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Abstract Introduction Major depressive disorder (MDD) is a prevalent and debilitating mental disorder that shares symptoms, genetics, and molecular changes in the brain with other psychiatric disorders, such as schizophrenia and bipolar disorder. Decreased brain pH, associated with increased lactate levels due to altered energy metabolism and neuronal hyperexcitation, has been consistently observed in schizophrenia and bipolar disorder. We recently demonstrated similar brain alterations in various animal models of neuropsychiatric disorders, including MDD. However, our understanding of brain pH alterations in human patients with MDD remains limited. Methods We conducted meta-analyses to assess postmortem brain pH in patients with MDD compared to control subjects, examining its relationships with recurrence of depressive episodes and illness duration, utilizing publicly available demographic data. Studies reporting individual raw pH data were identified through searches in the Stanley Medical Research Institute database, NCBI GEO database, PubMed, and Google Scholar. The data were analyzed using the random effects model, ANOVA, and ANCOVA. Results The random effects model, using 39 curated datasets (790 patients and 957 controls), indicated a significant decrease in brain pH in patients with MDD (Hedges’ g = −0.23, p = 0.0056). A two-way ANCOVA revealed that the effect of diagnosis on pH remained significant when considering covariates, including postmortem interval, age at death, and sex. Patients with recurrent episodes, but not a single episode, showed significantly lower pH than controls in both females and males (256 patients and 279 controls from seven datasets). Furthermore, a significant negative correlation was observed between brain pH and illness duration (115 patients from five datasets). Female preponderance of decreased pH was also found, possibly due to a longer illness duration and a higher tendency of recurrent episodes in females. Conclusion This study suggests a decrease in brain pH in patients with MDD, potentially associated with recurrent episodes and longer illness duration. As suggested from previous animal model studies, altered brain energy metabolism, leading to decreased pH, may serve as a potential transdiagnostic endophenotype for MDD and other neuropsychiatric disorders.

Abstract Chronic exposure to glucocorticoids in response to long-term stress is thought to be a risk factor for major depression. Depression is associated with disturbances in the gut microbiota composition and peripheral and central energy metabolism. However, the relationship between chronic glucocorticoid exposure, the gut microbiota, and brain metabolism remains largely unknown. In this study, we first investigated the effects of chronic corticosterone exposure on various domains of behavior in adult male C57BL/6J mice treated with the glucocorticoid corticosterone to evaluate them as an animal model of depression. We then examined the gut microbial composition and brain and plasma metabolome in corticosterone-treated mice. Chronic corticosterone treatment resulted in reduced locomotor activity, increased anxiety-like and depression-related behaviors, decreased rotarod latency, reduced acoustic startle response, decreased social behavior, working memory deficits, impaired contextual fear memory, and enhanced cued fear memory. Chronic corticosterone treatment also altered the composition of gut microbiota, which has been reported to be associated with depression, such as increased abundance of Bifidobacterium, Turicibacter, and Corynebacterium and decreased abundance of Barnesiella. Metabolomic data revealed that long-term exposure to corticosterone led to a decrease in brain neurotransmitter metabolites, such as serotonin, 5-hydroxyindoleacetic acid, acetylcholine, and gamma-aminobutyric acid, as well as changes in betaine and methionine metabolism, as indicated by decreased levels of adenosine, dimethylglycine, choline, and methionine in the brain. These results indicate that mice treated with corticosterone have good face and construct validity as an animal model for studying anxiety and depression with altered gut microbial composition and brain metabolism, offering new insights into the neurobiological basis of depression arising from gut-brain axis dysfunction caused by prolonged exposure to excessive glucocorticoids.

Brain functions are mediated via the complex interplay between several complex factors, and hence, identifying the underlying cause of an abnormality within a certain brain region can be challenging. In mitochondrial disease, abnormalities in brain function are thought to be attributed to accumulation of mitochondrial DNA (mtDNA) with pathogenic mutations; however, only few previous studies have directly demonstrated that accumulation of mutant mtDNA induced abnormalities in brain function. Herein, we examined the effects of mtDNA mutations on brain function via behavioral analyses using a mouse model with an A2748G point mutation in mtDNA tRNALeu(UUR). Our results revealed that mice with a high percentage of mutant mtDNA showed a characteristic trend toward reduced prepulse inhibition and memory-dependent test performance, similar to that observed in psychiatric disorders, such as schizophrenia; however, muscle strength and motor coordination were not markedly affected. Upon examining the hippocampus and frontal lobes of the brain, mitochondrial morphology was abnormal, and the brain weight was slightly reduced. These results indicate that the predominant accumulation of a point mutation in the tRNALeu(UUR) gene may affect brain functions, particularly the coordination of sensory and motor functions and memory processes. These abnormalities probably caused by both direct effects of accumulation of the mutant mtDNA in neuronal cells and indirect effects via changes of systemic extracellular environments. Overall, these findings will lead to a better understanding of the pathogenic mechanism underlying this complex disease and facilitate the development of optimal treatment methods.

Abstract Background Intramuscular adipose tissue (IMAT) formation derived from muscle fibro‐adipogenic progenitors (FAPs) has been recognized as a pathological feature of sarcopenia. This study aimed to explore whether genetic and pharmacological gastric inhibitory polypeptide (GIP) receptor antagonism suppresses IMAT accumulation and ameliorates sarcopenia in mice. Methods Whole body composition, grip strength, skeletal muscle weight, tibialis anterior (TA) muscle fibre cross‐sectional area (CSA) and TA muscle IMAT area were measured in young and aged male C57BL/6 strain GIP receptor (Gipr)‐knockout (Gipr−/−) and wild‐type (Gipr+/+) mice. FAPs isolated from lower limb muscles of 12‐week‐old Gipr+/+ mice were cultured with GIP, and their differentiation into mature adipocytes was examined. Furthermore, TA muscle IMAT area and fibre CSA were measured in untreated Gipr−/− mice and GIP receptor antagonist‐treated Gipr+/+ mice after glycerol injection into the TA muscles. Results Body composition analysis revealed that 104‐week‐old Gipr−/− mice had a greater proportion of lean tissue mass (73.7 ± 1.2% vs. 66.5 ± 2.7%, P

Abstract Brain-enriched guanylate kinase-associated protein (BEGAIN) is highly enriched in the post-synaptic density (PSD) fraction and was identified in our previous study as a protein associated with neuropathic pain in the spinal dorsal horn. PSD protein complexes containing N-methyl-d-aspartate receptors are known to be involved in neuropathic pain. Since these PSD proteins also participate in learning and memory, BEGAIN is also expected to play a crucial role in this behavior. To verify this, we first examined the distribution of BEGAIN in the brain. We found that BEGAIN was widely distributed in the brain and highly expressed in the dendritic regions of the hippocampus. Moreover, we found that BEGAIN was concentrated in the PSD fraction of the hippocampus. Furthermore, immunoelectron microscopy confirmed that BEGAIN was localized at the asymmetric synapses. Behavioral tests were performed using BEGAIN-knockout (KO) mice to determine the contribution of BEGAIN toward learning and memory. Spatial reference memory and reversal learning in the Barns circular maze test along with contextual fear and cued fear memory in the contextual and cued fear conditioning test were significantly impaired in BEGAIN-KO mice compared to with those in wild-type mice. Thus, this study reveals that BEGAIN is a component of the post-synaptic compartment of excitatory synapses involved in learning and memory.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer’s disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Abstract Disordered sleep is a global social problem and an established significant risk factor for psychological and metabolic diseases. We profiled non-targeted metabolites in saliva from mouse models of chronic sleep disorder (CSD). We identified 288 and 55 metabolites using CE-FTMS and LC-TOFMS, respectively, among which concentrations of 58 (CE-FTMS) and three (LC-TOFMS) were significantly changed by CSD. Pathway analysis revealed that CSD significantly suppressed glycine, serine and threonine metabolism. Arginine and proline metabolic pathways were among those that were both upregulated and downregulated. Pathways of alanine, aspartate and glutamate metabolism, genetic information processing, and the TCA cycle tended to be downregulated, whereas histidine metabolism tended to be upregulated in mice with CSD. Pyruvate, lactate, malate, succinate and the glycemic amino acids alanine, glycine, methionine, proline, and threonine were significantly decreased, whereas 3-hydroxybutyric and 2-hydroxybutyric acids associated with ketosis were significantly increased, suggesting abnormal glucose metabolism in mice with CSD. Increases in the metabolites histamine and kynurenic acid that are associated with the central nervous system- and decreased glycine, might be associated with sleep dysregulation and impaired cognitive dysfunction in mice with CSD. Our findings suggested that profiling salivary metabolites could be a useful strategy for diagnosing CSD.

Abstract Glycine receptors (GlyRs) are ligand-gated chloride channels comprising alpha (α1–4) and β subunits. The GlyR subunits play major roles in the mammalian central nervous system, ranging from regulating simple sensory information to modulating higher-order brain function. Unlike the other GlyR subunits, GlyR α4 receives relatively little attention because the human ortholog lacks a transmembrane domain and is thus considered a pseudogene. A recent genetic study reported that the GLRA4 pseudogene locus on the X chromosome is potentially involved in cognitive impairment, motor delay and craniofacial anomalies in humans. The physiologic roles of GlyR α4 in mammal behavior and its involvement in disease, however, are not known. Here we examined the temporal and spatial expression profile of GlyR α4 in the mouse brain and subjected Glra4 mutant mice to a comprehensive behavioral analysis to elucidate the role of GlyR α4 in behavior. The GlyR α4 subunit was mainly enriched in the hindbrain and midbrain, and had relatively lower expression in the thalamus, cerebellum, hypothalamus, and olfactory bulb. In addition, expression of the GlyR α4 subunit gradually increased during brain development. Glra4 mutant mice exhibited a decreased amplitude and delayed onset of the startle response compared with wild-type littermates, and increased social interaction in the home cage during the dark period. Glra4 mutants also had a low percentage of entries into open arms in the elevated plus-maze test. Although mice with GlyR α4 deficiency did not show motor and learning abnormalities reported to be associated in human genomics studies, they exhibited behavioral changes in startle response and social and anxiety-like behavior. Our data clarify the spatiotemporal expression pattern of the GlyR α4 subunit and suggest that glycinergic signaling modulates social, startle, and anxiety-like behaviors in mice.

Abstract The serotonin transporter (5-HTT) plays a critical role in the regulation of serotonin neurotransmission. Mice genetically deficient in 5-HTT expression have been used to study the physiological functions of 5-HTT in the brain and have been proposed as a potential animal model for neuropsychiatric and neurodevelopmental disorders. Recent studies have provided evidence for a link between the gut-brain axis and mood disorders. However, the effects of 5-HTT deficiency on gut microbiota, brain function, and behavior remain to be fully characterized. Here we investigated the effects of 5-HTT deficiency on different types of behavior, the gut microbiome, and brain c-Fos expression as a marker of neuronal activation in response to the forced swim test for assessing depression-related behavior in male 5-HTT knockout mice. Behavioral analysis using a battery of 16 different tests showed that 5-HTT−/− mice exhibited markedly reduced locomotor activity, decreased pain sensitivity, reduced motor function, increased anxiety-like and depression-related behavior, altered social behavior in novel and familiar environments, normal working memory, enhanced spatial reference memory, and impaired fear memory compared to 5-HTT+/+ mice. 5-HTT+/− mice showed slightly reduced locomotor activity and impaired social behavior compared to 5-HTT+/+ mice. Analysis of 16S rRNA gene amplicons showed that 5-HTT−/− mice had altered gut microbiota abundances, such as a decrease in Allobaculum, Bifidobacterium, Clostridium sensu stricto, and Turicibacter, compared to 5-HTT+/+ mice. This study also showed that after exposure to the forced swim test, the number of c-Fos-positive cells was higher in the paraventricular thalamus and lateral hypothalamus and was lower in the prefrontal cortical regions, nucleus accumbens shell, dorsolateral septal nucleus, hippocampal regions, and ventromedial hypothalamus in 5-HTT−/− mice than in 5-HTT+/+ mice. These phenotypes of 5-HTT−/− mice partially recapitulate clinical observations in humans with major depressive disorder. The present findings indicate that 5-HTT-deficient mice serve as a good and valid animal model to study anxiety and depression with altered gut microbial composition and abnormal neuronal activity in the brain, highlighting the importance of 5-HTT in brain function and the mechanisms underlying the regulation of anxiety and depression.

Major depressive disorder (depression) is a leading cause of disability. The severity of depression is affected by many factors, one of which being comorbidity with diabetes mellitus (DM). The comorbidity of depression with DM is a major public health concern due to the high incidence of both conditions and their mutually exacerbating pathophysiology. However, the mechanisms by which DM exacerbates depression remain largely unknown, and elucidating these regulatory mechanisms would contribute to a significant unmet clinical need. We generated a comorbid mouse model of depression and DM (comorbid model), which was extensively compared with depression and DM models. Depressive and anhedonic phenotypes were more severe in the comorbid model. We thus concluded that the comorbid model recapitulated exacerbated depression-related behaviors comorbid with DM in clinic. RNA sequencing analysis of prefrontal cortex tissue revealed that the brain pH homeostasis gene set was one of the most affected in the comorbid model. Furthermore, brain pH negatively correlated with anhedonia-related behaviors in the depression and comorbid models. By contrast, these correlations were not detected in DM or control group, neither of which had been exposed to chronic stress. This suggested that the addition of reduced brain pH to stress-exposed conditions had synergistic and aversive effects on anhedonic phenotypes. Because brain pH was strongly correlated with brain lactate level, which correlated with blood glucose levels, these findings highlight the therapeutic importance of glycemic control not only for DM, but also for psychiatric problems in patients with depression comorbid with DM.

Abstract Although dyslipidemia in the brain has been implicated in neurodegenerative disorders, the molecular mechanisms underlying its pathogenesis have been largely unclear. PDZD8 is a lipid transfer protein and mice deficient in PDZD8 (PDZD8-KO mice) manifest abnormal accumulation of cholesteryl esters (CEs) in the brain due to impaired lipophagy, the degradation system of lipid droplets. Here we show the detailed mechanism of PDZD8-dependent lipophagy. PDZD8 transports cholesterol to lipid droplets (LDs), and eventually promotes fusion of LDs and lysosomes. In addition, PDZD8-KO mice exhibit growth retardation, hyperactivity, reduced anxiety and fear, increased sensorimotor gating, and impaired cued fear conditioned memory and working memory. These results indicate that abnormal CE accumulation in the brain caused by PDZD8 deficiency affects emotion, cognition and adaptive behavior, and that PDZD8 plays an important role in the maintenance of brain function through lipid metabolism.

Abstract Calcineurin (Cn), a phosphatase important for synaptic plasticity and neuronal development, has been implicated in the etiology and pathophysiology of neuropsychiatric disorders, including schizophrenia, intellectual disability, autism spectrum disorders, epilepsy, and Alzheimer’s disease. Forebrain-specific conditional Cn knockout mice have been known to exhibit multiple behavioral phenotypes related to these disorders. In this study, we investigated whether Cn mutant mice show pseudo-immaturity of the dentate gyrus (iDG) in the hippocampus, which we have proposed as an endophenotype shared by these disorders. Expression of calbindin and GluA1, typical markers for mature DG granule cells (GCs), was decreased and that of doublecortin, calretinin, phospho-CREB, and dopamine D1 receptor (Drd1), markers for immature GC, was increased in Cn mutants. Phosphorylation of cAMP-dependent protein kinase (PKA) substrates (GluA1, ERK2, DARPP-32, PDE4) was increased and showed higher sensitivity to SKF81297, a Drd1-like agonist, in Cn mutants than in controls. While cAMP/PKA signaling is increased in the iDG of Cn mutants, chronic treatment with rolipram, a selective PDE4 inhibitor that increases intracellular cAMP, ameliorated the iDG phenotype significantly and nesting behavior deficits with nominal significance. Chronic rolipram administration also decreased the phosphorylation of CREB, but not the other four PKA substrates examined, in Cn mutants. These results suggest that Cn deficiency induces pseudo-immaturity of GCs and that cAMP signaling increases to compensate for this maturation abnormality. This study further supports the idea that iDG is an endophenotype shared by certain neuropsychiatric disorders.

Hydrogen ion (H+) is one of the most potent intrinsic neuromodulators in the brain in terms of concentration. Changes in H+ concentration, expressed as pH, are thought to be associated with various biological processes, such as gene expression, in the brain. Accumulating evidence suggests that decreased brain pH is a common feature of several neuropsychiatric disorders, including schizophrenia, bipolar disorder, autism spectrum disorder, and Alzheimer's disease. However, it remains unclear whether gene expression patterns can be used as surrogates for pH changes in the brain. In this study, we performed meta-analyses using publicly available gene expression datasets to profile the expression patterns of pH-associated genes, whose expression levels were correlated with brain pH, in human patients and mouse models of major central nervous system (CNS) diseases, as well as in mouse cell-type datasets. Comprehensive analysis of 281 human datasets from 11 CNS disorders revealed that gene expression associated with decreased pH was over-represented in disorders including schizophrenia, bipolar disorder, autism spectrum disorders, Alzheimer's disease, Huntington's disease, Parkinson's disease, and brain tumors. Expression patterns of pH-associated genes in mouse models of neurodegenerative disease showed a common time course trend toward lower pH over time. Furthermore, cell type analysis identified astrocytes as the cell type with the most acidity-related gene expression, consistent with previous experimental measurements showing a lower intracellular pH in astrocytes than in neurons. These results suggest that the expression pattern of pH-associated genes may be a surrogate for the state- and trait-related changes in pH in brain cells. Altered expression of pH-associated genes may serve as a novel molecular mechanism for a more complete understanding of the transdiagnostic pathophysiology of neuropsychiatric and neurodegenerative disorders.

Abstract Aim Capric acid (also known as decanoic acid or C10) is one of the fatty acids in the medium‐chain triglycerides (MCTs) commonly found in dietary fats. Although dietary treatment with MCTs is recently of great interest for the potential therapeutic effects on neuropsychiatric disorders, the effects of oral administration of C10 on behavior remain to be examined. This study investigated acute and chronic effects of oral administration of C10 on locomotor activity and anxiety‐like and depression‐related behaviors in adult male C57BL/6J mice. Methods To explore the acute effects of C10 administration, mice were subjected to a series of behavioral tests in the following order: light/dark transition, open field, elevated plus maze, Porsolt forced swim, and tail suspension tests, 30 minutes after oral gavage of either vehicle or C10 solution (30 mmol/kg dose in Experiment 1; 0.1, 0.3, 1.0, 3.0 mmol/kg doses in Experiment 2). Next, to examine chronic effects of C10, mice repeatedly administered with either vehicle or C10 solution (0.3, 3.0 mmol/kg doses per day, for 21 days, in Experiment 3) were subjected to behavioral tests without oral administration immediately before each test. Results The mice administrated with the high dose of C10 (30 mmol/kg) showed lower body weights, shorter distance traveled, and more anxiety‐like behavior than vehicle‐treated mice, and the results reached study‐wide statistical significance. The C10 administration at a lower dose of 0.3 mmol/kg had no significant effects on body weights and induced nominally significantly longer distance traveled than vehicle administration. Repeated administration of C10 at a dose of 3.0 mmol/kg for more than 21 days caused lower body weights and decreased depression‐related behavior, although the behavioral differences did not reach study‐wide significance. Conclusions Although these results suggest dose‐dependent effects of oral administration of capric acid on locomotor activity and anxiety‐like and depression‐related behaviors, further study will be needed to replicate the findings and explore the underlying brain mechanisms.

Abstract Aim Experimental animals, such as non-human primates (NHPs), mice, Zebrafish, and Drosophila, are frequently employed as models to gain insights into human physiology and pathology. In developmental neuroscience and related research fields, information about the similarities of developmental gene expression patterns between animal models and humans is vital to choose what animal models to employ. Here, we aimed to statistically compare the similarities of developmental changes of gene expression patterns in the brains of humans with those of animal models frequently used in the neuroscience field. Methods The developmental gene expression datasets that we analyzed consist of the fold-changes and P values of gene expression in the brains of animals of various ages compared with those of the youngest postnatal animals available in the dataset. By employing the running Fisher algorithm in a bioinformatics platform, BaseSpace, we assessed similarities between the developmental changes of gene expression patterns in the human (Homo sapiens) hippocampus with those in the dentate gyrus (DG) of the rhesus monkey (Macaca mulatta), the DG of the mouse (Mus musculus), the whole brain of Zebrafish (Danio rerio), and the whole brain of Drosophila (D. melanogaster). Results Among all possible comparisons of different ages and animals in developmental changes in gene expression patterns within the datasets, those between rhesus monkeys and mice were highly similar to those of humans with significant overlap P-value as assessed by the running Fisher algorithm. There was the highest degree of gene expression similarity between 40–59-year-old humans and 6–12-year-old rhesus monkeys (overlap P-value = 2.1 × 10− 72). The gene expression similarity between 20–39-year-old humans and 29-day-old mice was also significant (overlap P = 1.1 × 10− 44). Moreover, there was a similarity in developmental changes of gene expression patterns between 1–2-year-old Zebrafish and 40–59-year-old humans (Overlap P-value = 1.4 × 10− 6). The overlap P-value of developmental gene expression patterns between Drosophila and humans failed to reach significance (30 days Drosophila and 6–11-year-old humans; overlap P-value = 0.0614). Conclusions These results indicate that the developmental gene expression changes in the brains of the rhesus monkey, mouse, and Zebrafish recapitulate, to a certain degree, those in humans. Our findings support the idea that these animal models are a valid tool for investigating the development of the brain in neurophysiological and neuropsychiatric studies.

Introduction: Lumbar spondylolysis is usually single level, and only a few multiple-level cases have been reported. We investigated the frequency of multiple-level spondylolysis and the bone union rates among growth-stage children with lower back pain (LBP). Methods: The subjects were growth-stage children examined for LBP between April 2013 and December 2018. All patients with LBP persisting for at least 2 weeks and severe enough to make playing sports difficult underwent lumbar plain radiogram, computed tomography, and magnetic resonance imaging. The cases diagnosed as multiple-level spondylolysis and classified as early or progressive stage received conservative treatment to achieve bone union. Results: A total of 782 growth-stage children were examined for LBP. Of them, 243 children (31.1%) were diagnosed with lumbar spondylolysis. Of these 243 children, 23 (9.5%) children had multiple-level spondylolysis. Of the children diagnosed with multiple-level spondylolysis, most children (87.0%) had pars defects in the early or progressive stage in which bone union could be expected. Most children (78.3%) had pars defects in the terminal stage and combined with these defects, had pars defects in the early or progressive stage at a different spinal level. Twenty children diagnosed with multiple-level spondylolysis who also had pars defects in the early or progressive stage received conservative treatment for bone union, which was achieved in 31 of 39 sites (79.5%). The bone union rate by stage was 92.9% (26 of 28 sites) in the early stage and 45.5% (5 of 11 sites) in the progressive stage. Conclusions: In cases of multiple-level spondylolysis, bone union is likely to be achieved with conservative treatment when the pars defects are in the early or progressive stage. Therefore, the first choice of treatment should be conservative treatment to achieve bone union, the same for single-level spondylolysis.

Summary: The human vesicular monoamine transporter 1 (VMAT1) harbors unique substitutions (Asn136Thr/Ile) that affect monoamine uptake into synaptic vesicles. These substitutions are absent in all known mammals, suggesting their contributions to distinct aspects of human behavior modulated by monoaminergic transmissions, such as emotion and cognition. To directly test the impact of these human-specific mutations, we introduced the humanized residues into mouse Vmat1 via CRISPR/Cas9-mediated genome editing and examined changes at the behavioral, neurophysiological, and molecular levels. Behavioral tests revealed reduced anxiety-related traits of Vmat1Ile mice, consistent with human studies, and electrophysiological recordings showed altered oscillatory activity in the amygdala under anxiogenic conditions. Transcriptome analyses further identified changes in gene expressions in the amygdala involved in neurodevelopment and emotional regulation, which may corroborate the observed phenotypes. This knock-in mouse model hence provides compelling evidence that the mutations affecting monoaminergic signaling and amygdala circuits have contributed to the evolution of human socio-emotional behaviors.

Abstract Aims Neurogranin (NRGN) is a postsynaptic protein kinase substrate that binds calmodulin in the absence of calcium. Recent studies suggest that NRGN is involved in neuropsychiatric disorders, including schizophrenia, ADHD, and Alzheimer's disease. Previous behavioral studies of Nrgn knockout (Nrgn KO) mice identified hyperactivity, deficits in spatial learning, impaired sociability, and decreased prepulse inhibition, which suggest that these mice recapitulate some symptoms of neuropsychiatric disorders. To further validate Nrgn KO mice as a model of neuropsychiatric disorders, we assessed multiple domains of behavioral phenotypes in Nrgn KO mice using a comprehensive behavioral test battery including tests of homecage locomotor activity and nesting behavior. Methods Adult Nrgn KO mice (28‐54 weeks old) were subjected to a battery of comprehensive behavioral tests, which examined general health, nesting behavior, neurological characteristics, motor function, pain sensitivity, locomotor activity, anxiety‐like behavior, social behavior, sensorimotor gating, depression‐like behavior, and working memory. Results The Nrgn KO mice displayed a pronounced decrease in nesting behavior, impaired motor function, and elevated pain sensitivity. While the Nrgn KO mice showed increased locomotor activity in the open field test, these mice did not show hyperactivity in a familiar environment as measured in the homecage locomotor activity test. The Nrgn KO mice exhibited a decreased number of transitions in the light‐dark transition test and decreased stay time in the center of the open field test, which is consistent with previous reports of increased anxiety‐like behavior. Interestingly, however, these mice stayed on open arms significantly longer than wild‐type mice in the elevated plus maze. Consistent with previous studies, the mutant mice exhibited decreased prepulse inhibition, impaired working memory, and decreased sociability. Conclusions In the current study, we identified behavioral phenotypes of Nrgn KO mice that mimic some of the typical symptoms of neuropsychiatric diseases, including impaired executive function, motor dysfunction, and altered anxiety. Most behavioral phenotypes that had been previously identified, such as hyperlocomotor activity, impaired sociability, tendency for working memory deficiency, and altered sensorimotor gating, were reproduced in the present study. Collectively, the behavioral phenotypes of Nrgn KO mice detected in the present study indicate that Nrgn KO mice are a valuable animal model that recapitulates a variety of symptoms of neuropsychiatric disorders, such as schizophrenia, ADHD, and Alzheimer's disease.

Abstract The 15q13.3 microdeletion syndrome is a genetic disorder characterized by a wide spectrum of psychiatric disorders that is caused by the deletion of a region containing 7 genes on chromosome 15 (MTMR10, FAN1, TRPM1, MIR211, KLF13, OTUD7A, and CHRNA7). The contribution of each gene in this syndrome has been studied using mutant mouse models, but no single mouse model recapitulates the whole spectrum of human 15q13.3 microdeletion syndrome. The behavior of Trpm1 −/− mice has not been investigated in relation to 15q13.3 microdeletion syndrome due to the visual impairment in these mice, which may confound the results of behavioral tests involving vision. We were able to perform a comprehensive behavioral test battery using Trpm1 null mutant mice to investigate the role of Trpm1, which is thought to be expressed solely in the retina, in the central nervous system and to examine the relationship between TRPM1 and 15q13.3 microdeletion syndrome. Our data demonstrate that Trpm1 −/− mice exhibit abnormal behaviors that may explain some phenotypes of 15q13.3 microdeletion syndrome, including reduced anxiety-like behavior, abnormal social interaction, attenuated fear memory, and the most prominent phenotype of Trpm1 mutant mice, hyperactivity. While the ON visual transduction pathway is impaired in Trpm1 −/− mice, we did not detect compensatory high sensitivities for other sensory modalities. The pathway for visual impairment is the same between Trpm1 −/− mice and mGluR6 −/− mice, but hyperlocomotor activity has not been reported in mGluR6 −/− mice. These data suggest that the phenotype of Trpm1 −/− mice extends beyond that expected from visual impairment alone. Here, we provide the first evidence associating TRPM1 with impairment of cognitive function similar to that observed in phenotypes of 15q13.3 microdeletion syndrome.

As an important neurotransmitter, glutamate acts in over 90% of excitatory synapses in the human brain. Its metabolic pathway is complicated, and the glutamate pool in neurons has not been fully elucidated. Tubulin polyglutamylation in the brain is mainly mediated by two tubulin tyrosine ligase-like (TTLL) proteins, TTLL1 and TTLL7, which have been indicated to be important for neuronal polarity. In this study, we constructed pure lines of Ttll1 and Ttll7 knockout mice. Ttll knockout mice showed several abnormal behaviors. Matrix-assisted laser desorption/ionization (MALDI) Imaging mass spectrometry (IMS) analyses of these brains showed increases in glutamate, suggesting that tubulin polyglutamylation by these TTLLs acts as a pool of glutamate in neurons and modulates some other amino acids related to glutamate.

Abstract The elevated plus maze test is a widely used test for assessing anxiety-like behavior and screening novel therapeutic agents in rodents. Previous studies have shown that a variety of internal factors and procedural variables can influence elevated plus maze behavior. Although some studies have suggested a link between behavior and plasma corticosterone levels, the relationships between them remain unclear. In this study, we investigated the effects of experience with a battery of behavioral tests, the wall color of the closed arms, and illumination level on the behavior and plasma corticosterone responses in the elevated plus maze in male C57BL/6J mice. Mice were either subjected to a series of behavioral tests, including assessments of general health and neurological function, a light/dark transition test, and an open field test, or left undisturbed until the start of the elevated plus maze test. The mice with and without test battery experience were allowed to freely explore the elevated plus maze. The other two independent groups of naïve mice were tested in mazes with closed arms with different wall colors (clear, transparent blue, white, and black) or different illumination levels (5, 100, and 800 lx). Immediately after the test, blood was collected to measure plasma corticosterone concentrations. Mice with test battery experience showed a lower percentage of open arm time and entries and, somewhat paradoxically, had lower plasma corticosterone levels than the mice with no test battery experience. Mice tested in the maze with closed arms with clear walls exhibited higher open arm exploration than mice tested in the maze with closed arms with black walls, while there were no significant differences in plasma corticosterone levels between the different wall color conditions. Illumination levels had no significant effects on any measure. Our results indicate that experience with other behavioral tests and different physical features of the maze affect elevated plus maze behaviors. Increased open arm time and entries are conventionally interpreted as decreased anxiety-like behavior, while other possible interpretations are considered: open arm exploration may reflect heightened anxiety and panic-like reaction to a novel situation under certain conditions. With the possibility of different interpretations, the present findings highlight the need to carefully consider the test conditions in designing experiments and drawing conclusions from the behavioral outcomes in the elevated plus maze test in C57BL/6J mice.

Abstract Protrudin is a protein that resides in the membrane of the endoplasmic reticulum and is highly expressed in the nervous system. Although mutations in the human protrudin gene (ZFYVE27, also known as SPG33) give rise to hereditary spastic paraplegia (HSP), the physiological role of the encoded protein has been largely unclear. We therefore generated mice deficient in protrudin and subjected them to a battery of behavioral tests designed to examine their intermediate phenotypes. The protrudin-deficient mice were found to have a reduced body size and to manifest pleiotropic behavioral abnormalities, including hyperactivity, depression-like behavior, and deficits in attention and fear-conditioning memory. They exhibited no signs of HSP, however, consistent with the notion that HSP-associated mutations of protrudin may elicit neural degeneration, not as a result of a loss of function, but rather as a result of a gain of toxic function. Overall, our results suggest that protrudin might play an indispensable role in normal neuronal development and behavior.

Abstract Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid β (Aβ) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aβ deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency.

Abstract Aims Restraint stress is one of the most widely used experimental methods for generating rodent models of stress‐induced neuropsychiatric disorders, such as depression and anxiety. Although various types of restraint apparatuses have been used to expose animals to stress, the magnitudes of the effects of stress exposure via different types of restraint apparatuses on physiology and behavior have not been compared in the same environment. Here, we investigated the effects of stress exposure via two types of restraint apparatuses on body weight, locomotor activity, anxiety‐ and depression‐related behaviors, and plasma corticosterone levels in mice. Methods Adult male BALB/cAJcl mice were restrained by placing them in either a well‐ventilated plastic conical tube or a tapered plastic film envelope for 6 hours per day for 10 or 21 consecutive days. Mice were weighed during and after the stress period and were subjected to a battery of behavioral tests, including light/dark transition, open field, elevated plus maze, Porsolt forced swim, tail suspension, and sucrose preference tests, starting on the day after the last stress session. Plasma corticosterone levels were measured in another cohort of mice on the 1st and the 21st stress sessions and after the Porsolt forced swim test. Results Exposure to repeated stress via the two above mentioned types of restraint apparatuses caused body weight loss, heightened locomotor activity, altered immobility during forced swim, and increased plasma corticosterone levels, and some of these results differed between the restraint stress protocols. Film‐restraint–stressed mice had significantly lower body weights than tube‐restraint–stressed mice. Film‐restraint–stressed mice exhibited significantly higher or lower immobility during forced swim than tube‐restraint–stressed mice, depending on the test time. Additionally, the stress‐induced increase in plasma corticosterone levels was found to be higher in film‐restraint–stressed mice than in tube‐restraint–stressed mice. Conclusion Our results indicate that film‐restraint stress has more pronounced effects on body weight, depression‐related behavior, and corticosterone response than tube‐restraint stress in mice. These findings may help guide which restraint stress procedures to use, depending on the objectives of a given study, in generating animal models of stress‐induced neuropsychiatric disorders.

Abstract Tsukushi (TSK)—a small, secreted, leucine-rich-repeat proteoglycan—interacts with and regulates essential cellular signaling cascades. However, its functions in the mouse inner ear are unknown. In this study, measurement of auditory brainstem responses, fluorescence microscopy, and scanning electron microscopy revealed that TSK deficiency in mice resulted in the formation of abnormal stereocilia in the inner hair cells and hearing loss but not in the loss of these cells. TSK accumulated in nonprosensory regions during early embryonic stages and in both nonprosensory and prosensory regions in late embryonic stages. In adult mice, TSK was localized in the organ of Corti, spiral ganglion cells, and the stria vascularis. Moreover, loss of TSK caused dynamic changes in the expression of key genes that drive the differentiation of the inner hair cells in prosensory regions. Finally, our results revealed that TSK interacted with Sox2 and BMP4 to control stereocilia formation in the inner hair cells. Hence, TSK appears to be an essential component of the molecular pathways that regulate inner ear development.

Abstract A reproducibility crisis is a situation where many scientific studies cannot be reproduced. Inappropriate practices of science, such as HARKing, p-hacking, and selective reporting of positive results, have been suggested as causes of irreproducibility. In this editorial, I propose that a lack of raw data or data fabrication is another possible cause of irreproducibility. As an Editor-in-Chief of Molecular Brain, I have handled 180 manuscripts since early 2017 and have made 41 editorial decisions categorized as “Revise before review,” requesting that the authors provide raw data. Surprisingly, among those 41 manuscripts, 21 were withdrawn without providing raw data, indicating that requiring raw data drove away more than half of the manuscripts. I rejected 19 out of the remaining 20 manuscripts because of insufficient raw data. Thus, more than 97% of the 41 manuscripts did not present the raw data supporting their results when requested by an editor, suggesting a possibility that the raw data did not exist from the beginning, at least in some portions of these cases. Considering that any scientific study should be based on raw data, and that data storage space should no longer be a challenge, journals, in principle, should try to have their authors publicize raw data in a public database or journal site upon the publication of the paper to increase reproducibility of the published results and to increase public trust in science.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ-aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the “odorant binding” pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring.

Abstract Bipolar disorder is a major mental illness characterized by severe swings in mood and activity levels which occur with variable amplitude and frequency. Attempts have been made to identify mood states and biological features associated with mood changes to compensate for current clinical diagnosis, which is mainly based on patients’ subjective reports. Here, we used infradian (a cycle > 24 h) cyclic locomotor activity in a mouse model useful for the study of bipolar disorder as a proxy for mood changes. We show that metabolome patterns in peripheral blood could retrospectively predict the locomotor activity levels. We longitudinally monitored locomotor activity in the home cage, and subsequently collected peripheral blood and performed metabolomic analyses. We then constructed cross-validated linear regression models based on blood metabolome patterns to predict locomotor activity levels of individual mice. Our analysis revealed a significant correlation between actual and predicted activity levels, indicative of successful predictions. Pathway analysis of metabolites used for successful predictions showed enrichment in mitochondria metabolism-related terms, such as “Warburg effect” and “citric acid cycle.” In addition, we found that peripheral blood metabolome patterns predicted expression levels of genes implicated in bipolar disorder in the hippocampus, a brain region responsible for mood regulation, suggesting that the brain–periphery axis is related to mood-change-associated behaviors. Our results may serve as a basis for predicting individual mood states through blood metabolomics in bipolar disorder and other mood disorders and may provide potential insight into systemic metabolic activity in relation to mood changes.

Abstract It is agreed upon that adult hippocampal neurogenesis (AHN) occurs in the dentate gyrus (DG) in rodents. However, the existence of AHN in humans, particularly in elderly individuals, remains to be determined. Recently, several studies reported that neural progenitor cells, neuroblasts, and immature neurons were detected in the hippocampus of elderly humans, based on the expressions of putative markers for these cells, claiming that this provides evidence of the persistence of AHN in humans. Herein, we briefly overview the phenomenon that we call “dematuration,” in which mature neurons dedifferentiate to a pseudo-immature status and re-express the molecular markers of neural progenitor cells and immature neurons. Various conditions can easily induce dematuration, such as inflammation and hyper-excitation of neurons, and therefore, the markers for neural progenitor cells and immature neurons may not necessarily serve as markers for AHN. Thus, the aforementioned studies have not presented definitive evidence for the persistence of hippocampal neurogenesis throughout adult life in humans, and we would like to emphasize that those markers should be used cautiously when presented as evidence for AHN. Increasing AHN has been considered as a therapeutic target for Alzheimer’s disease (AD); however, given that immature neuronal markers can be re-expressed in mature adult neurons, independent of AHN, in various disease conditions including AD, strategies to increase the expression of these markers in the DG may be ineffective or may worsen the symptoms of such diseases.

Abstract Very‐long‐chain fatty acids, with a chain length of >C20, are abundant in myelin sphingolipids. Recently, a de novo mutation in the ELOVL1 gene, which encodes fatty acid elongase, was identified in patients with neurocutaneous disorders involving skin ichthyosis and multiple neurological abnormalities, including hypomyelination, spastic paraplegia, and high‐frequency deafness. However, the consequences of ELOVL1 deficiency for lipid composition and detailed pathological changes in the brain remain unclear. Here, we analyzed Elovl1 mutant mice as a model of human ELOVL1 deficiency. The mice exhibited a decreased postnatal survival rate, and some died of startle epilepsy. The acyl chain length of sphingolipids such as galactosylceramides, sulfatides, sphingomyelins, and ceramides in the brains of these mice was markedly shortened. Moreover, the mice exhibited reduced levels of galactosylceramides, which are important for myelin formation and stability. Electron microscope analysis of the corpus callosum in Elovl1 mutant mice revealed modest hypomyelination, especially in large‐diameter axons. Furthermore, behavioral testing of the mice revealed deficits such as poorer motor coordination and reduced acoustic startle response to high‐intensity stimulus. These findings provide clues to the molecular mechanism of the neurological symptoms of patients with the ELOVL1 mutation.

Summary: Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function.

Abstract Aims Synaptic Ras GTPase‐activating protein 1 (SYNGAP1) regulates synaptic plasticity through AMPA receptor trafficking. SYNGAP1 mutations have been found in human patients with intellectual disability (ID) and autism spectrum disorder (ASD). Almost every individual with SYNGAP1‐related ID develops epilepsy, and approximately 50% have ASD. SYNGAP1‐related ID is estimated to account for at least 1% of ID cases. In mouse models with Syngap1 mutations, strong cognitive and affective dysfunctions have been reported, yet some findings are inconsistent across studies. To further understand the behavioral significance of the SYNGAP1 gene, we assessed various domains of behavior in Syngap1 heterozygous mutant mice using a behavioral test battery. Methods Male mice with a heterozygous mutation in the Syngap1 gene (Syngap1−/+ mice) created by Seth Grant's group were subjected to a battery of comprehensive behavioral tests, which examined general health, and neurological screens, rotarod, hot plate, open field, light/dark transition, elevated plus maze, social interaction, prepulse inhibition, Porsolt forced swim, tail suspension, gait analysis, T‐maze, Y‐maze, Barnes maze, contextual and cued fear conditioning, and home cage locomotor activity. To control for type I errors due to multiple‐hypothesis testing, P‐values below the false discovery rate calculated by the Benjamini‐Hochberg method were considered as study‐wide statistically significant. Results Syngap1−/+ mice showed increased locomotor activity, decreased prepulse inhibition, and impaired working and reference spatial memory, consistent with preceding studies. Impairment of context fear memory and increased startle reflex in Syngap1 mutant mice could not be reproduced. Significant decreases in sensitivity to painful stimuli and impaired motor function were observed in Syngap1−/+ mice. Decreased anxiety‐like behavior and depression‐like behavior were noted, although increased locomotor activity is a potential confounding factor of these phenotypes. Increased home cage locomotor activity indicated hyperlocomotor activity not only in specific behavioral test conditions but also in familiar environments. Conclusion In Syngap1−/+ mice, we could reproduce most of the previously reported cognitive and emotional deficits. The decreased sensitivity to painful stimuli and impaired motor function that we found in Syngap1−/+ mice are consistent with the common characteristics of patients with SYNGAP‐related ID. We further confirmed that the Syngap1 heterozygote mouse recapitulates the symptoms of ID and ASD patients.

Abstract The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely used to treat depression and anxiety disorders. Chronic FLX treatment reportedly induces cellular responses in the brain, including increased adult hippocampal and cortical neurogenesis and reversal of neuron maturation in the hippocampus, amygdala, and cortex. However, because most previous studies have used rodent models, it remains unclear whether these FLX-induced changes occur in the primate brain. To evaluate the effects of FLX in the primate brain, we used immunohistological methods to assess neurogenesis and the expression of neuronal maturity markers following chronic FLX treatment (3 mg/kg/day for 4 weeks) in adult marmosets (n = 3 per group). We found increased expression of doublecortin and calretinin, markers of immature neurons, in the hippocampal dentate gyrus of FLX-treated marmosets. Further, FLX treatment reduced parvalbumin expression and the number of neurons with perineuronal nets, which indicate mature fast-spiking interneurons, in the hippocampus, but not in the amygdala or cerebral cortex. We also found that FLX treatment increased the generation of cortical interneurons; however, significant up-regulation of adult hippocampal neurogenesis was not observed in FLX-treated marmosets. These results suggest that dematuration of hippocampal neurons and increased cortical neurogenesis may play roles in FLX-induced effects and/or side effects. Our results are consistent with those of previous studies showing hippocampal dematuration and increased cortical neurogenesis in FLX-treated rodents. In contrast, FLX did not affect hippocampal neurogenesis or dematuration of interneurons in the amygdala and cerebral cortex.

Abstract Pregnancy and lactation are characterized by dramatic changes in the endocrine system and brain in mammalian females. These changes, with stress before pregnancy, are potential risk factors for the development of postpartum depression (PPD). A valid animal model of PPD is needed to understand the neurobiological basis of the depressive state of females. To explore a mouse model of PPD, we first assessed anxiety-like and depression-related behaviors in nulliparous (virgin), nonlactating primiparous, and lactating primiparous females in four inbred strains of mice (C57BL/6J, C57BL/6JJcl, BALB/cAnNCrlCrlj, and BALB/cAJcl). Pups from the nonlactating female group were removed one day after parturition to examine the effects of physical interaction with pups on the postpartum behaviors. Second, we investigated the additional effects of prepregnancy stress (restraint stress for 6 h/day for 21 days) on postpartum behaviors in the BALB/cAJcl strain. We found that females of the two BALB/c substrains showed decreased locomotor activity and increased anxiety-like and depression-related behaviors compared with females of the two C57BL/6 substrains. Behavioral differences were also observed between the two substrains of each strain. Additionally, pregnancy- and lactation-dependent behavioral differences were found in some strains: lactating BALB/cAJcl females traveled shorter distance than the females of the other reproductive state groups, while nonlactating and lactating BALB/cAJcl and C57BL/6J females showed increased depression-related behavior compared with nulliparous females. Lactating BALB/cAJcl and C57BL/6JJcl females exhibited decreased sucrose preference or anhedonia-like behavior compared with nulliparous and nonlactating females, although these results did not reach statistical significance after correction for multiple testing. An additional independent experiment replicated the marked behavioral changes in lactating BALB/cAJcl females. Moreover, increased anxiety-like behavior was observed in lactating BALB/cAJcl females that experienced prepregnancy stress. These results suggest genetic contributions to the regulation of anxiety-like and depression-related behaviors in female mice. Furthermore, this study suggests that pregnancy and lactation cause decreased locomotor activity and increased depression-related behaviors, which was consistently found in our results, and that prepregnancy stress enhances anxiety-like behavior in the BALB/cAJcl strain. The inbred strain of female mice may be used as a potential model of PPD to further study the genetic and neurobiological mechanisms underlying the development of this disorder.

Abstract Aim Aging is thought to coincide with gradual and progressive changes in brain function and behavior over the lifetime. Our previous meta‐analytic study reported age‐related behavioral changes from young to middle age in male C57BL/6J mice. However, the previous study had some limitations that could affect the generalizability of the findings because of the potential influence of genetic and environmental factors on behavior, in addition to a lack of information regarding the behaviors of old‐aged mice. Here, to investigate age‐related behavioral changes from young to old age in mice, we analyzed the behaviors of male C57BL/6J mice from four different age groups (8, 47, 73, and 99 weeks of age at the beginning of the experiment) from a colony that had been maintained in a genetically controlled condition based on The Jackson Laboratory's Genetic Stability Program in an environmentally controlled animal facility. Methods We used a battery of behavioral tests, including the light/dark transition, open field, elevated plus maze, hot plate, social interaction, rotarod, three‐chamber social approach, prepulse inhibition, Porsolt forced swim, T‐maze, Barnes maze, tail suspension, and fear‐conditioning tests. Results Some behavioral changes occurred between young and middle age, and further changes in various behaviors were observed in old age. Decreased locomotor activity and increased anxiety‐like behavior were found in old‐aged mice compared to those in young and middle‐aged mice in the light/dark transition test. Similarly, an age‐dependent decrease in locomotor activity was observed in the open field test and the elevated plus maze test, while there was an age‐dependent increase in the time spent in the center area in the open field test and there were no significant differences among age groups in behavioral measures of anxiety in the elevated plus maze test. Decreases in motor performance and the auditory startle response were found in middle‐aged mice compared to those in young mice. Similar behavioral changes and increased pain sensitivity, decreased social novelty preference, reduced working and spatial memory, and impaired cued fear memory were observed in old‐aged mice compared to those in young mice. Prepulse inhibition was higher in middle‐aged mice than in young and old‐aged mice. Age‐related changes in depression‐related behavior were dependent on the type of test and the test time period. Conclusions This study generally confirmed our previous report regarding age‐related behavioral changes from young to middle age and expanded the previous observations by examining the behaviors of old‐aged mice. Our results show age‐related changes in a wide range of behaviors in mice from young to old age. Most behaviors showed gradual changes with advancing age, but some types of behaviors, such as vertical activity, prepulse inhibition, and depression‐related behavior, did not show unidirectional changes with age. These findings provide basic information about the behavioral characteristics of young, middle‐aged, and aged male C57BL/6J mice.

Abstract Aims The molecular and cellular mechanisms underlying the antidepressant effects of fluoxetine in the brain are not fully understood. Emerging evidence has led to the hypothesis that chronic fluoxetine treatment induces dematuration of certain types of mature neurons in rodents. These studies have focused on the properties of typical molecular and/or electrophysiological markers for neuronal maturation. Nevertheless, it remains unknown whether dematuration‐related phenomena are present at the genome‐wide gene expression level. Methods Based on the aforementioned hypothesis, we directly compared transcriptome data between fluoxetine‐treated adult mice and those of naive infants in the hippocampus and medial prefrontal cortex (mPFC) to assess similarities and/or differences. We further investigated whether fluoxetine treatment caused dematuration in these brain regions in a hypothesis‐free manner using a weighted gene co‐expression network analysis (WGCNA). Results Gene expression patterns in fluoxetine‐treated mice resembled those in infants in the mPFC and, to a large extent, in the hippocampus. The gene expression patterns of fluoxetine‐treated adult mice were more similar to those of approximately 2‐week‐old infants than those of older mice. WGCNA confirmed that fluoxetine treatment was associated with maturation abnormalities, particularly in the hippocampus, and highlighted respective co‐expression modules for maturity and immaturity marker genes in the hippocampus in response to fluoxetine treatment. Conclusions Our results strongly support the hypothesis that chronic fluoxetine treatment induces dematuration in the adult mouse brain from a transcriptomic standpoint. Detection of discrete transcriptomic regulatory networks related to fluoxetine treatment may help to further elucidate the mechanisms of antidepressant action.

Abstract Aim Tryptophan hydroxylase 2 (Tph2) is a rate‐limiting enzyme for the biosynthesis of 5‐hydroxytryptamine (5‐HT, serotonin). Previous studies have reported that C1473G polymorphism of the murine Tph2 gene leads to decreased 5‐HT levels in the brain and abnormal behavioral phenotypes, such as impaired anxiety‐ and depression‐like behaviors. In this study, to confirm the effect of the C1473G polymorphism on mouse phenotypes, we conducted a comprehensive battery of behavioral tests and measured the amounts of brain free amino acids involved in the production of 5‐HT. Methods We obtained C57BL/6J congenic mice that were homozygous for the 1473G allele of Tph2 (1473G) and subjected them and their wild‐type littermates (1473C) to a battery of behavioral tests. Using reverse‐phase high‐performance liquid chromatography (HPLC), we measured the amounts of free amino acids in the 5‐HT and epinephrine synthetic/metabolic pathways in the frontal cortex, hippocampus, striatum, and midbrain. Results We failed to detect significant differences between genotypes in depression‐like behaviors, anxiety‐like behaviors, social behaviors, sensorimotor gaiting, or learning and memory, while 1473G mice exhibited a nominally significant impairment in gait analysis, which failed to reach study‐wide significance. In the HPLC analysis, there were no significant differences in the amounts of 5‐HT, dopamine, norepinephrine, and epinephrine in the frontal cortex, hippocampus, striatum, and midbrain. Conclusion Our findings do not support the idea that congenic C57BL/6J mice carrying the 1473G allele may represent an animal model of mood disorder under normal conditions without stress.

Abstract Background Mutations of the SCN2A gene encoding a voltage-gated sodium channel alpha-II subunit Nav1.2 are associated with neurological disorders such as epilepsy, autism spectrum disorders, intellectual disability, and schizophrenia. However, causal relationships and pathogenic mechanisms underlying these neurological defects, especially social and psychiatric features, remain to be elucidated. Methods We investigated the behavior of mice with a conventional or conditional deletion of Scn2a in a comprehensive test battery including open field, elevated plus maze, light-dark box, three chambers, social dominance tube, resident-intruder, ultrasonic vocalization, and fear conditioning tests. We further monitored the effects of the positive allosteric modulator of AMPA receptors CX516 on these model mice. Results Conventional heterozygous Scn2a knockout mice (Scn2a KO/+) displayed novelty-induced exploratory hyperactivity and increased rearing. The increased vertical activity was reproduced by heterozygous inactivation of Scn2a in dorsal-telencephalic excitatory neurons but not in inhibitory neurons. Moreover, these phenotypes were rescued by treating Scn2a KO/+ mice with CX516. Additionally, Scn2a KO/+ mice displayed mild social behavior impairment, enhanced fear conditioning, and deficient fear extinction. Neuronal activity was intensified in the medial prefrontal cortex of Scn2a KO/+ mice, with an increase in the gamma band. Conclusions Scn2a KO/+ mice exhibit a spectrum of phenotypes commonly observed in models of schizophrenia and autism spectrum disorder. Treatment with the CX516 ampakine, which ameliorates hyperactivity in these mice, could be a potential therapeutic strategy to rescue some of the disease phenotypes.

Summary: Haploinsufficiency of SETD5 is implicated in syndromic autism spectrum disorder (ASD), but the molecular mechanism underlying the pathological role of this protein has remained unclear. We have now shown that Setd5+/– mice manifest ASD-related behavioral phenotypes and that the expression of ribosomal protein genes and rDNA is disturbed in the brain of these mice. SETD5 recruited the HDAC3 complex to the rDNA promoter, resulting in removal of the histone mark H4K16ac and its reader protein TIP5, a repressor of rDNA expression. Depletion of SETD5 attenuated rDNA expression, translational activity, and neural cell proliferation, whereas ablation of TIP5 in SETD5-deficient cells rescued these effects. Translation of cyclin D1 mRNA was specifically down-regulated in SETD5-insufficient cells. Our results thus suggest that SETD5 positively regulates rDNA expression via an HDAC3-mediated epigenetic mechanism and that such regulation is essential for translation of cyclin D1 mRNA and neural cell proliferation. : Behavior Genetics; Molecular Genetics; Behavioral Neuroscience; Molecular Neuroscience Subject Areas: Behavior Genetics, Molecular Genetics, Behavioral Neuroscience, Molecular Neuroscience

Abstract The dentate gyrus of the hippocampus has been implicated in the pathophysiological basis of neuropsychiatric disorders including schizophrenia. We have identified several mouse models of neuropsychiatric disorders with robust molecular and functional defects in the dentate gyrus. Among them, mice lacking Schnurri-2 (Shn2 or HIVEP2) have been proposed as a model of schizophrenia and intellectual disability. Shn2 knockout mice exhibit behavioral abnormalities resembling symptoms of schizophrenia and HIVEP2-related intellectual disability as well as marked functional alterations in the soma and output synapse of the dentate granule cells (GCs). Although robust abnormalities were also observed in the dendritic spine morphology in the GCs, their functional correlates remain unknown. In the present study, we performed electrophysiological analyses of synaptic transmission at the medial perforant path (MPP) input onto the GCs in Shn2 knockout mice. While the basal synaptic efficacy was preserved, short-term synaptic depression induced by paired-pulse or low-frequency stimulation was reduced in the mutant mice. High-frequency tetanic stimulation induced lasting synaptic potentiation in both wild-type and mutant mice. However, the decaying synaptic potentiation shortly after the tetanic stimulation was significantly reduced in the mutant mice. These results indicate that the Shn2 deficiency attenuates bidirectional short-term synaptic plasticity at the MPP-GC synapse, thereby rendering the synapse more static. Our finding further supports a possible role of the dentate gyrus dysfunction in pathophysiology of schizophrenia and may also provide important information in interpreting morphology changes of the brain synapses in neuropsychiatric disorders.

Abstract Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.

Abstract Aim Indoleamine 2,3‐dioxygenase 1 (IDO1) metabolizes the essential amino acid tryptophan into kynurenine derivatives, which are involved in neural activity via the kynurenine pathway (KP). IDO1 is an initial rate‐limiting enzyme in the KP and is activated by stress and/or inflammation. The perturbation of IDO1 activity, which causes KP imbalance, is associated with psychiatric and neurological disorders. It has been reported that wild‐type (WT) mice under inflammatory conditions show increased anxiety‐like behavior and decreased novel object recognition, whereas Ido1 knockout (KO) mice do not display these behaviors. However, the behavioral phenotypes of Ido1 KO mice have not yet been fully examined under non‐inflammatory conditions. Methods We subjected Ido1 KO mice to a comprehensive behavioral test battery under normal conditions. Results Ido1 KO mice and WT mice showed similar locomotor activity, anxiety‐like behavior, social behavior, depression‐like behavior, and fear memory. In the T‐maze test, Ido1 KO mice exhibited weak but nominally significant impairment in the working memory task of the T‐maze, but this result failed to reach study‐wide significance. Conclusions Ido1 KO mice did not show any clear behavioral abnormalities under normal conditions. Further studies may be necessary to investigate their behavioral phenotype under inflammatory conditions due to their known roles in inflammation.

Abstract Aim Maturation abnormalities of the brain cells have been suggested in several neuropsychiatric disorders, including schizophrenia, bipolar disorder, autism spectrum disorders, and epilepsy. In this study, we examined the expression patterns of neuronal maturation markers in the brain of a mouse model of dementia with Lewy body-linked mutant β-synuclein (βS), especially in the hippocampus, to explore whether such brain abnormalities occur in neurodegenerative disorders as well. Methods Quantitative PCR (qPCR) and immunohistochemical analyses were performed using the hippocampus of 14-month-old P123H βS transgenic (Tg) mice to evaluate the expression of molecular markers for maturation of dentate granule cells. Results Based on qPCR results, expression of Tdo2 and Dsp (markers of mature granule cells) was decreased and that of Drd1a (a marker of immature granule cells) was increased in the hippocampus of P123H βS Tg mice compared to that in wild-type controls. Immunohistochemical analysis revealed decreased expression of mature granule cell markers Calb1 and Gria1, along with increased expression of the microglial marker Iba1, in the hippocampal dentate gyrus region of P123H βS Tg mice. P123H βS Tg mice exhibited immature-like neuronal molecular expression patterns and microgliosis in the hippocampus. Pseudo-immaturity of dentate granule cells, associated with neuroinflammation, may be a shared endophenotype in the brains of at least a subgroup of patients with neuropsychiatric disorders and neurodegenerative diseases.

Abstract Prepulse inhibition (PPI) is the suppression of a startle reflex response to a startle stimulus that occurs when a weak prepulse stimulus precedes the startle stimulus. PPI is measured to assess sensorimotor gating across species, including humans and rodents. Reduced PPI, which is thought to reflect dysfunction of sensorimotor gating, is reported in patients with psychiatric disorders, such as schizophrenia, bipolar disorder, and post-traumatic stress disorder (PTSD), and in animal models of these disorders. Individual differences in basal startle reactivity occur even in a genetically homogenous group of animals; however, there is limited information regarding whether basal levels of the startle response are associated with variations in PPI levels. Here, to explore the relationship between an acoustic startle response (ASR) and PPI, we performed a meta-analysis of data obtained from more than 1300 C57BL/6J male mice on the influence of an ASR to 110- and 120-dB startle stimuli on the PPI levels of the ASR at 74- and 78-dB prepulse intensities. Examination of scatter plots of the ASR amplitudes and PPI levels followed by correlation analyses indicated that there is no simple linear relationship between the two measures; when mice were divided into three groups on the basis of their startle amplitudes, there were positive correlations between the amplitude of the ASR to the 110-dB stimulus and PPI levels in a group of mice that showed lower ASR amplitudes among the genetically homogenous group, whereas no significant correlations were identified in groups of mice that showed intermediate and higher ASR amplitudes. As indicated by the correlation analysis, the lowest responders to the 110-dB stimulus exhibited lower levels of PPI than the intermediate or higher responders. In contrast, for the 120-dB stimulus, a negative correlation was identified between the amplitude of the ASR to the 120-dB stimulus and the PPI levels in the groups of mice that showed intermediate or higher ASR amplitudes. Lower and intermediate responders showed higher levels of PPI than higher responders to the 120-dB stimulus. These findings suggest that basal startle reactivity may affect PPI levels in male C57BL/6J mice, thus representing one potential confounding factor that may confuse the interpretation of PPI results. These findings emphasize the importance of careful examination of startle reactivity to ensure a reliable assessment of PPI.

Abstract Aims Tryptophan 2,3‐dioxygenase (TDO2) is an initial rate‐limiting enzyme of the kynurenine (Kyn) pathway in tryptophan (Trp) metabolism. The Trp‐degrading enzymes, TDO2 and indoleamine 2,3‐dioxygenase, are activated by stress and/or inflammation. Dysregulation of Trp metabolism, which causes shifts in the balance between Kyn and serotonin (5‐HT) pathways, is associated with psychiatric and neurological disorders. In genetic studies, single‐nucleotide polymorphisms in the TDO2 gene were shown to be involved in psychiatric disorders, such as schizophrenia and depression. It has been reported that targeted deletion of the Tdo2 gene in mice resulted in reduced anxiety‐like behavior, enhanced exploratory activity and cognitive performance, and increased levels of Trp and 5‐HT in the hippocampus and midbrain. However, the effect of Tdo2 gene deletion on behavioral phenotypes has not yet been investigated extensively. Materials & Methods We conducted tests to further examine the behavioral effects of knockout (KO) of Tdo2 in mice. Results Deletion of Tdo2 resulted in seemingly lower anxiety‐like behavior, higher locomotor activity, and abnormal gait pattern in mice, though none of them reached study‐wide statistical significance. Tdo2 deficiency had no significant effects on other behaviors, such as prepulse inhibition, and depression‐like and social behaviors. Discussion and Conclusion He lack of clear phenotypes in Tdo2KO mice in this study might be due to the absence of stress and inflammatory conditions, which could induce expression of Tdo2 mRNA. Further studies are necessary to elucidate the roles of Tdo2 in behavioral phenotypes related to psychiatric disorders.

Abstract Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

Abstract Background Aluminum (Al) is considered to be a neurotoxic metal, and excessive exposure to Al has been reported to be a potential risk factor for neurodegenerative diseases. Al ammonium sulfate is one of the Al compounds that is widely used as a food additive. However, the effects of the oral administration of Al ammonium sulfate on physical development and behavior remain to be examined. Methods In this study, we investigated the effects of the administration of Al ammonium sulfate 12‐water dissolved in drinking water (0.075 mg/mL) beginning in adolescence on various types of behavior in adult female C57BL/6J mice through a battery of behavioral tests (low‐dose experiment; Experiment 1). We further examined the behavioral effects of the oral administration of a higher dose of the Al compound in drinking water (1 mg/mL) beginning in the prenatal period on behavior in adult male and female mice (high‐dose experiment; Experiment 2). Results In the low‐dose experiment, in which females’ oral intake of Al was estimated to be 0.97 mg Al/kg/d as adults, Al‐treated females exhibited an increase in total arm entries in the elevated plus maze test, an initial decrease and subsequent increase in immobility in the forced swim test, and reduced freezing in the fear conditioning test approximately 1 month after the conditioning session compared with vehicle‐treated females (uncorrected P < .05). However, the behavioral differences did not reach a statistically significant level after correction for multiple testing. In the high‐dose experiment, in which animals’ oral intakes were estimated to be about ten times higher than those in the low‐dose experiment, behavioral differences found in the low‐dose experiment were not observed in high‐dose Al‐treated mice, suggesting that the results of the low‐dose experiment might be false positives. Additionally, although high‐dose Al‐treated females exhibited increased social contacts with unfamiliar conspecifics and impaired reference memory performance, and high‐dose Al‐treated mice exhibited decreases in prepulse inhibition and in correct responses in the working memory task (uncorrected P < .05), the differences in any of the behavioral measures did not reach the significance level after correction for multiple testing. Conclusion Our results show that long‐term oral exposure to Al ammonium sulfate at the doses used in this study may have the potential to induce some behavioral changes in C57BL/6J mice. However, the behavioral effects of Al were small and statistically weak, as indicated by the fact that the results failed to reach the study‐wide significance level. Thus, further study will be needed to replicate the results and reevaluate the behavioral outcomes of oral intake of Al ammonium sulfate.

Abstract Aim We have conducted a series of behavioral tests, which cover a broad range of behavioral domains, on various strains of genetically engineered mice. For the behavioral screening, we have been using Image J plugins that we developed for most of the tests in the battery. Our behavioral analysis system with the plugins enables systematic and automated image analysis of behavior. The plugins are freely available on the “Mouse Phenotype Database” website (http://www.mouse-phenotype.org/software.html). Here, we release the source code of the plugins in a Git repository with the aim of promoting their use and expanding their functionality. Methods We published the source code of the Image J plugins for behavioral analysis at Git repository (https://github.com/neuroinformatics). The source code for light/dark transition, elevated plus maze, open filed, T‐maze, and fear conditioning tests was made publicly available in the repository. Conclusions The source code of the plugins for the behavioral tests as well as the pre‐compiled binaries can be freely obtained. The open source code could promote the development and modification of the plugins for additional behavioral indices in these tests and for other behavioral tests.

Abstract Accumulating evidence suggests that subcellular-scale structures such as dendritic spine and mitochondria may be involved in the pathogenesis/pathophysiology of schizophrenia and intellectual disability. Previously, we proposed mice lacking Schnurri-2 (Shn2; also called major histocompatibility complex [MHC]-binding protein 2 [MBP-2], or human immunodeficiency virus type I enhancer binding protein 2 [HIVEP2]) as a schizophrenia and intellectual disability model with mild chronic inflammation. In the mutants’ brains, there are increases in C4b and C1q genes, which are considered to mediate synapse elimination during postnatal development. However, morphological properties of subcellular-scale structures such as dendritic spine in Shn2 knockout (KO) mice remain unknown. In this study, we conducted three-dimensional morphological analyses in subcellular-scale structures in dentate gyrus granule cells of Shn2 KO mice by serial block-face scanning electron microscopy. Shn2 KO mice showed immature dendritic spine morphology characterized by increases in spine length and decreases in spine diameter. There was a non-significant tendency toward decrease in spine density of Shn2 KO mice over wild-type mice, and spine volume was indistinguishable between genotypes. Shn2 KO mice exhibited a significant reduction in GluR1 expression and a nominally significant decrease in SV2 expression, while PSD95 expression had a non-significant tendency to decrease in Shn2 KO mice. There were significant decreases in dendrite diameter, nuclear volume, and the number of constricted mitochondria in the mutants. Additionally, neuronal density was elevated in Shn2 KO mice. These results suggest that Shn2 KO mice serve as a unique tool for investigating morphological abnormalities of subcellular-scale structures in schizophrenia, intellectual disability, and its related disorders.