimulus. MALTTs showed decreased direct social investigation of a FVB/NJ partner and increased nonsocial behaviors relative to WT littermates. During these social investigations USVs were simultaneously recorded. The presence of a MALTT mouse in the pair dramatically reduced the overall USVs emitted from the pair. In the second experiment, when pairs of only MALTT mice were analyzed the majority of pairs emitted no vocalizations at all. This suggests that the MALTT mouse was responsible for the lack of vocalizations during the social interactions when a “normal” partner mouse was present. Both pup and adolescent vocalizations have been analyzed where specific patterned frequency waveforms were identified [21
]. It will be of interest to determine if the MALTT line’s USVs are aberrant with regard to vocalization frequency pattern in addition to the alteration in ‘call number’.
Social interactions rely heavily on olfactory input [47
]. Data from the olfaction test clearly demonstrate that MALTT mice have intact olfactory function and can discriminate between non-social and social odors. However, the MALTT mice do appear to have a reduced “interest” in a first social odor. We believe this observation is consistent with reduced social interest and interactions of MALTT mice in the three-chamber and direct social interaction tests.
In addition to the social and communication deficits in the MALTT line, the males exhibit a clear and developmentally progressing circling stereotypy. In humans the repetitive feature in ASD may present as motor mannerisms, including whole body movements such as rocking. A clinical study also describes that children with ASD may “enjoy spinning or whirling their bodies” [71
]. In the MALTT line, a circling stereotypy typically develops between 3–6 weeks of age in the MALTT males and lasts throughout adulthood. The stereotypy appears spontaneously in the home-cage and is not a constant trait, but rather it seems to be exacerbated when the mice are disturbed (personal observation). Vestibular dysfunction does not appear to play a role in this behavioral abnormality, as is the case for many rodents that exhibit a rotation behavior. In addition, the MALTT mice do not have impaired hearing as measured using the ABR test. MALTT mice were also hyperactive in the open-field even prior to the age of onset for the stereotypic responding. Although it is unclear why the MALTT mice are more active, hyperactivity is consistent with an impairment in response inhibition. We are currently evaluating other potential assays for assessing stereotypic responses and response inhibition in the MALTT mice.
It is important to consider how secondary traits apparent in the MALTT line might have contributed to the observed social deficits and low vocalization levels. The MALTT line is hyperactive and it is possible that MALTT mice were so active during the direct social interaction test that it interfered with interactions. For example, if increased activity of MALTT mice interfered with interactions then the level of ‘passive’ interactions (i.e. those interactions initiated by the WT partner) in the MALTT:WT dyad would have been significantly lower compared to the ‘passive’ interactions in the WT:WT dyad; however, the levels of this type of interaction were similar in both types of dyads suggesting that the activity of the MALTT mice did not confound the interactions in the direct interaction test. In the three-chamber test, although the MALTTs spent less time at the partner partition than WTs, they spent an equivalent amount of time at the object partition, and the number of entries into both chambers was equivalent between MALTT and WT mice indicating that their activity levels were similar during this type of social interaction test. Aggression and stereotypical circling behaviors could also affect social interactions. Therefore, all social tasks were performed using juveniles between 23 and 28 days of age. While the MALTTs showed a clear circling stereotypy at 45 days, there were no significant genotype differences observed at 17, 22, or 28 days of age. In addition, we observed no circling behavior during either the direct social interaction test or the three-chamber test. Similarly, the tests for social interaction were performed prior to the age-of-onset for aggressive behaviors in the MALTT mice, and we did not observe aggressive responses during the juvenile direct social interaction test. Finally, although isolated housing is routinely utilized prior to a variety of social tests, it is still possible that MALTTs differentially responded to the isolated housing than WTs. At this point it cannot be eliminated as a contributing factor, although, since the single-housing was limited to only a few days, we believe the differential impact, if any, may have been minimized.
A wide range of additional behaviors are associated with ASD including cognitive impairments, hyperactivity, abnormal anxiety, seizures, aggressiveness, and odd responses to sensory stimuli, including oversensitivity to sounds or being touched [2
]. MALTT male mice exhibit increased audiogenic seizures, reduced sensorimotor gating, and increased sensitivity to tactile stimulation. Sensory function is involved in all three of these behaviors and suggests that the MALTT line may be especially amenable to understanding a link or common pathology between sensory abnormalities and ASD core feature symptoms. One juncture at which the sensory hypersensitivity may intersect the observed social abnormalities is the heightened aggression. Elevated aggressive responses have been observed in the BTBR mouse line, and it was suggested that after prolonged investigation by a partner mouse the BTBR mouse may have experienced a “sensory overload” resulting in aggressive attack [20
]. It is possible that a similar explanation might underlie the MALTT line aggression. In a third sensory-mediated test 100% of MALTT males tested exhibited full erratic seizures in response to a loud auditory stimulus. Audiogenic seizures rarely occur in humans, but epileptic spontaneous seizures occur in the ASD population at a rate as high as 30% [53
]. Interestingly, similar sensory abnormalities, including increased tactile sensitivity, heat sensitivity, and susceptibility to PTZ-seizure were recently described in the Gabrb3 mouse model of ASD [72
]. In the Pten condition null mouse model of ASD, reciprocal social interaction deficits, occurrence of spontaneous seizure, and impaired PPI have been reported [73
]. Similarly, susceptibility to audiogenic seizure and abnormal PPI response occurs in the Fragile X mouse model, currently being investigated for insights into ASD due to an overlap in clinical populations [74
]. ASD mouse models exhibiting both core and secondary phenotypes provide a tool for examining the extent to which a similar underlying pathology is responsible for the concomitant features.
We believe that the behavioral pattern observed in the MALTT line of mice suggest that they represent a novel mouse model with abnormalities in assays for each of the core features of ASD, in addition to multiple other phenotypes consistent with secondary abnormalities observed in some individuals with ASD.
The MALTT line was generated by a random insertional mutation rather than based on a single human population-identified genetic abnormality. This novel mouse line highlights the fact that although it is currently common to discount non-coding region duplications and deletions, they may not necessarily be benign. Certainly, insertions, rearrangements, duplications, and deletions do not only cause gene copy number effects, but they can affect coding region and non-coding region chromatin structure and status and nearby and long-distance transcription factor binding [76
]. Direct analysis of the effects of these types of changes is difficult in the human disease population because the effects may largely be at the level of RNA expression and quality samples are rare and challenging to obtain. However, with the establishment of the AGRE database these types of studies are now more readily being undertaken [41
] in human samples from lymphoblast cell lines. Currently, rodent models are especially amenable to these types of analysis because of the ease of access to preserved chromatin status and RNA expression patterns.
Results from our molecular studies have identified several genes that may be involved in the autism-related phenotypes seen in the MALTT line. In the MALTT mice, the strongest gene expression change is the dramatic upregulation of Fam46d expression observed in both adult and newborn cortex. Fam46d is a protein of unknown function. However, it is a proposed cancer-testis antigen [78
], and homology and protein-protein interaction studies suggest FAM46 family proteins may be crucial for cellular signaling and potentially involved in the TGF-β signaling pathway [79
]. Importantly, Fam46d probes were included in an RNA expression microarray screen of lymphoblastoid cells from a population of humans with fragile X also meeting the criteria for autism, and Fam46d was found to be upregulated [41
]. A close homologue, Fam46c was also found to be differentially expressed in the autism-affected versus control populations. Taken together our data and those of Nishimura et al. support the hypothesis that this gene family, and specifically Fam46d, may in some cases be involved in the expression of ASD-related behaviors. Our data also suggest low-level upregulation of the nearby genes 2610002M06Rik, Gm732, Brwd3, and Hmgn5 occurred in MALTT male cortex, although this upregulation relative to WT was not observed in newborn cortex. Whether these genes contribute to the murine behavioral output will require future systematic analyses. While the striking upregulation of Fam46d, which is located near the transgene insertion site, is compelling, future studies will be necessary to directly manipulate Fam46d to better understand its’ role in the types of ASD-related responses we have documented in the MALTT mice.
Finally, the brain morphological survey indicated that MALTT male brain weights were significantly lower in adults. A wide range of autism studies have described varying morphological abnormalities in numerous brain regions, and it should be noted that no clear and consistent abnormalities have thus far been identified. However, post-mortem and imaging studies have most frequently described the frontal lobes, amygdala, and cerebellum as differing in ASD populations compared to controls [82
]. Our preliminary histopathological examination of the brain revealed no major malformations. Future detailed studies will be necessary to assess for cellular organization and composition.
The molecular mechanisms underlying ASD still remain elusive. ASD is an incredibly heterogeneous disorder, and it will undoubtedly require a variety of models to elucidate common downstream mechanisms resulting in similar behavioral outcomes. Mouse models, such as the MALTT mice, which replicate the behavioral phenotypes of ASD are an important tool for identification of novel genetic abnormalities and molecular mechanisms that might improve our understanding of the etiology of ASD.