Because mutations in TSC patients have been reported to be clustered in the region of the tsc2
gene encoding the GAP domain of TSC2 (Maheshwar et al., 1997
), ΔRG transgenic mouse model of TSC provide an opportunity to assess the neurological consequences of mutations in TSC2-GAP domain and their correlation with the neuropsychiatric phenotypes observed in human TSC patients and humans with autism.
Studies in children and adolescents suggest that autism may be seen as inappropriate or indiscriminate approaches to strangers instead of lack of social interaction (Loveland et al., 2001
). In mice, this behavior is measured as equal or less exploration of a novel social target over a familiar one (Crawley, 2004
; Moy et al., 2007
). Initial social behavior studies using the three-chambered social task (Silverman et al., 2010b
) failed to produce sociability and social novelty preference in the wild-type mice in our studies (Supplementary Fig.1A and 1B
). Based on this observation and the fact that ΔRG mice exhibit anxiety-like phenotypes (Ehninger and Silva, 2010
) that could confound the interpretation of the social behavior experiments (Carter et al., 2011
; Silverman et al., 2010b
), we decided to modify the three-chambered social task to include longer habituation sessions (30 min). This modified version of the task was able to produce normal social behaviors in wild-type mice (). In addition, we found that although ΔRG mice exhibit normal social preference, they exhibit inappropriate social behavior when exposed to novel social experiences by equally exploring both familiar and novel social targets (). This inappropriate social behavior was not a consequence of either a failure in social learning or a failure in recognizing novelty. ΔRG mice showed normal social recognition learning of the familiar social target 24 h after their initial encounter (). In addition, ΔRG mice had deficits in a direct reciprocal social task, a more natural social behavior (), although it should be noted that the three-minute test session we used is short compared to 10-minute sessions used in most social interaction studies (Roullet and Crawley, 2011
; Silverman et al., 2010b
). Interestingly, the average time of total direct reciprocal social interactions of wild-type mice using session test lengths of 10 minutes or more (Matsuo et al., 2009
; McFarlane et al., 2008
; Radyushkin et al., 2009
) is 85 seconds, similar to the behavior of the wild-type mice (90 seconds, 50% interacting time) in our studies. In addition, ΔRG mice exhibited less interaction with a freely moving target mouse, perhaps due to increased levels of social anxiety caused by direct contact with the target mouse. In support of this argument, increased levels of anxiety behaviors in ΔRG mice were reported previously (Ehninger and Silva, 2010
). Moreover, the most common psychiatric disorder in children with autism is social anxiety (Simonoff et al., 2008
Although TSC often has been categorized as a cortical disorder, other brain regions, such as the cerebellum are also affected in TSC patients (Ertan et al., 2010
; Ridler et al., 2007
). Consistent with this idea, clinical studies of autistic children have suggested a correlation between lower performance in motor coordination skills and a high score in autistic diagnostic criteria (Halayem et al., 2010
). It has been shown that ΔRG mice suffer from cerebellar developmental problems, including enhanced proliferation and failure in migration of cerebellar granule cells (Bhatia et al., 2009
; Govindarajan et al., 2005
), consistent with the pathology of TSC in humans (Crino and Henske, 1999
). We observed that ΔRG mice have mild, but significant motor learning impairments as measured with an accelerating rotarod paradigm (). In contrast to our findings with ΔRG mice, TSC2 heterozygous knockout mice have been reported to have normal motor skills (Ehninger et al., 2008
). In addition, no cerebellar abnormality has been reported in TSC2 heterozygous knockout mice, suggesting that disruption of the GAP domain of TSC2 might specifically impact the cerebellum. Moreover, recent study using conditional deletion of TSC2 in the cerebellum, showed increased purkinje cell apoptosis, as well as motor skills deficits (Reith et al., 2011
The motor learning impairments observed in ΔRG mice are likely related to cerebellar deficits, and it is possible that the social deficits observed in ΔRG mice also are related to cerebellar dysfunction. Clinical evidence suggests an involvement of the cerebellum in the impaired attentional and orienting skills, stereotypical behaviors, impaired social interactions and impaired communication in children with TSC and autism (Asano et al., 2001
; Courchesne, 2004
; Courchesne et al., 2004
). Similarly, a gabrb3−/−
mouse model of autism, which also displays cerebellar abnormalities, exhibits social interaction and attentional function deficits (DeLorey et al., 2008
It was shown previously that ΔRG mice have normal spatial memory in a Morris water maze task, but a minor impairment throughout the acquisition of the task and during a probe trial test (Ehninger and Silva, 2010
). Similarly, in our studies we observed that the ΔRG mice exhibit normal spatial learning, but a mild impairment was observed on the last day of acquisition of the Morris water maze task (). In contrast to previous studies (Ehninger and Silva, 2010
), we found that ΔRG mice have a significant preference for the target quadrant, indicating normal spatial memory. The protocols used in these studies are slightly different in that spaced training was used in the former study, and a combination of massed and spaced training was used in the latter study. It has been shown that rodents trained with spaced trials perform better and have better long-lasting memories of the platform location than those trained with massed trials (Commins et al., 2003
; Spreng et al., 2002
). Moreover, we also examined the latency to first find the platform zone during the probe trial and found that ΔRG mice reached a plateau in their performance as reflected by a significantly higher latency to find the platform zone (). However, this plateau in performance did not affect the spatial reference memory of the ΔRG mice ().
The ΔRG mice were shown to have normal fear responses to the training context but were not able to discriminate between distinct contexts using a context fear discrimination paradigm (Ehninger and Silva, 2010
). Herein, we report that ΔRG mice have normal contextual fear memory that persists up to seven days after the initial training (). Because it has been suggested that amygdala dysfunction is an important component of autism (Baron-Cohen et al., 2000
; Corbett et al., 2009
; Monk et al., 2010
; Schultz et al., 2000
), it would be interesting to investigate whether cued fear conditioning and partial reinforcement fear conditioning learning are altered in ΔRG mice resulting in indiscriminate fear responses as well.
Other mouse models of TSC have been used to investigate the behavioral consequences of decreasing TSC1 and TSC2 protein levels. TSC2 heterozygous knockout mice were shown to have normal social preference tendencies (Ehninger et al., 2008
), similar to what we observed with ΔRG mice (). This suggests that the functionality of TSC2-GAP domain is not involved in mediating social preference in mice. However, the previous studies did not determine whether the TSC2 heterozygous knockout mice have alterations in social novelty or reciprocal social interactions. On the other hand, of TSC1 heterozygous knockout mice were shown to have reduced social approach in a reciprocal direct social interaction task (Goorden et al., 2007
), similar to our findings with ΔRG mice (). Because both TSC1 and TSC2 proteins form a complex that confers stability to both proteins (Chong-Kopera et al., 2006
; Henske, 2003
; Krymskaya and Shipley, 2003
; Nellist et al., 1999
), decreasing TSC1 protein levels might affect the stability of the TSC1/TSC2 heterodimer. A decrease in TSC1/TSC2 heterodimer stability might compromise the GAP activity of TSC2, which would be similar to disruptive mutations in TSC2-GAP domain as in the ΔRG mice. Taken together, these findings suggest that normal function of the GAP domain of TSC2 is required for normal reciprocal social interactions.
Both TSC1 and TSC2 heterozygous knockout mice have impaired spatial memory in the Morris water maze task, with the impairments being more severe in the TSC1 heterozygous knockout mice (Ehninger et al., 2008
; Goorden et al., 2007
). Taken together with the alterations we observed in the ΔRG mice (), these differences in spatial learning and memory in TSC model mice suggest a differential role or function for the two proteins (Orlova and Crino, 2010
) during hippocampus-dependent spatial memory, and that the GAP activity of TSC2 is not engaged in these processes. In addition, both ΔRG mice and TSC2 heterozygous knockout mice have generalized contextual fear responses (Ehninger et al., 2008
; Ehninger and Silva, 2010
), possibly due to decreased GAP activity in TSC2 in both mouse lines. However, TSC1 heterozygous knockout mice have significantly lower contextual fear memory (Goorden et al., 2007
). These findings suggest a differential role for TSC1 and TSC2 in associative fear learning and memory.
Our studies herein indicate that ΔRG mice exhibit some behavioral phenotypes associated with core symptoms of autism, including social interaction deficits and mild impairments in motor learning skills. Mutations in the GAP domain of TSC2 affect specific aspects of social behavior in ΔRG mice, particularly those engaging novel experiences and reciprocal interactions. The inappropriate social approach observed in ΔRG mice represent a failure for the mice to adapt socially. Moreover, the decrease in reciprocal social interactions displayed by the ΔRG mice might be an indication of social anxiety in these mice. Finally, abnormal cerebellar development observed previously in ΔRG mice is correlated with deficits in motor learning and memory. Thus, the ΔRG transgenic mouse is a mouse model of TSC that could be exploited to further investigate the role of the GAP domain of TSC2 in social anxiety, as well as its potential role in the cerebellum for social interactions and attentional functions that are relevant to autism associated with TSC.
- ΔRG mice exhibit deficits in preference for social novelty and social reciprocal interaction.
- ΔRG mice exhibit a mild, but significant impairment in rotarod motor learning.
- ΔRG mice do not exhibit either repetitive or perseverative behaviors.