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Social behavior, defined as any mode of communication between conspecifics is regulated by a widespread network comprising multiple brain structures. The anterior cingulate cortex (ACC) serves as a hub region interconnected with several brain regions involved in social behavior. Because the ACC coordinates various behaviors, it is important to focus on a subpopulation of neurons that are potentially involved in social behavior to clarify the precise role of the ACC in social behavior. In this study, we aimed to analyze the roles of a social stimulus-responsive subpopulation of neurons in the ACC in social behavior in mice. We demonstrated that a subpopulation of neurons in the ACC was activated by social stimuli and that silencing the social stimulus-responsive subpopulation of neurons in the ACC significantly impaired social interaction without affecting locomotor activity or anxiety-like behavior. Our current findings highlight the importance of the social stimulus-responsive subpopulation of neurons in the ACC for social behavior and the association between ACC dysfunction and impaired social behavior, which sheds light on therapeutic interventions for psychiatric conditions., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adeno-associated virus (AAV) vectors are potential tools for cell-type-selective gene delivery to the central nervous system. Although cell-type-specific enhancers and promoters have been identified for AAV systems, there is limited information regarding the effects of AAV genomic components on the selectivity and efficiency of gene expression. Here, we offer an alternative strategy to provide specific and efficient gene delivery to a targeted neuronal population by optimizing recombinant AAV genomic components, named TAREGET (TransActivator-Regulated Enhanced Gene Expression within Targeted neuronal populations). We established this strategy in oxytocinergic neurons and showed that the TAREGET enabled sufficient gene expression to label long-projecting axons in wild-type mice. Its application to other cell types, including serotonergic and dopaminergic neurons, was also demonstrated. These results demonstrate that optimization of AAV expression cassettes can improve the specificity and efficiency of cell-type-specific gene expression and that TAREGET can renew previously established cell-type-specific promoters with improved performance., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Chronic social isolation increases the risk of mental health problems, including cognitive impairments and depression. While subanesthetic ketamine is considered effective for cognitive impairments in patients with depression, the neural mechanisms underlying its effects are not well understood. Here we identified unique activation of the anterior insular cortex (aIC) as a characteristic feature in brain-wide regions of mice reared in social isolation and treated with (R)-ketamine, a ketamine enantiomer. Using fiber photometry recording on freely moving mice, we found that social isolation attenuates aIC neuronal activation upon social contact and that (R)-ketamine, but not (S)-ketamine, is able to counteracts this reduction. (R)-ketamine facilitated social cognition in social isolation-reared mice during the social memory test. aIC inactivation offset the effect of (R)-ketamine on social memory. Our results suggest that (R)-ketamine has promising potential as an effective intervention for social cognitive deficits by restoring aIC function., (© 2024. The Author(s).)

Alternatives to ketamine without psychotomimetic properties for the treatment of depression have attracted much attention. Here, we examined the anti-despair and anti-anhedonia effects of the ketamine metabolites (S)-norketamine ((S)-NK), (R)-NK, (2S,6S)-hydroxynorketamine, and (2R,6R)-hydroxynorketamine in a mouse model of depression induced by social isolation. All ketamine metabolites examined had acute (30 min after administration) anti-despair-like effects in the forced swim test, but only (S)-NK showed a long-lasting (1 week) effect. Additionally, only (S)-NK improved reduced motivation both 30 min and 24 h after injection in the female encounter test. These results suggest that (S)-NK has potent and long-lasting antidepressant-like effects., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

The medial prefrontal cortex (mPFC) is associated with various behavioral controls via diverse projections to cortical and subcortical areas of the brain. Dysfunctions and modulations of this circuitry are related to the pathophysiology of schizophrenia and its pharmacotherapy, respectively. Clozapine is an atypical antipsychotic drug used for treatment-resistant schizophrenia and is known to modulate neuronal activity in the mPFC. However, it remains unclear which prefrontal cortical projections are activated by clozapine among the various projection targets. To identify the anatomical characteristics of neurons activated by clozapine at the mesoscale level, we investigated the brain-wide projection patterns of neurons with clozapine-induced c-Fos expression in the mPFC. Using a whole-brain imaging and virus-mediated genetic tagging of activated neurons, we found that clozapine-responsive neurons in the mPFC had a wide range of projections to the mesolimbic, amygdala and thalamic areas, especially the mediodorsal thalamus. These results may provide key insights into the neuronal basis of the therapeutic action of clozapine.

Major depressive disorder (MDD) is among the most common mental illnesses. Serotonergic (5-HT) neurons are central to the pathophysiology and treatment of MDD. Repeatedly recalling positive episodes is effective for MDD. Stimulating 5-HT neurons of the dorsal raphe nucleus (DRN) or neuronal ensembles in the dorsal dentate gyrus (dDG) associated with positive memories reverses the stress-induced behavioral abnormalities. Despite this phenotypic similarity, their causal relationship is unclear. This study revealed that the DRN 5-HT neurons activate dDG neurons; surprisingly, this activation was specifically observed in positive memory ensembles rather than neutral or negative ensembles. Furthermore, we revealed that dopaminergic signaling induced by activation of DRN 5-HT neurons projecting to the ventral tegmental area mediates an increase in active coping behavior and positive dDG ensemble reactivation. Our study identifies a role of DRN 5-HT neurons as specific reactivators of positive memories and provides insights into how serotonin elicits antidepressive effects., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

We recently reported that a neuronal population in the claustrum (CLA) identified under exposure to psychological stressors plays a key role in stress response processing. Upon stress exposure, the main inputs to the CLA come from the basolateral amygdala (BLA); however, the upstream brain regions that potentially regulate both the CLA and BLA during stressful experiences remain unclear. Here by combining activity-dependent viral retrograde labeling with whole brain imaging, we analyzed neurons projecting to the CLA and BLA activated by exposure to social defeat stress. The labeled CLA projecting neurons were mostly ipsilateral, excluding the prefrontal cortices, which had a distinctly labeled population in the contralateral hemisphere. Similarly, the labeled BLA projecting neurons were predominantly ipsilateral, aside from the BLA in the opposite hemisphere, which also had a notably labeled population. Moreover, we found co-labeled double-projecting single neurons in multiple brain regions such as the ipsilateral ectorhinal/perirhinal cortex, entorhinal cortex, and the contralateral BLA. These results suggest that CLA and BLA receive inputs from neuron collaterals in various brain regions during stress, which may regulate the CLA and BLA forming in a stress response circuitry., (© 2022. The Author(s).)

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by specific social symptoms, restricted interests, stereotyped repetitive behaviors, and delayed language development. The 3q29 microdeletion (3q29del), a recurrent copy number variant, confers a high risk for ASD and schizophrenia, and serves as an important pathological model for investigating the molecular pathogenesis of a large number of neurodevelopmental and psychiatric conditions. Recently, mouse models carrying a deletion of the chromosomal region corresponding to the human 3q29 region (Df/+ mice) were generated and demonstrated neurodevelopmental and psychiatric conditions associated behavioral abnormalities, pointing to the relevance of Df/+ mice as a model for these conditions with high construct and face validity. Currently, the molecular pathogenesis of these behavioral phenotypes in Df/+ mice remains unclear. The oxytocin (OXT) system plays a central role in social behavior across species and has a potential role in ASD. In this study, to elucidate the molecular mechanisms behind impaired social behavior in Df/+ mice, we investigated the possible involvement of OXT signaling in impaired social behavior in Df/+ mice. We demonstrated that OXT administration restored the impaired social behavior in Df/+ mice. We also demonstrated that the number of OXT-positive cells in the paraventricular nucleus (PVN) was significantly lower in Df/+ mice than in wild-type (WT) littermates. Consistent with this, the level of OXT peptide in the cerebral cortex of Df/+ mice was lower than in WT littermates. Our study may provide important insights into the molecular pathophysiological basis of neurodevelopmental and psychiatric conditions, including ASD., (© 2022. The Author(s).)

The processing of stress responses involves brain-wide communication among cortical and subcortical regions; however, the underlying mechanisms remain elusive. Here, we show that the claustrum (CLA) is crucial for the control of stress-induced anxiety-related behaviors. A combined approach using brain activation mapping and machine learning showed that the CLA activation serves as a reliable marker of exposure to acute stressors. In TRAP2 mice, which allow activity-dependent genetic labeling, chemogenetic activation of the CLA neuronal ensemble tagged by acute social defeat stress (DS) elicited anxiety-related behaviors, whereas silencing of the CLA ensemble attenuated DS-induced anxiety-related behaviors. Moreover, the CLA received strong input from DS-activated basolateral amygdala neurons, and its circuit-selective optogenetic photostimulation temporarily elicited anxiety-related behaviors. Last, silencing of the CLA ensemble during stress exposure increased resistance to chronic DS. The CLA thus bidirectionally controls stress-induced emotional responses, and its inactivation can serve as a preventative strategy to increase stress resilience.

An increasing body of evidence suggests that impaired synapse development and function are associated with schizophrenia; however, the underlying molecular pathophysiological mechanism of the disease remains largely unclear. We conducted a family-based study combined with molecular and cellular analysis using induced pluripotent stem cell (iPSC) technology. We generated iPSCs from patients with familial schizophrenia, differentiated these cells into neurons, and investigated the molecular and cellular phenotypes of the patient's neurons. We identified multiple altered synaptic functions, including increased glutamatergic synaptic transmission, higher synaptic density, and altered splicing of dopamine D2 receptor mRNA in iPSC-derived neurons from patients. We also identified patients' specific genetic mutations using whole-exome sequencing. Our findings support the notion that altered synaptic function may underlie the molecular and cellular pathophysiology of schizophrenia, and that multiple genetic factors cooperatively contribute to the development of schizophrenia., (© 2021. The Author(s).)

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterized by core symptoms of impaired social behavior and communication. Recent studies have suggested that the oxytocin system, which regulates social behavior in mammals, is potentially involved in ASD. Mouse models of ASD provide a useful system for understanding the associations between an impaired oxytocin system and social behavior deficits. However, limited studies have shown the involvement of the oxytocin system in the behavioral phenotypes in mouse models of ASD. We have previously demonstrated that a mouse model that carries the ASD patient-derived de novo mutation in the pogo transposable element derived with zinc finger domain (POGZ WT/Q1038R mice), showed ASD-like social behavioral deficits. Here, we have explored whether oxytocin (OXT) administration improves impaired social behavior in POGZ WT/Q1038R mice and found that intranasal oxytocin administration effectively restored the impaired social behavior in POGZ WT/Q1038R mice. We also found that the expression level of the oxytocin receptor gene (OXTR) was low in POGZ WT/Q1038R mice. However, we did not detect significant changes in the number of OXT-expressing neurons between the paraventricular nucleus of POGZ WT/Q1038R mice and that of WT mice. A chromatin immunoprecipitation assay revealed that POGZ binds to the promoter region of OXTR and is involved in the transcriptional regulation of OXTR. In summary, our study demonstrate that the pathogenic mutation in the POGZ, a high-confidence ASD gene, impairs the oxytocin system and social behavior in mice, providing insights into the development of oxytocin-based therapeutics for ASD.

The dopamine system plays an important role in regulating many brain functions, including the motor function. The blockade of dopamine receptors results in a serious motor dysfunction, such as catalepsy and Parkinsonism. However, the neuronal mechanism underlying the drug-induced motor dysfunction is not well understood. Here, we examine brain-wide activation patterns in Fos-enhanced green fluorescent protein reporter mice that exhibit cataleptic behavior induced by SCH39166, a dopamine D1-like receptor antagonist, and raclopride, a dopamine D2-like receptor antagonist. Support vector classifications showed that the orbital cortex (ORB) and striatum including the caudoputamen (CP) and nucleus accumbens (ACB), prominently contribute to the discrimination between brains of the vehicle-treated and both SCH39166- and raclopride-treated mice. Interregional correlations indicated that the increased functional connectivity of functional networks, including the ORB, CP, and ACB, is the common mechanism underlying SCH39166- and raclopride-induced cataleptic behavior. Moreover, the distinct mechanisms in the SCH39166- and raclopride-induced cataleptic behaviors are the decreased functional connectivity between three areas above and the cortical amygdala, and between three areas above and the anterior cingulate cortex, respectively. Thus, the alterations of functional connectivity in diverse brain regions, including the ORB, provide new insights on the mechanism underlying drug-induced movement disorders.

Clinical and preclinical studies have shown that the N-methyl-d-aspartate receptor antagonist ketamine exerts rapid and long-lasting antidepressant effects. Although ketamine metabolites might also have potential antidepressant properties, controversial results have been reported for (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) in particular, and there is little information regarding the effects of other ketamine metabolites. Here we aimed to compare the effects of (R)-norketamine ((R)-NK), (S)-NK, (2R,6R)-HNK, and (2S,6S)-HNK in a mouse model of depression induced by chronic corticosterone (CORT) injection. None of the ketamine metabolites at doses up to 20 mg/kg showed antidepressant-like activity in naïve male C57BL6/J mice. Chronic CORT treatment increased immobility in the forced swim test and caused anhedonic-like behaviors in the female encounter test. A single administration of (S)-NK and (2S,6S)-HNK dose-dependently reduced the enhanced immobility at 30 min after injection in chronic CORT-treated mice, while (R)-NK or (2R,6R)-HNK did not. Additionally, (S)-NK and (2S,6S)-HNK, but not (R)-NK or (2R,6R)-HNK, improved chronic CORT-induced anhedonia at 24 h after the injection. These results suggest that (S)-ketamine metabolites (S)-NK and (2S,6S)-HNK have potent acute and sustained antidepressant effects in rodents., Competing Interests: Declaration of competing interest Dr. Kenji Hashimoto is the inventor of filed patent applications ‘The use of (R)-ketamine in the treatment of psychiatric diseases’ and ‘(S)-norketamine and a salt thereof as pharmaceutical’ by Chiba University. Dr. Kenji Hashimoto also declares that he has received research support and consultant fees from Sumitomo Dainippon Pharma Co., Ltd., Otsuka Pharmaceutical Co., Ltd., and Taisho Pharmaceutical Co., Ltd. The other authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Detailed spatial information of low-molecular weight compound distribution, especially in the brain, is crucial to understanding their mechanism of actions. Imaging techniques that can directly visualize drugs in the brain at a high resolution will complement existing tools for drug distribution analysis. Here, we performed surface-enhanced Raman scattering (SERS) imaging using a bioorthogonal alkyne tag to visualize drugs directly in situ at a high resolution. Focusing on the selective serotonin reuptake inhibitor S-citalopram (S-Cit), which possesses a nitrile group, we substituted an alkynyl group into its structure and synthesized alkynylated S-Cit (Alk-S-Cit). The brain transitivity and the serotonin reuptake inhibition of Alk-S-Cit were not significantly different as compared with S-Cit. Alk-S-Cit was visualized in the coronal mouse brain section using SERS imaging with silver nanoparticles. Furthermore, SERS imaging combined with fluorescence microscopy allowed Alk-S-Cit to be visualized in the adjacent neuronal membranes, as well as in the brain vessel and parenchyma. Therefore, our multimodal imaging technique is an effective method for detecting low-molecular weight compounds in their original tissue environment and can potentially offer additional information regarding the precise spatial distribution of such drugs.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder, characterized by impaired social interaction, repetitive behavior and restricted interests. Although the molecular etiology of ASD remains largely unknown, recent studies have suggested that de novo mutations are significantly involved in the risk of ASD. We and others recently identified spontaneous de novo mutations in PKD2, a protein kinase D family member, in sporadic ASD cases. However, the biological significance of the de novo PKD2 mutations and the role of PKD2 in brain development remain unclear. Here, we performed functional analysis of PKD2 in cortical neuron development using in utero electroporation. PKD2 is highly expressed in cortical neural stem cells in the developing cortex and regulates cortical neuron development, including the neuronal differentiation of neural stem cells and migration of newborn neurons. Importantly, we determined that the ASD-associated de novo mutations impair the kinase activity of PKD2, suggesting that the de novo PKD2 mutations can be a risk factor for the disease by loss of function of PKD2. Our current findings provide novel insight into the molecular and cellular pathogenesis of ASD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

3q29 microdeletion, a rare recurrent copy number variant (CNV), greatly confers an increased risk of psychiatric disorders, such as schizophrenia and autism spectrum disorder (ASD), as well as intellectual disability. However, disease-relevant cellular phenotypes of 3q29 deletion syndrome remain to be identified. To reveal the molecular and cellular etiology of 3q29 deletion syndrome, we generated a mouse model of human 3q29 deletion syndrome by chromosome engineering, which achieved construct validity. 3q29 deletion (Df/+) mice showed reduced body weight and brain volume and, more importantly, impaired social interaction and prepulse inhibition. Importantly, the schizophrenia-related impaired prepulse inhibition was reversed by administration of antipsychotics. These findings are reminiscent of the growth defects and neuropsychiatric behavioral phenotypes in patients with 3q29 deletion syndrome and exemplify that the mouse model achieves some part of face validity and predictive validity. Unbiased whole-brain imaging revealed that neuronal hyperactivation after a behavioral task was strikingly exaggerated in a restricted region of the cortex of Df/+ mice. We further elucidated the cellular phenotypes of neuronal hyperactivation and the reduction of parvalbumin expression in the cortex of Df/+ mice. Thus, the 3q29 mouse model provides invaluable insight into the disease-causative molecular and cellular pathology of psychiatric disorders.

Background: Although recent studies provide insight into the molecular mechanisms of the effects of ketamine, the antidepressant mechanism of ketamine enantiomers and their metabolites is not fully understood. In view of the involvement of mechanisms other than the N-methyl-D-aspartate receptor in ketamine's action, we investigated the effects of (R)-ketamine, (S)-ketamine, (R)-norketamine [(R)-NK], (S)-NK, (2R,6R)-hydroxynorketamine [(2R,6R)-HNK], and (2S,6S)-HNK on monoaminergic neurotransmission in the prefrontal cortex of mice., Methods: The extracellular monoamine levels in the prefrontal cortex were measured by in vivo microdialysis., Results: (R)-Ketamine and (S)-ketamine acutely increased serotonin release in a dose-dependent manner, and the effect of (R)-ketamine was greater than that of (S)-ketamine. In contrast, (S)-ketamine caused a robust increase in dopamine release compared with (R)-ketamine. Both ketamine enantiomers increased noradrenaline release, but these effects did not differ. (2R,6R)-HNK caused a slight but significant increase in serotonin and noradrenaline but not dopamine release. (S)-NK increased dopamine and noradrenaline but not serotonin release. Differential effects between (R)-ketamine and (S)-ketamine were also observed in a lipopolysaccharide-induced model of depression. An α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4- tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), attenuated (S)-ketamine-induced, but not (R)-ketamine-induced serotonin release, whereas NBQX blocked dopamine release induced by both enantiomers. Local application of (R)-ketamine into the prefrontal cortex caused a greater increase in prefrontal serotonin release than that of (S)-ketamine., Conclusions: (R)-Ketamine strongly activates the prefrontal serotonergic system through an AMPA receptor-independent mechanism. (S)-Ketamine-induced serotonin and dopamine release was AMPA receptor-dependent. These findings provide a neurochemical basis for the underlying pharmacological differences between ketamine enantiomers and their metabolites., (© The Author(s) 2019. Published by Oxford University Press on behalf of CINP.)

Alterations in pituitary adenylate cyclase-activating polypeptide (PACAP), a multifunctional neuropeptide, and its receptors have been identified as risk factors for certain psychiatric disorders, including schizophrenia. Increasing evidence from human genetic and animal model studies suggest an association between various psychiatric disorders and altered dendritic spine morphology. In the present study, we investigated the role of exogenous and endogenous PACAP in spine formation and maturation. PACAP modified the density and morphology of PSD-95-positive spines in primary cultured hippocampal neurons. Notably, PACAP increased the levels of microRNA (miR)-132 and decreased expression of corresponding miR-132 target genes and protein expression of p250GAP, a miR-132 effector known to be involved in spine morphology regulation. In corroboration, PSD-95-positive spines were reduced in PACAP-deficient ( PACAP -/- ) mice versus WT mice. Golgi staining of hippocampal CA1 neurons revealed a reduced spine densities and atypical morphologies in the male PACAP -/- mice. Furthermore, viral miR-132 overexpression reversed the reduction in hippocampal spinal density in the male PACAP -/- mice. These results indicate that PACAP signaling plays a critical role in spine morphogenesis possibly via miR-132. We suggest that dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through its effects on spine formation. SIGNIFICANCE STATEMENT Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling dysfunction and dendritic spine morphology alterations have recently been suggested as important pathophysiological mechanisms underlying several psychiatric and neurological disorders. In this study, we investigated whether PACAP regulates dendritic spine morphogenesis. In a combination of pharmacological and viral gain- and loss-of-function approaches in vitro and in vivo experiments, we found PACAP to increase the size and density of dendritic spines via miR-132 upregulation. Together, our data suggest that a dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through abnormal spine formation., (Copyright © 2019 the authors.)

Here, we describe an optimized and detailed protocol for block-face serial microscopy tomography (FAST). FAST enables high-speed serial section fluorescence imaging of fixed brains at an axonal spatial resolution and subsequent image data processing. It renders brain-wide anatomical and functional analyses, including structural profiling of nuclear-stained brain at the single-cell level, cell-type-specific mapping with reporter animal brains and neuronal tracing with anterograde/retrograde labeling. Light-sheet fluorescence microscopy of cleared brains is advantageous in regard to imaging speed, but its spatial resolution is generally limited, whereas the opposite is true for conventional confocal microscopy. FAST offers a solution to overcome these technical limitations. This protocol describes detailed procedures for assembling the FAST hardware, sample preparation, imaging and image processing. A single imaging session takes as little as 2.4 h per mouse brain, and sample preparation requires 1 to several days, depending on pretreatments; however, multiple samples can be prepared simultaneously. We anticipate that FAST will contribute to unbiased and hypothesis-free approaches for a better understanding of brain systems.

Brain functions are performed by highly interconnected neurons distributed across the whole brain. Imaging of the whole brain at subcellular resolution is crucial for precise understanding of the pathological and therapeutic mechanisms of neuropsychiatric disorders; however, microscopic imaging of the whole brain remains a challenge due to the trade-offs between imaging speed and spatial resolution. To overcome this, we have recently developed block-face serial microscopy tomography (FAST), which is a novel serial-section imaging system using high-speed spinning-disk confocal microscopy. FAST enables high-throughput imaging of whole mouse brains (2.4 h per brain at maximum speed) and can be applied to nonhuman primate whole brains and human postmortem brains. Whole-brain neuronal activation mapping using FAST and Arc-dVenus mice reveals differences in brain-wide activation patterns between acute and chronic stress exposure. These applications of FAST are expected to contribute to unbiased and hypothesis-free analyses for understanding the anatomical and functional relationships of the brain underlying disease and pharmacotherapy.

The neuronal activity forms the basis of functional circuits and brain functions. To understand how the brain operates, recording of neural activity at micro-, meso-, and macro-scales is required. Recently, improved optical microscopic technology helps us to develop a whole-brain imaging system at a single-cell resolution. The combination of a whole-brain imaging system and a reporter system of neuronal activation enables a whole-brain mapping of neuronal activity. In this review, we first describe the high-speed and scalable whole-brain imaging system including our recently developed system, named FAST, and then present the instances of whole-brain mapping of neuronal activity and its analytical methods.

A pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, PAC1R, is coupled with multiple signal transduction pathways including stimulation of adenylate cyclase, phospholipase C and extracellular-signal regulated kinase (ERK)1/2. PAC1R has been shown to exert its long-lasting and potent signals via β-arrestin1 and β-arrestin2. However, the precise roles of the two β-arrestin isoforms in PACAP-PAC1R signaling remain unclear. Here we examined the interaction between the two β-arrestin isoforms and PAC1R, β-arrestin-dependent PAC1R subcellular localization and ERK1/2 activation. Upon PACAP stimulation, although PAC1R similarly interacted with β-arrestin1 and β-arrestin2 in HEK293T cells, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained in the cell surface regions in HeLa cells and mouse primary cultured neurons. Silencing of β-arrestin2 blocked PACAP-induced PAC1R internalization and ERK1/2 phosphorylation, but silencing of β-arrestin1 increased ERK1/2 phosphorylation. These results show that β-arrestin1 and β-arrestin2 exert differential actions on PAC1R internalization and PAC1R-dependent ERK1/2 activation, and suggest that the two β-arrestin isoforms may be involved in fine and precise tuning of the PAC1R signaling pathways.

Mitochondrial dysfunction in the nigrostriatal dopaminergic system is a critical hallmark of Parkinson's disease (PD). Mitochondrial toxins produce cellular and behavioural dysfunctions resembling those in patients with PD Causative gene products for familial PD play important roles in mitochondrial function. Therefore, targeting proteins that regulate mitochondrial integrity could provide convincing strategies for PD therapeutics. We have recently identified a novel 13-kDa protein (p13) that may be involved in mitochondrial oxidative phosphorylation. In the current study, we examine the mitochondrial function of p13 and its involvement in PD pathogenesis using mitochondrial toxin-induced PD models. We show that p13 overexpression induces mitochondrial dysfunction and apoptosis. p13 knockdown attenuates toxin-induced mitochondrial dysfunction and apoptosis in dopaminergic SH-SY5Y cells via the regulation of complex I. Importantly, we generate p13 -deficient mice using the CRISPR/Cas9 system and observe that heterozygous p13 knockout prevents toxin-induced motor deficits and the loss of dopaminergic neurons in the substantia nigra. Taken together, our results suggest that manipulating p13 expression may be a promising avenue for therapeutic intervention in PD., (© 2018 The Authors.)

Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glutamatergic N-methyl-d-aspartate (NMDA) receptors play critical roles in several neurological and psychiatric diseases. Blockade by noncompetitive NMDA receptor antagonist leads to psychotomimetic effects; however, the brain regions responsible for the effects are not well understood. Here, we determined the specific brain regions responsive to MK-801, a noncompetitive NMDA receptor antagonist, by mapping Arc expression as an indicator of neuronal activity using Arc::dVenus reporter mice. MK-801 increased dVenus expression predominantly in the orbitofrontal cortex (OFC) and, as expected, induced a marked hyperlocomotion. Local OFC lesions selectively attenuated the early phase (0-30 min) of MK-801-induced hyperlocomotion. Further, clozapine, an atypical antipsychotic, effectively attenuated both the MK-801-induced dVenus expression in the OFC and hyperlocomotion. These results suggest that the OFC may be critically involved in NMDA receptor-mediated psychotic-like behavioral abnormalities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

There is growing evidence and a consensus in the field that most pediatric brain tumors originate from stem cells, of which radial glial cells constitute a subtype. Here we show that orthotopic transplantation of human radial glial (RG) cells to the subventricular zone of the 3rd ventricle--but not to other transplantation sites--of the brain in immunocompromised NOD-SCID mice, gives rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. Importantly, the observed tumorigenic transformation was accompanied by aspects of an epithelial to mesenchymal transition (EMT)-like process. It is also noteworthy that the tumors are highly invasive, and that they effectively recruit mouse endothelial cells for angiogenesis. These results are significant for several reasons. First, they show that malignant transformation of radial glial cells can occur in the absence of specific mutations or inherited genomic alterations. Second, they demonstrate that the same radial glial cells may either give rise to brain tumors or differentiate normally depending upon the microenvironment of the specific region of the brain to which the cells are transplanted. In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal radial glial cells to a malignant phenotype.

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activities including modulation of synaptic plasticity and memory, hippocampal neurogenesis, and neuroprotection, most of which are shared with brain-derived neurotrophic factor (BDNF). Therefore, the aim of this study was to compare morphological effects of PACAP and BDNF on primary cultured hippocampal neurons. At days in vitro (DIV) 3, PACAP increased neurite length and number to similar levels by BDNF, but vasoactive intestinal polypeptide showed much lower effects. In addition, PACAP increased axon, but not dendrite, length, and soma size at DIV 3 similarly to BDNF. The PACAP antagonist PACAP6-38 completely blocked the PACAP-induced increase in axon, but not dendrite, length. Interestingly, the BDNF-induced increase in axon length was also inhibited by PACAP6-38, suggesting a mechanism involving PACAP signaling. K252a, a TrkB receptor inhibitor, inhibited axon outgrowth induced by PACAP and BDNF without affecting dendrite length. These results indicate that in primary cultured hippocampal neurons, PACAP shows morphological actions via its cognate receptor PAC1, stimulating neurite length and number, and soma size to a comparable extent as BDNF, and that the increase in total neurite length is ascribed to axon outgrowth.

The serotonin 5-HT(7) receptor has been linked to various psychiatric disorders, including schizophrenia, anxiety and depression, and is antagonized by antipsychotics such as risperidone, clozapine and lurasidone. In this study, we examined whether inhibiting the 5-HT(7) receptor could reverse behavioral abnormalities in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP), an experimental mouse model for psychiatric disorders such as schizophrenia. The selective 5-HT(7) antagonist SB-269970 effectively suppressed abnormal jumping behavior in PACAP-deficient mice. SB-269970 tended to alleviate the higher immobility in the forced swim test in PACAP-deficient mice, although SB-269970 reduced the immobility also in wild-type mice. In addition, we found that mutant mice had impaired performance in the Y-maze test, which was reversed by SB-269970. In the mutant mouse brain, 5-HT(7) protein expression did not differ from wild-type mice. In primary embryonic hippocampal neurons, the 5-HT(7) agonist AS19 increased neurite length and number. Furthermore, SB-269970 significantly inhibited the increase in neurite extension mediated by the 5-HT(1A/7) agonist 8-OH-DPAT. These results indicate that 5-HT(7) receptor blockade ameliorates psychomotor and cognitive deficits in PACAP-deficient mice, providing additional evidence that the 5-HT(7) receptor is a rational target for the treatment of psychiatric disorders.

Kinetic and physicochemical properties of hamster liver diacetyl reductase have been examined. The results of kinetic studies on the reduction of diacetyl and NADPH to acetoin and NADP+ suggest that the reaction follows an Ordered Bi Bi mechanism in which NADPH binds first before diacetyl. The enzyme is a tetrameric glycoprotein of single subunits of a molecular weight of 23,500 with a sedimentation coefficient of 6.0S. The enzyme does not contain Zn, Cu, or Fe. The amino acid composition revealed an unusually low proportion of proline residues (0.9%). p-Chloromercuriphenylsulfonate and phenylglyoxal inactivated the enzyme, but the presence of NADPH prevented the loss of activity due to thiol and arginine modification. The enzyme transferred the pro 4S hydrogen atom of NADPH to the substrate and the binding of the enzyme to NADPH resulted in a red shift of the ultraviolet absorption spectrum of the cofactor.