Septin-5 is a GTP-binding protein implicated in synaptic vesicle exocytosis and 22q11.2 deletion–related neuropsychiatric disorders. We recently showed that Septin5-deficient (Septin5−/−) mice display intact hippocampal spine ultrastructure, but marked deficits in both recent and remote contextual fear memory, whereas cued fear memory is preserved. Building on these findings, we asked whether Septin-5 is required for baseline forms of hippocampus-dependent spatial and object recognition memories, or more selectively for novelty-dependent memory stabilization. Using congenic Septin5−/− mice, we performed a behavioural test battery including hippocampus-dependent spatial and object recognition tasks. Septin5−/− mice showed normal performance in T-maze (spontaneous and forced alternation), Barnes maze (acquisition and recent/remote spatial reference memory), and object location memory. After 5-min training in the novel object recognition task, short-term recognition memory was indistinguishable between genotypes. Together with our previous report that long-term object recognition after 15-min training is intact in Septin5−/− mice, these results indicate that Septin-5 is dispensable for a broad set of hippocampus-dependent spatial and object recognition memories despite contextual fear deficits. In contrast, Septin5−/− mice exhibited a selective deficit in behavioural tagging: in wild-type mice, novelty exploration 30 min after 5-min object training converted an otherwise labile trace into a 24-h memory, whereas this novelty-induced stabilization was absent in Septin5−/− mice. Thus, Septin-5 is not required for baseline performance in hippocampus-dependent spatial and object recognition tasks, but is implicated in novelty-dependent stabilization of weak hippocampal memories under the established 10-min novelty exposure condition, consistent with a contribution to synaptic tagging–like processes.

Abstract Constitutive heterozygosity of Tbx1, a T-box transcription factor gene located within the 22q11.2 deletion region, results in behavioral deficits and altered composition of myelinated axons in the fimbria, together with reduced levels of an oligodendrocyte precursor cell (OPC) marker, in mice. However, the cellular origins of these effects and the extent to which axonal changes causally contribute to behavioral impairments remain unclear. We hypothesized that Tbx1 deficiency specifically within the oligodendrocyte lineage contributes to myelin and behavioral phenotypes. To test this hypothesis, we first demonstrated through in vitro siRNA knockdown that Tbx1 regulates both OPCs and mature oligodendrocytes. Subsequently, we assessed the impact of Tbx1 heterozygosity initiated in OPCs on behavioral and myelin phenotypes in male conditional PdgfrαCre;Tbx1 +/flox mice. These mice exhibited Cre-mediated recombination in Pdgfrα-expressing brain regions and in the OPC progeny within the fimbria. At one month of age, the mutants displayed a higher rate of spontaneous alternation at the longest inter-trial interval in the T-maze compared to their wild-type littermates—an effect that was dissipated at two months. No significant phenotypic abnormalities were observed in conditional PdgfrαCre;Tbx1 +/flox mice regarding neonatal ultrasonic vocalizations, social interaction, novel object approach, anxiety-like behavior (elevated plus maze), or open-field locomotion and thigmotaxis. Electron microscopic analysis revealed a compositional shift in myelinated axons within the fimbria of adult male mutants, characterized by an increased number of myelinated axons in the 300–800 nm diameter range and a decreased number in the ~ 1,200 nm and ~ 1,400 nm ranges, with myelin thickness remaining unchanged across diameters. These findings indicate that Tbx1 heterozygosity in the oligodendrocyte lineage leads to a selective shift towards smaller myelinated axons in the fimbria and a transiently higher level of capacity for working memory and cognitive flexibility. However, it does not replicate the full spectrum of myelination abnormalities or the broader cognitive and social deficits observed in constitutive Tbx1 heterozygotes, suggesting that Tbx1 deficiency in non-oligodendrocyte lineage cells may lead to altered myelination and neurodevelopmental behavioral impairments.

Mental illnesses associated with high-risk copy number variations (CNVs) are characterized by incomplete penetrance and variable severity, with their underlying mechanisms remaining inadequately understood. We hypothesized that such phenotypic variability is evident from the neonatal stage and is, at least in part, attributable to individual differences in the expression levels of CNV-encoded genes in the brain. We conducted an analysis of the quantitative and functional structure of neonatal social communication, assessed post-pubertal social interaction, and evaluated the brain expression levels of genes within the same cohort of a mouse model of paternal human 15q11-13 duplication, a high-risk factor variably associated with neurodevelopmental disorders. Subsequently, computational methods were utilized to identify predictive variables for the variability of post-pubertal social interaction. Mice harboring the 15q11-13 duplication exhibited distinctive call sequences characterized by diverse connections, which lacked the incentive value necessary for effective social communication with mother mice. The neonatal call sequences and the expression levels of Magel2 , along with, to a lesser extent, Herc2 and Ndn , in the prefrontal cortex of the 15q11-13 duplication model were predictive of post-pubertal social interaction. Our findings demonstrate that variability in post-pubertal social interaction-a dimensional characteristic of neurodevelopmental disorders-can be predicted by the variability of neonatal social communication and is influenced by the expression levels of specific CNV-encoded genes in the prefrontal cortex. This computational approach has the potential to predict the developmental trajectories of various dimensions of mental illness among CNV carriers in humans and to identify CNV-encoded driver genes in preclinical models, thereby providing potential mechanistic bases for the development of gene-based therapeutic strategies.

Septin-3 and Septin-5 are components of the septin cytoskeleton highly expressed in the nervous system, yet the extent of their shared and distinct roles is not fully understood. We recently demonstrated that Septin-3 regulates late-phase long-term potentiation (L-LTP)-dependent invasion of smooth endoplasmic reticulum (sER) into dentate gyrus (DG) spines. Septin3−/− mice exhibit normal synaptic ultrastructure in the hippocampal DG, CA3, and CA1, yet the fraction of sER-containing spines is reduced; behaviorally, they show selective deficits in 1-day object recognition and 1-day contextual fear memory, whereas cued fear conditioning and contextual memory tested at 1 month are intact. Here, using adult male Septin5−/− mice, we tested whether morphological and behavioral phenotypes identified in Septin3−/− mice are shared or subunit-specific. Electron microscopy showed no detectable differences in synapse density, spine volume, and postsynaptic density (PSD) area in the hippocampal DG, CA3, and CA1, with an unchanged fraction of sER-containing spines relative to wild-type littermates. Behaviorally, Septin5−/− mice were impaired in recent (1 day) and remote (1 month) contextual fear memory, but were normal in 1-day novel object recognition memory and in recent and remote cued fear memory. The shared and distinct structural and behavioral phenotypes observed in Septin5−/− and Septin3−/− mice suggest either sER-independent common mechanisms or subunit-specific ones for recent contextual fear memory deficit, and indicate a Septin-5-dependent contribution to remote contextual fear memory.

The hemizygous deletion of human chromosome 22q11.2 leads to cognitive and social deficits, as well as psychiatric disorders. Constitutive deletion of Tbx1, a T-box transcription factor gene encoded within this chromosomal region, results in deficits in various cognitive and social behaviors and altered formation of oligodendrocytes, myelin, and myelinated axons in the fimbria of adult mice. Given that Tbx1 is present in neonatal stem cells and oligodendrocytes, the present study was designed to test the hypothesis that Tbx1 heterozygosity in the oligodendrocyte lineage contributes to behavioral phenotypes relevant to neurodevelopmental disorders and ultrastructural changes in myelinated axons in the fimbria. To this end, we conditionally induced Tbx1 heterozygosity in the oligodendrocyte lineage in PdgfrαCre;Tbx1+/flox mice. This mouse line showed recombination in brain regions known to express Pdgfra. Male Pdgfra;Cre;Tbx1+/flox mice performed better in spontaneous alternation in a T-maze than wild-type littermates at 1, but not 2 months of age; otherwise, mutant and wild-type littermates were indistinguishable in neonatal ultrasonic vocalization, peripubertal and postpubertal social interaction, novel object approach, anxiety-related behaviors in an elevated plus maze, and motor activity and thigmotaxis in an inescapable open field. The fimbria of male Pdgfra;Cre;Tbx1+/flox mice showed an increase in the number of myelinated axons in a range between 300 nm and 800 nm diameter and a decrease in the myelinated axons around 1,200 nm and 1,400 nm; the thickness of myelin was unchanged across all diameters. Our findings show that Tbx1 heterozygosity in oligodendrocyte precursor cells or their progeny selectively improves spontaneous alternation and shifts the proportion of myelinated axons toward small- to medium-sized axons, suggesting that the compositional shift in axons in the fimbria induced by Tbx1 heterozygosity in the oligodendrocyte lineage might underlie improved cognition relevant to neurodevelopmental disorders. ### Competing Interest Statement The authors have declared no competing interest. National Institue of Health, R01MH099660, R01GM63074, F30MH134482 The University of Texas Health Science Center at San Antonio, https://ror.org/02f6dcw23, T32R004545, T32NS082145, T32GM113896, T32GM145432 Society for Neuroscience, https://ror.org/04410kq54, R25NS089462

Professor Noboru Hiroi is a faculty member in the Departments of Pharmacology, Cellular and Integrative Physiology, and Cell Systems & Anatomy at the University of Texas Health Science Center at San Antonio (UT Health San Antonio), USA. He joined his current institution in 2019 after working at Albert Einstein College of Medicine in New York for 21 years. His current work is focused on the cellular and developmental origins of the dimensions of neuropsychiatric disorders in genetically engineered mouse and cell models. Professor Hiroi is happy to provide our readers with reflections on his life and career.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and changes in brain structures. However, because CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how each gene encoded in the 22q11.2 region contributes to structural alterations, associated mental illnesses, and their dimensions. Our previous studies identified Tbx1, a T-box family transcription factor encoded in the 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes and behavioral alterations relevant to affected structures in congenic Tbx1 heterozygous mice. Our data showed that the volumes of the anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were most robustly reduced in Tbx1 heterozygous mice. In an amygdala-dependent task, Tbx1 heterozygous mice were impaired in their ability to learn the incentive value of a social partner. The volumes of the primary and secondary auditory cortexes were increased, and acoustic, but not non-acoustic, sensorimotor gating was impaired in Tbx1 heterozygous mice. Our findings identify the brain’s regional volume alterations and their relevant behavioral dimensions associated with Tbx1 heterozygosity.

Schizophrenia is a common chronic psychiatric disorder that causes age-related dysfunction. The life expectancy in patients with schizophrenia is ≥10 years shorter than that in the general population because of the higher risk of other diseases, such as cardiovascular diseases. Aging studies based on DNA methylation status have received considerable attention. Several epigenetic age accelerations and predicted values of aging-related proteins (GrimAge and GrimAge2 components) have been analyzed in multiple diseases. However, no studies have investigated up to GrimAge and GrimAge2 components between patients with schizophrenia and controls. Therefore, we aimed to conduct multiple regression analyses to investigate the association between schizophrenia and epigenetic age accelerations and GrimAge and GrimAge2 components in seven cohorts. Furthermore, we included patients with first-episode psychosis whose illness duration was often shorter than schizophrenia in our analysis. We integrated these results with meta-analyses, noting the acceleration of GrimAge, GrimAge2, and DunedinPACE, and increase in adrenomedullin, beta-2 microglobulin, cystatin C, and plasminogen activation inhibitor-1 levels, in patients with schizophrenia or first-episode psychosis. These results corroborated the finding that patients with schizophrenia had an increased risk of diabetes, cardiovascular disease, and cognitive dysfunction from a biological perspective. Patients with schizophrenia and first-episode psychosis showed differences in the results when compared with controls. Such analyses may lead to the development of novel therapeutic targets to patients with schizophrenia or relevant diseases from the perspective of aging in the future.

Microglia and brain-derived neurotrophic factor (BDNF) are essential for the neuroplasticity that characterizes critical developmental periods. The experience-dependent development of social behaviors-associated with the medial prefrontal cortex (mPFC)-has a critical period during the juvenile period in mice. However, whether microglia and BDNF affect social development remains unclear. Herein, we aimed to elucidate the effects of microglia-derived BDNF on social behaviors and mPFC development. Mice that underwent social isolation during p21-p35 had increased Bdnf in the microglia accompanied by reduced adulthood sociability. Additionally, transgenic mice overexpressing microglial Bdnf-regulated using doxycycline at different time points-underwent behavioral, electrophysiological, and gene expression analyses. In these mice, long-term overexpression of microglial BDNF impaired sociability and excessive mPFC inhibitory neuronal circuit activity. However, administering doxycycline to normalize BDNF from p21 normalized sociability and electrophysiological function in the mPFC, whereas normalizing BDNF from later ages (p45-p50) did not normalize electrophysiological abnormalities in the mPFC, despite the improved sociability. To evaluate the possible role of BDNF in human sociability, we analyzed the relationship between adverse childhood experiences and BDNF expression in human macrophages, a possible proxy for microglia. Results show that adverse childhood experiences positively correlated with BDNF expression in M2 but not M1 macrophages. In summary, our study demonstrated the influence of microglial BDNF on the development of experience-dependent social behaviors in mice, emphasizing its specific impact on the maturation of mPFC function, particularly during the juvenile period. Furthermore, our results propose a translational implication by suggesting a potential link between BDNF secretion from macrophages and childhood experiences in humans.

Fragile X syndrome is caused by monogenic silencing of the FMR1 gene and is characterized by high rates of autism spectrum disorder. A previous study demonstrated that prepartum administration of bumetanide, a chloride transporter blocker, normalized neonatal vocalization in non-congenic Fmr1 knockout (KO) pups. However, the genuine contribution of Fmr1 deletion to this phenotype in a congenic Fmr1 KO mouse model and the long-lasting effect of prepartum bumetanide administration on postpubertal social interaction remains unclear. The current study aimed to determine the impact of prepartum bumetanide administration on vocalization at postnatal day 7 and social interaction at 6 and 8 weeks of age in a congenic Fmr1 KO mouse model in which the genetic backgrounds were homogeneous between KO and wild-type (WT) littermates. Moreover, we applied a computational analytical algorithm and determined predictive variables of neonatal vocalization for postpubertal social interaction. Our data showed that (1) KO mice exhibited altered numbers and sequences of distinct call types during neonatal vocalization and reduced social interaction at 6 weeks, (2) select sets of neonatal vocalization variables predicted postpubertal social interaction levels, and (3) bumetanide restored neonatal vocalization in KO pups but nonspecifically reduced social interaction in WT and KO mice at 6 weeks. These data indicate that Fmr1 deletion selectively impacts distinct elements of neonatal vocalization and postpubertal social interaction. Additionally, bumetanide selectively restores neonatal vocalization but has a transient nonspecific negative impact on subsequent postpubertal social interaction.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Infant crying is a communicative behavior impaired in neurodevelopmental disorders (NDDs). Because advanced paternal age is a risk factor for NDDs, we performed computational approaches to evaluate how paternal age affected vocal communication and body weight development in C57BL/6 mouse offspring from young and aged fathers. Analyses of ultrasonic vocalization (USV) consisting of syllables showed that advanced paternal age reduced the number and duration of syllables, altered the syllable composition, and caused lower body weight gain in pups. Pups born to young fathers had convergent vocal characteristics with a rich repertoire, whereas those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Additional analyses revealed that some pups from aged fathers displayed atypical USV trajectories. Thus, our study indicates that advanced paternal age has a significant effect on offspring's vocal development. Our computational analyses are effective in characterizing altered individual diversity.

Autism spectrum disorder (ASD) is often signaled by atypical cries during infancy. Copy number variants (CNVs) provide genetically identifiable cases of ASD, but how early atypical cries predict a later onset of ASD among CNV carriers is not understood in humans. Genetic mouse models of CNVs have provided a reliable tool to experimentally isolate the impact of CNVs and identify early predictors for later abnormalities in behaviors relevant to ASD. However, many technical issues have confounded the phenotypic characterization of such mouse models, including systematically biased genetic backgrounds and weak or absent behavioral phenotypes. To address these issues, we developed a coisogenic mouse model of human proximal 16p11.2 hemizygous deletion and applied computational approaches to identify hidden variables within neonatal vocalizations that have predictive power for postpubertal dimensions relevant to ASD. After variables of neonatal vocalizations were selected by least absolute shrinkage and selection operator (Lasso), random forest, and Markov model, regression models were constructed to predict postpubertal dimensions relevant to ASD. While the average scores of many standard behavioral assays designed to model dimensions did not differentiate a model of 16p11.2 hemizygous deletion and wild-type littermates, specific call types and call sequences of neonatal vocalizations predicted individual variability of postpubertal reciprocal social interaction and olfactory responses to a social cue in a genotype-specific manner. Deep-phenotyping and computational analyses identified hidden variables within neonatal social communication that are predictive of postpubertal behaviors.

Rare gene variants confer a high level of penetrance to neurodevelopmental disorders, but their developmental origin and cellular substrates remain poorly understood. To address this limitation, we explored the role of TBX1, a gene encoded in a rare copy number variant, in cell and mouse models. Here, we report that neonatal Tbx1 deficiency contributes to defective peripubertal social behavior and impairs the proliferation of neonatal neural stem/progenitor cells. Moreover, TBX1 transcriptionally regulates genes linked to post-embryonic neurogenesis and neurodevelopmental disorders associated with other rare gene variants. Our data indicate a precise time window and cell type through which the social dimension is altered by a gene encoded in a rare CNV and provide a potential common mechanistic basis for a group of neurodevelopmental disorders. One-Sentence Summary Tbx1, a gene affecting neonatal stem cell proliferation, influences peripubertal social behavior.

Copy number variants (CNVs) have provided a reliable entry point to identify the structural correlates of atypical cognitive development. Hemizygous deletion of human chromosome 22q11.2 is associated with impaired cognitive function; however, the mechanisms by which the CNVs contribute to cognitive deficits via diverse structural alterations in the brain remain unclear. This study aimed to determine the cellular basis of the link between alterations in brain structure and cognitive functions in mice with a heterozygous deletion of Tbx1 , one of the 22q11.2-encoded genes. Ex vivo whole-brain diffusion-tensor imaging (DTI)–magnetic resonance imaging (MRI) in Tbx1 heterozygous mice indicated that the fimbria was the only region with significant myelin alteration. Electron microscopic and histological analyses showed that Tbx1 heterozygous mice exhibited an apparent absence of large myelinated axons and thicker myelin in medium axons in the fimbria, resulting in an overall decrease in myelin. The fimbria of Tbx1 heterozygous mice showed reduced mRNA levels of Ng2 , a gene required to produce oligodendrocyte precursor cells. Moreover, postnatal progenitor cells derived from the subventricular zone, a source of oligodendrocytes in the fimbria, produced fewer oligodendrocytes in vitro. Behavioral analyses of these mice showed selectively slower acquisition of spatial memory and cognitive flexibility with no effects on their accuracy or sensory or motor capacities. Our findings provide a genetic and cellular basis for the compromised cognitive speed in patients with 22q11.2 hemizygous deletion.

Deletions in 22q11.2 human chromosome are known to be associated with psychiatric disorders, such as intellectual disability, schizophrenia, autism spectrum disorder, and anxiety disorders. This copy number variation includes a 3.0 Mb deletion and a nested proximal 1.5 Mb hemizygous deletion in the same region. Evidence indicates that the distal 22q11.2 region outside the nested 1.5 Mb deletion also might be contributory in humans. However, the precise genetic architecture within the distal region responsible for psychiatric disorders remains unclear, and this issue cannot be experimentally evaluated beyond the correlation in humans. As CRKL (CRK-like Proto-Oncogene, Adaptor Protein) is one of the genes encoded in the distal 22q11.2 segment and its homozygous deletion causes physical phenotypes of 22q11.2 hemizygous deletion, we tested the hypothesis that its murine homolog Crkl contributes to behavioral phenotypes relevant to psychiatric disorders in mice. Congenic Crkl heterozygosity reduced thigmotaxis, an anxiety-related behavior, in an inescapable open field, but had no apparent effect on social interaction, spontaneous alternation in a T-maze, anxiety-like behavior in an elevated plus maze, or motor activity in an open field. Our data indicate that the heterozygosity of murine Crkl does not recapitulate social deficits, working memory deficits, repetitive behavior traits or hyperactivity of human 22q11.2 hemizygous deletion. Moreover, while 22q11.2 hemizygous deletion is associated with high levels of phobia and anxiety in humans, our data suggest that Crkl heterozygosity rather acts as a protective factor for phobia-like behavior in an open field.

How the intrinsic sequence structure of neonatal mouse pup ultrasonic vocalization (USV) and maternal experiences determine maternal behaviors in mice is poorly understood. Our previous work showed that pups with aTbx1heterozygous (HT) mutation, a genetic risk for autism spectrum disorder (ASD), emit altered call sequences that do not induce maternal approach behaviors in C57BL6/J mothers. Here, we tested how maternal approach behaviors induced by wild-type and HT USVs are influenced by the mother's experience in raising pups of these two genotypes. The results showed that wild-type USVs were effective in inducing maternal approach behaviors when mothers raised wild-type but not HT pups. The USVs of HT pups were ineffective regardless of whether mothers raised HT or wild-type pups. However, the sequence structure of pup USVs had no effect on the general, non-directional incentive motivation of maternal behaviors. Our data show how the mother's experience with a pup with a genetic risk for ASD alters the intrinsic incentive values of USV sequences in maternal approach behaviors.