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Assessment of Region-Specific TBR1 Haplo-insufficiency and Overexpression in Autism Spectrum Disorder Mouse Models

Applebey, Courtney, Verma
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045

Autism Spectrum Disorder (ASD) is a compilation of developmental disorders characterized by impairments in social communication, intellectual disability, speech deficits, anxiety, and repetitive and restricted patterns of behavior. Although the lives of individuals with ASD are often disrupted by these symptoms, they are managed only with an assortment of behavioral therapies and psychiatric drugs. A variety of ASD risk genes – genes that are more susceptible to mutations leading to ASD – have been shown to contribute to varying degrees of ASD behavior. TBR1 is an ASD risk gene that encodes for a transcription factor widely expressed in the brain. Loss of function mutations in one copy of TBR1 results in dysregulation of other genes by TBR1, causing ASD behaviors. Few studies have characterized the behavioral phenotype of TBR1 haploinsufficiency in animal models. Behaviors associated with Rett’s syndrome, one of the disorders of the ASD spectrum, have been shown to be region-specific in terms of behavior. Presently, it is unknown whether mutations in TBR1 are associated with region-specific ASD-like behaviors. Thus, we predict the behaviors stemming from TBR1 haploinsufficiency are mediated by specific regions of the brain. If this is the case, TBR1 will be overexpressed in a region-specific manner using wild-type TBR1 mice to evaluate possible side effects for overexpression as a potential therapy. Finally, if negative side effects do not occur, TBR1 will be overexpressed in pre-established ASD mouse models in addition to TBR1 knockouts. Ultimately, this will allow for the selective rescue of life-impairing ASD symptoms, while retaining other desired traits.





Autism Spectrum Disorder is a compilation of neuropsychiatric disorders characterized by behavioral deficits (1) including impairments in speech and social skills, repetitive behaviors, anxiety, developmental delay, etc. (2). These impairments vary in severity and functionality (3) and are implicated with underlying mutations (1). Brain regions, such as the prefrontal cortex and hippocampus, are commonly implicated in these behaviors with the prefrontal cortex playing a role in executive functioning, cognitive flexibility, inhibition of perseveration, and repetitive behaviors (4). Furthermore, the hippocampus has been shown to be implicated in social memory (5). Additionally, the amygdala has been found to be the source of anxiety and impairment in associative learning (6, 7). These behaviors are thought to be facilitated by synaptic imbalance in excitation and inhibition (8), partly induced by genetic mutations (1). For example, mutations in a ASD risk gene, SHANK3B, which encodes a protein involved in synapse formation and dendritic spine maturation, have been shown to increase excitatory postsynaptic current frequencies in the hippocampus (9). Loss of function mutations in another risk gene, SCN2A, have been found to decrease spiking potential in excitatory neurons showing an inhibitory effect (10).

As a member of the T-box gene family, TBR1 is encoded by a neuron-specific transcription factor involved in regulating development (11). It has been shown to regulate discrete steps in axon differentiation during development, specifically post-mitotic neurons. Loss of both TBR1 alleles is related to abnormal differentiation of early-born cortical neurons and impaired axonal migration (12). De novo loss-of-function mutations in one copy of the TBR1 allele have been functionally linked to ASD (10, 13, 14). In addition, these mutations in TBR1 account for 1 percent of the mutations associated with ASD (15).

 TBR1 also regulates several high-confidence ASD risk genes in the developing neocortex, functioning as both a regulator and a repressor, as seen in the cases of two ASD-risk genes AUST2 and FEZF2 (16). Though the loss of both alleles impairs axonal migration, the majority of ASD patients have loss of function mutations in one of the two copies of TBR1 (17,18,). This defines TBR1 haploinsufficiency within mouse models. Clinically, TBR1 haploinsufficiency is characterized by ASD symptoms such as intellectual disability, aggressive–impulsive behavior, and speech deficits; however, clinical reports of these behaviors are inconsistent (19,20). Only recently has a preclinical behavioral model of TBR1 haploinsufficiency (TBR1+/−) been characterized in mice (7). These mice display certain ASD-like behaviors, including impairments in associative memory, cognitive flexibility, sociability, and communication.

Notably, this study determined some of these symptoms could be attributed to the amygdala by agonizing amygdalar NMDA receptors and ameliorating some of the associative memory and social interaction deficits. This suggests ASD-like behaviors caused by TBR1 haploinsufficiency may be attributed to specific of regions of the brain. Indeed, this pattern has been observed in Rett syndrome, a disorder within the autism spectrum, causing anxiety, intellectual delay, and motor impairment (21). The deletion of MECP2 in inhibitory GABA neurons has been shown to replicate Rett-like behaviors (22.) Rett-like behaviors such as anxiety and aggression have been shown to be region-specific, through deletion of MECP2 from Sim1-expressing neurons (23). This suggests specific brain regions control specific behaviors.

TBR1 is expressed broadly throughout the cerebral cortex during development and in neurons that differentiate into the olfactory bulb, entorhinal cortex, hippocampus, thalamus, striatum, hypothalamus (11, 16), prefrontal cortex (24), and the amygdala (7) The hippocampus, prefrontal cortex, and amygdala have been implicated in impairments to social behavior and memory (6). The amygdala interacts with regions such as the prefrontal cortex, hippocampus, striatum, thalamus, hypothalamus, and cerebellum (25). The prefrontal cortex expresses genes such as FOXP2 that are regulated by TBR1 and mutations in this gene are related to language impairment (15). TBR1 also regulates the expression of GRIN2B, another high-confidence ASD gene that encodes a subunit of the NMDA receptor (26). Based on the following associations, these regions may mediate region-specific responses following TBR1 manipulations.

We predict specific regions of the brain mediate ASD-like behaviors as a result of TBR1 haploinsufficiency. Thus, selectively deleting one functional copy of the TBR1 in mouse models will result in animals displaying ASD-like behaviors mediated only by that region. If behaviors are not region-specific, animals will display ASD-like behaviors regardless of the where TBR1 is knocked out.




Broader Relevance: ASD affects 1 out of 68 children (2) in the United States. Research advancements in ASD could lead to clinical testing and eventually provide therapeutic outlets for those affected by ASD. While the ultimate goal of this lab is to develop a cure for autism and its related phenotypes, we would like to note that ASD symptoms that are not detrimental to functioning and enjoyment of life do not necessitate a cure. Thus, we wish to find region-specific sources of behaviors that may allow for alleviation of some, but not necessarily all, symptoms. In addition, implementation of undergraduates in the laboratory will increase the chances of finding a cure for ASD. The success in this project will provide additional resources to the scientific community as well as families affected by ASD.


Intellectual Relevance: The TBR1 gene is a de novo risk gene that has been implicated with autism-like phenotype in about 1 percent of ASD cases (15). These de novo variants, typically consisting of missense or truncated mutations, affect subcellular localization, transcription and interactions with proteins (24). These mutations, in effect, have been found to lead to the onset of ASD phenotype (14). Effects of region-specific TBR1 knockouts and overexpression remain elusive. The central aim of our proposal is to examine the effects of region-specific TBR1 knockouts, TBR1 overexpression in region-specific knockout models, and TBR1 overexpression in known ASD mouse models. If region-specific TBR1 knockouts yield ASD phenotype, this would allow for a clearer understanding of the brain regions associated with various ASD phenotypes. In addition, if region-specific TBR1 overexpression is achieved in TBR1 knockouts and pre-established ASD mouse models, this would provide new potential routes for possible ASD therapies.


Specific Aims


The goal of this project is to identify region-specific targets to allow for treatment of the ASD-behavioral deficits. This requires the sources of these deficits to be identified and effects of overexpression to be characterized in mice to identify potential side effects. The regions used will include the amygdala, hippocampus, prefrontal cortex, and cells expressing NMDARs. Finally, overexpression of TBR1 can then be evaluated as a treatment by overexpressing TBR1 in other ASD mouse models. In addition, a secondary goal of this proposal is to determine whether the effects of site-specific TBR1 manipulation are due to active TBR1 action, or effects that occurred only during development.

  1. Delete one TBR1 allele in a region-specific manner to evaluate the source of region-specific behaviors: Behavioral and neural activity assays will be used to assess the brain-specific sources of behavior and the underlying neural effects of TBR1 haploinsufficiency, as seen in (23).
  2. Overexpress TBR1 in a region-specific manner to ascertain potential side effects: Where deficits are identified in Aim 1, TBR1 will be overexpressed in wild-type. Prior literature has shown the possibility of overexpression causing ASD-like symptoms (27). Ideal results would allow overexpression to be used as a potential treatment.
  3. Overexpress TBR1 in known ASD mouse models and TBR1+/− mice to see whether these phenotypes can be rescued: This aim will be contingent upon mouse models resembling wild-type TBR1 mouse models rather than displaying negative side effects after TBR1 overexpression in Aim 2. Here, TBR1 will overexpressed in known ASD mouse models in which the genes CHD8, SHANK3, and FMR1 have been mutated. The purpose of this aim is to view rescue effects after overexpression as therapeutic approach for ASD treatment. This will be done employing the overexpression methods discussed in (28).


Research Methods and Design


  1. Delete one TBR1 allele in a region-specific manner to evaluate the source of region-specific behaviors.

Rationale: Prior studies have selectively knocked out genes involved in the developmental disorder, Rett syndrome, from specific regions in the brain to determine the regions’ relative contributions to physiological and behavioral symptoms of Rett’s syndrome (23). Similarly, we plan to generate mouse models with region-specific knockouts to determine the contribution of each region to TBR1 phenotype. As autism is thought to be related to an imbalance of excitatory or inhibitory neuronal activity, we also intend to assess putative spontaneous neural activity (8). A secondary aim includes evaluating the effects of region-specific TBR1 haploinsufficiency during development or adulthood to determine whether these effects are dependent on a critical period.

  1. Generation of region-specific TBR1+/- knockout mouse lines:

To generate site-specific conditional knock-outs, animals carrying two floxed TBR1 alleles will be bred with transgenic mouse lines expressing Cre under the control of a promoter; the resulting generation of animals with one floxed TBR1 allele will be utilized for further study. Both TBR1 alleles will also be deleted and neural activity evaluated, but behavior cannot be characterized as it is lethal (24). Cre will be linked to the α-CAMKII promoter, selectively affecting the CA1 region of the hippocampus (29), and the Lypd1 promoter in the NR149 mouse line, selectively affecting expression of pyramidal neurons within infralimbic region of the prefrontal cortex (30) and the NR1 promoter, which mediates expression of a subunit that is present in all four NMDARs (31, 32). However, as the entire amygdala was previously implicated in TBR1 behavioral deficits (7), viral injection will be used to inject Cre recombinase into the genome of that region. This method has been used to delete BDNF in the hippocampus (33). Negative controls consist of wild-type siblings with floxed TBR1 genes, but not expressing Cre. Amygdala-specific control animals will also undergo surgeries, but the virus will express GFP-alone.  A positive control animal with whole brain TBR1+/- knockouts, as opposed to region specific knockouts will also be generated as previous, but Cre recombinase expression will be link to the TBR1 promoter. (B) To generate temporal conditional knock-outs in cells expressing NMDA receptors and throughout the entire brain, tamoxifen will be used to induce Cre recombinase during development or adulthood. In region-specific models, these viral injection surgeries will be conducted during development or adulthood (35).

  1. Confirm the region-specific expression of TBR1:

To confirm expression of the virus after physiological or behavioral testing, the animal will be sacrificed and in situ hybridization analysis of Cre recombinase, whose expression will also be linked to the GFP gene, (36) and TBR1 mRNA will be conducted to demonstrate accurate location of protein expression. To examine quantity of proteins, RT-qPCR be conducted, and Western blots will allow for the experimental TBR1 levels to be compared to wild-type and positive control animals in both adult mice and during development (37, 38). Statistical significances will be tested using one-way ANOVAs or non-parametric Kruskal-Wallis tests if the distribution does not meet normality.

Prediction: If the desired models are generated efficiently, then expression of TBR1 will be reduced to approximately half the amount protein expressed in WT mice homozygous for TBR1, but greater than the full TBR1 knockout. As shown via in situ hybridization, this dysregulation will be specific to the selected regions. If the methodology was not efficient, TBR1 expression and the genes it regulates will resemble wild-type expression.

  1. Examine the overall impact of TBR1 knockout on neural activity:

Ca2+ imaging will be used to evaluate population activity (39) while whole cell patch-clamp recordings of excitatory post-synaptic output will be used to assess the activity from CA1 pyramidal neurons, pyramidal prefrontal cortex neurons, and neurons expressing NMDARs, including within the amygdala. (7). Statistical significance will be tested using one-way ANOVAs and non-parametric Kruskal-Wallis tests. 

Prediction: As ASD is a disorder of activation and inhibition (8) we expect regions to be overactive or underactive relative to wild-type. Regions containing NMDARs, including the amygdala and hippocampus, and tissues expressing TBR1 will be overactive, as NMDARS have been shown to be hyperactive in TBR1 haploinsufficient mice (7, 40). However, we predict the prefrontal cortex may be underactive, as its activity is often required for inhibition of certain ASD behavior (4). As TBR1 acts primarily during development, we suggest that conditional knockouts during adulthood will resemble wild-type (24).

  1. Characterize the behavioral effects of TBR1 haploinsufficiency:

We will use multiple behavioral assays to evaluate symptoms characteristic to ASD. Repetitive behaviors will be assessed by examining patterns of grooming, and marble burying, a repetitive digging behavior (40). Impairment in social interaction will be evaluated using the social transmission of flavor preference (41) and the three chambered social approach tests (42). Language impairment will be assessed using vocalization in mice removed from their mothers (43) and vocalized index of social recognition (44). Tests of associative memory will be conducted using fear conditioning (45) and conditioned taste aversion (46). Cognitive Flexibility will be assessed using reversal learning in the Morris water maze (47) and two-choice digging task (48). Finally, anxiety-related behaviors will be evaluated using elevated plus-maze and open field tests (49, 50). Statistical significances will be evaluated using one-way ANOVA, two-way ANOVA, and repeated measures ANOVAs or nonparametric Kruskal-Wallis.

Prediction: We suggest that each brain region will mediate some behavioral aspects of TBR1 ASD-like symptoms, such that only specific behavioral deficits will occur when the source of that behavior is manipulated. TBR1 knockout in the amygdala may mediate anxiety-like and impairment in associative memory, as it is related to associative learning and anxiety (7, 15). As CA1 pyramidal neurons are related to social memory, it may mediate impaired social behaviors (29).  Finally, as NMDARs are expressed in the amygdala and hippocampus, impairment in this tissue may mediate all three behaviors (6, 7, 31). As the prefrontal cortex mediates perseveration and executive functioning, (4) TBR1 knockouts may be the source of repetitive behaviors and impaired cognitive flexibility. It may also mediate language impairment, as genes TBR1 regulate, like FOXP2 that impair language are predominately expressed in the prefrontal cortex (15). However, if these behaviors are not region-specific, all knockout may express all ASD-like behaviors. It is also plausible one region may mediate all behaviors, thus only one knockout will express all ASD-like behaviors and the other knockouts will resemble wild-type.

  1. Overexpress TBR1 in a region-specific manner to ascertain potential side effects.

Rationale: If haploinsufficiency displays the predicted ASD behavioral phenotypes from Aim 1, then an overexpression mouse model will provide insight into potential therapeutic rescue (Aim 3). No difference in expression from wild type and overexpressed mice in all brain regions will suggest upregulation of TBR1 as a potential therapy. Identification of cell type and function associated with TBR1 overexpression will help characterize ASD symptoms, which include large suites of genes. Developing mice and adult mice will also specify the importance of therapy at proper times in a life cycle.

  1. Generation of region-specific TBR1 overexpression mouse lines:

We will use Cre mediated recombination in mice with one loxP-flanked STOP codon within a tissue specific promoter. (A) To generate site-specific conditional overexpression, animals carrying a one floxed stop codon allele will inherit a Cre recombinase expressing an ASD target gene promoter into either the amygdala, hippocampus, and prefrontal cortex, and whole-brain. The promoters and cell types from Aim 1 will be used to overexpress TBR1 in specific brain regions, and viral injection will comprise the Amygdala target area. All brain regions and NMDA cell types will attribute the same neuronal and behavioral function as in aim 1. This method has been used to knockout and overexpress target genes in brain-region specific mouse models (28). Negative controls will contain a floxed stop codon gene without the Cre mediated expression.  Mice focusing on the amygdala region will undergo surgery as in Aim 1. A positive control will consist of mice with whole brain TBR1+/- overexpression, as opposed to region specific overexpression, and Cre mediation will express the region-specific promoter as in Aim 1. (B) To generate temporal conditional knock-outs in cells expressing NMDA receptors and throughout the entire brain, tamoxifen will be used to induce Cre recombinase during development or adulthood. In region-specific models, these viral injection surgeries will be conducted during development or adulthood (26).

  1. Confirm the region-specific overexpression of TBR1:

Region specific overexpression coordinates NMDAR treatment, which will cause the TBR1 gene to be upregulated specifically in the amygdala, hippocampus, and the prefrontal cortex (26). In situ hybridization of Cre recombinase and TBR1 RNA will confirm the overexpression of targeted proteins (Aim 1). mRNA sequencing will provide a comparison between the wild type mice and positive control as exhibited in Aim 1. All analyses include the comparison of adult to developing mice. Statistical significances will be tested using one-way ANOVAs or non-parametric Kruskal-Wallis tests if the distribution does not meet normality.

Prediction: Overexpressed mouse models containing two alleles for TBR1 overexpression will show no difference in protein quantification from the wild type (40). The lack of differential expression will imply that over expression of our tissue specific promoter might restore function normal function in TBR1 knockout mice (40). Differences in protein expression from wildtype to overexpressed mice suggest lack of therapeutic potential in neuronal function. The differences in expression at the developing stage versus adulthood will suggest a timeframe to target for therapeutic purposes. MECP2 overexpression leads to normalized expression within in situ and Western blot models (27).

  1. Examine the overall impact of TBR1 overexpression on neural activity:

Ca2+ imaging will assess the activity of neurons (Easton). Patch cell recordings will provide post synaptic channel evaluations for the CA1 pyramidal neurons, which could coordinate signaling through targeted brain regions. (See Aim 1 for more details) Statistical significances will be tested using one-way ANOVAs (Aim 1A) and two-way ANOVA (Aim 1B) and non-parametric Kruskal-Wallis tests.

Prediction: Calcium levels compared between wildtype and overexpressed mice will show no difference if the potential for therapeutic rescue remains. Upregulation of TBR1 will display normal post synaptic channel signaling in all mouse models (40). Alternatively, difference in calcium levels and post synaptic signaling will suggest neuronal dysfunction in targeted brain regions. Previous studies involving Rett concluded overexpression as another contributor to dysfunction (40).

  1. Characterize the behavioral effects of TBR1 overexpression:

Language deficits, social recognition, associated memory, cognitive flexibility, and studies in anxiety-like behaviors provide observations for possible over expressive behavioral phenotypes. All behavioral test will replicate the procedures from Aim 1.

Prediction:  All behavioral tests will coordinate with brain specific regions as discussed in aim 1. However, overexpression of TBR1 in mice will exhibit similar behavioral phenotypes to wildtype mice (40). Alternatively, difference in behavioral phenotypes suggest upregulation of TBR1 causes a shift in NMDA receptor function at the synaptic level. In this case, closely related ASD genes and NMDA cell types may account for synapse dysfunction and ASD behavioral phenotypes from overexpression of TBR1(4). For example, Mecp2 overexpression mice displayed similar behavioral phenotypes compared to the Mecp2 knock-out mice (27).

  1. Overexpress TBR1 in known ASD mouse models and TBR1+/− mice to see whether these phenotypes can be rescued.

Rationale: We plan to overexpress TBR1 genes in two different experimental animal models. For the first animal model group, region-specific TBR1 overexpression will be implemented in TBR1+/- mice as seen in Aim 1A. In the second group, region-specific TBR1 overexpression will be implemented in pre-established ASD mice models with three different dysfunctional risk genes. In each of the sets of mice, we will view behavioral effects as a result of TBR1 overexpression to draw some conclusions on possible ASD therapeutic strategies.

  1. Generation of region-specific TBR1 overexpression:

TBR1 overexpression will be used to determine view possibilities of phenotype rescue. Similar experiments have previously shown that conditional gene overexpression can have result in rescued behaviors. Specifically, (51) demonstrated that the MECP2 gene, which is implicated with Rett’s syndrome, can be overexpressed using Cre-mediated recombination to rescue proper gene function, which also translates to baseline behavioral functioning (51). Here, we will employ the overexpression methodology as described in (28). In this study, researchers used the Cre-Lox system to cut the STOP codon of the target gene, Cox-2. The transgene is attached to a tissue-specific promoter, so that overexpression can be conditionally mediated. Upon cutting the STOP codon in TBR1, gene transcription and translation can continue to increase TBR1 expression in either the TBR1+/ - mouse models, or the ASD mouse models discussed in subaim 3B, section 2. In addition, the same controls will be used here as described in Aim 1A.

Prediction: Contingent upon TBR1 tissue-specific overexpression mouse models showing no adverse side effects in Aim 2, we should see normalized tissue-specific morphology in addition to significantly reduced engagements in ASD-related behaviors (see subaim 3D).

B1. Generation of tissue-specific TBR1+/- knockout mouse lines of Aim 1A:

Using the same methods as described in Aim 1A, we will generate region-specific TBR1 knockouts using the Cre/Lox system. Here, one functional TBR1 allele will be conditionally knocked-out in the amygdala, prefrontal cortex, hippocampus, and regions expressing NMDA receptor cells. Region-specific knockouts will be induced with Cre-mediated recombination and tamoxifen treatment.

B2. Generation of ASD Mouse Models:

Pre-established ASD mouse models will be adopted from other labs in which three different ASD-associated risk genes have been manipulated: CHD8, SHANK3, and FMR1. CHD8 mouse models adapted from (52) showed deficits in social interaction, anxiety, and increased repetitive behaviors. SHANK3 mouse models adapted from (9) showed decreased social sniffing and vocalizations, and depression. FMR1 mouse models from (53) showed anxiety-related behaviors during social interaction and hyperactivity. Here, the same controls will used as described in Aim 1A.

Prediction: Being that CHD8, SHANK3, and FMR1 are all risk genes of ASD and are linked with TBR1 regulation (14), overexpression treatment should relieve signs of ASD related behaviors to baseline behaviors in various behavioral assays. In fluorescent in situ hybridization, TBR1+/- overexpression mouse models should closely resemble wild type TBR1 fluorescence. Similarly, since ASD models resemble TBR1 knockouts – as seen by Aim 1 – we expect fluorescence in these mouse models to also resemble wild-type TBR1.

  1. Confirm the region-specific expression of TBR1:

We will employ whole cell patch clamp to examine neuronal changes upon subsequent TBR1 overexpression experiments in both TBR1+/- mouse models and ASD mouse models. Whole cell patch clamp will be utilized in a region-specific manner in both experimental groups. These regions include the amygdala, prefrontal cortex, hippocampus, and cells with NMDA receptors (11, 24). In addition to whole cell patch clamp, Ca2+ imaging will also be utilized to view differences in neuronal activation based on Ca2+ ratios. Furthermore, RT-qPCR will be used to view DNA . Statistical significances will be tested using one-way ANOVAs or non-parametric Kruskal-Wallis tests if the distribution does not meet normality.

Prediction: Normalizing TBR1 levels in the brain should lead to rescued TBR1 function (28, 51). These results should show evidence for region-specific rescue, which will be especially notable in ASD mouse models that have specific conditional deficits (9, 52, 53). In addition, calcium (Ca2+) imagining studies and RT-qPCR assays should reveal normalized levels of activation and DNA expression that resemble the TBR1 wild-type control. Alternatively, differences in Ca2+ levels and postsynaptic signaling away from baseline will suggest neuronal dysfunction in targeted brain regions.

  1. Characterize the behavioral effects of TBR1 overexpression in ASD Mouse Models:

For each of the regions examined, we will conduct specific behavioral assays to investigate the changes in social interaction, language impairment, and anxiety-like behaviors. In the TBR1+/- overexpression mouse models, the behavioral assays described in Aim 1D will be utilized here as well. The open field test will be used to view changes in anxiety related behaviors as seen in CHD8 mouse models (54). Secondly, the vocalized index test will be used to view changes after SHANK3 conditional overexpression since these mouse models show deficits in social vocalizations (9). Finally, the three-chambered box test will be utilized to view changes in social interaction for FMR1 overexpression mouse models since FMR1 mutations have been found to result in reduced social interaction (53).

Prediction: Similar to the described electrophysiological studies, behavioral assays should result in mouse models engaging in baseline levels of behaviors similar to TBR1 wild-type mice. If found, this will indicate possibilities in therapies for region-specific ASD behaviors. Alternatively, if overexpression studies are ineffective, ASD mouse models should show no changes in behaviors.



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