Precision medicine in psychiatry requires identifying risk factors, brain alterations, and behavioral phenotypes. Rare copy number variants (CNVs), including the 22q11.2 deletion, are robust risk factors for psychiatric disorders. The 22q11.2 deletion is associated with various brain volumetric changes in humans and mouse models, including reduced amygdala and increased striatal volumes. However, the specific contribution of individual genes within the 22q11.2 region remains unclear. Recent studies implicate *TBX1*, a T-box transcription factor, as a driver gene for several behavioral dimensions, including social interaction and memory. This study aimed to determine the impact of *Tbx1* heterozygosity on brain region volumes and associated behaviors in a congenic mouse model, thereby isolating the effects of *Tbx1* from other genes within the 22q11.2 region. Understanding the specific contribution of *TBX1* is crucial because ultra-rare *TBX1* variants are found in individuals with neuropsychiatric disorders independent of 22q11.2 deletions. While mouse models of whole 22q11.2 deletions are not ideal for isolating single gene effects, altering the dosage of *Tbx1* in mice provides a valuable model to study its contribution to specific phenotypes. Previous research demonstrated that *Tbx1* heterozygosity in mice impairs social interaction, communication, memory, and cognitive flexibility – dimensions commonly affected in individuals with 22q11.2 deletions and other neurodevelopmental disorders. This study builds upon this foundation by comprehensively evaluating brain structure and relevant behavioral phenotypes in a well-controlled congenic *Tbx1* heterozygous mouse model.

Extensive research links CNVs, particularly the 22q11.2 deletion, to psychiatric disorders. Studies have shown consistent volumetric brain alterations in individuals with 22q11.2 deletions, including reduced volumes in the amygdala, hippocampus, thalamus, and putamen, and increased volumes in the caudate nucleus and nucleus accumbens. However, disentangling the contributions of individual genes within the 22q11.2 locus to these structural and behavioral changes is a major challenge. Large-scale sequencing studies have identified ultra-rare variants of several 22q11.2 genes, including *TBX1*, in individuals with idiopathic schizophrenia and autism spectrum disorder (ASD). These findings, while suggestive, have limitations due to small sample sizes and the presence of multiple variants in affected individuals. Mouse models, while helpful, often lack the precision needed to dissect individual gene contributions. Existing research on *Tbx1* in mice supports its role in social behavior, memory, and cognitive flexibility, all relevant to neurodevelopmental disorders.

The study employed congenic *Tbx1* heterozygous (HT) mice and their wild-type (WT) littermates on a C57BL/6J background. The use of congenic mice minimized the confounding effects of genetic background variation. Ex vivo volumetric MRI was performed on adult mice to obtain detailed brain region volumes, allowing for more precise and reproducible measurements compared to in vivo scanning. A total of 19 atlas-based brain regions were analyzed. A voxel-based analysis was used to identify focal volume alterations within heterogeneous regions, such as the amygdala. To validate MRI findings, immunohistochemistry was performed to assess the size of calretinin-positive neuropil in the amygdalopiriform transition area. Behavioral tests included: 1) a place conditioning procedure using a social partner to assess social incentive learning; 2) an olfactory test to assess response to a social cue; and 3) acoustic and non-acoustic startle and prepulse inhibition (PPI) tests to evaluate sensorimotor gating. Sample sizes were determined by power analyses. Statistical analyses included ANOVAs, t-tests, and non-parametric tests as appropriate, with adjustments for multiple comparisons.

Volumetric MRI revealed that *Tbx1* heterozygosity did not affect whole-brain volume but resulted in significant focal volume reductions in the anterior and posterior portions of the right amygdala and surrounding cortical regions in *Tbx1* HT mice compared to WT mice. Voxel-based analysis supported these findings. Immunohistochemistry confirmed a reduction in calretinin-positive neuropil in the amygdalopiriform transition area of *Tbx1* HT mice. In a social incentive learning task, *Tbx1* HT mice were impaired in their ability to learn the incentive value of a social partner, exhibiting a decreased conditioned preference for the compartment previously associated with a social partner. The size of the auditory cortices and adjacent temporal association area were significantly increased in *Tbx1* HT mice, which correlated with impaired acoustic but not non-acoustic sensorimotor gating as measured by PPI. Olfactory responses to social cues were normal in *Tbx1* HT mice.

The findings demonstrate that *Tbx1* heterozygosity leads to highly specific and demarcated structural alterations in the brain, affecting the amygdala and auditory cortex in opposite directions. These structural alterations are associated with impaired social incentive learning and acoustic sensorimotor gating. The observed deficits in social incentive learning are consistent with the known role of the amygdala in processing social information and reward. The amygdala volume reduction in conjunction with the impaired social incentive learning suggests that *Tbx1* plays a critical role in the development and function of the amygdala. The increase in auditory cortex volume and concomitant deficit in acoustic PPI could be related to previously reported hearing loss in *Tbx1* deficient mice, though the precise relationship needs further investigation. The lack of effects on non-acoustic sensorimotor gating suggests a selective impact of *Tbx1* on auditory processing. These results support a significant role for *TBX1* in the development and function of brain regions critical for social behavior and sensory processing, providing further insight into the genetic basis of neuropsychiatric disorders associated with 22q11.2 deletions. This study further highlights the importance of using approaches beyond simple volumetric analysis to detect subtle structural changes in the brain.

This study demonstrates that *Tbx1* heterozygosity in mice leads to focal volume alterations in the amygdala and auditory cortex, and these structural changes are associated with impaired social incentive learning and acoustic sensorimotor gating. The findings highlight *Tbx1*'s specific contribution to the neurodevelopmental phenotypes associated with 22q11.2 CNVs. Future research could focus on investigating the cellular mechanisms underlying the observed structural changes and the precise interaction between *Tbx1* and other genes in the 22q11.2 region. Further studies could also examine the developmental trajectory of these changes and explore potential therapeutic interventions.

The study used only mice, limiting the direct translation to human conditions. The sample size, while determined by power analysis, might not have captured all subtle brain region volume differences. The study focused on a limited set of behavioral tests; future studies should explore a broader range of behaviors to fully characterize the phenotype. The study did not investigate sex differences, despite the lack of sex differences in prevalence of certain neuropsychiatric disorders in 22q11.2 deletion carriers. Additional investigations are necessary to determine if this lack of sex differences holds true at the molecular and cellular levels. Although parametric testing was used for most analyses, a non-parametric analysis was used for non-acoustic PPI.