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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

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.

DOI： 10.1016/j.neuron.2017.05.017

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Language：English   Publisher：Biological and Pharmaceutical Bulletin  

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.

DOI： 10.1248/bpb.b23-00835

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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.

DOI： 10.1016/j.jphs.2023.12.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

Abstract

Associative learning is crucial for adapting to environmental changes. The encoding of associative learning involves the dorso-medial prefrontal cortex (dmPFC), and is underpinned by interactions within the resident neuronal population. However, the nature of this population coding is poorly understood. Here we developed a pipeline for computational dissection and longitudinal two-photon imaging of neural population activities in the mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC topology. Through regularized regression methods and graphical modeling, we found fear conditioning organized neuronal ensembles encoding conditioned responses (CR), with enhancing their coactivity, functional connectivity, and association with conditioned stimuli (CS). This suggests that fear conditioning drives dmPFC reorganization to generate novel associative circuits for CS-to-CR transformation. Importantly, neurons strongly responding to unconditioned stimuli (US) during conditioning anterogradely became a hub of the CR ensemble. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on an activity-dependent hub-network formation within the dmPFC.

Teaser

Optical and computational dissection uncovered how prefrontal cortical networks are rewired to encode new associative memory

Significance statement

Animals learn to adapt to changing environments. Associative learning is one of the simplest types of learning that has been intensively studied over the past century. Recent development in molecular, genetic, and optogenetic methods has enabled the identification of a neural population encoding the associative memory in the brain. However, it remains unclear how information is stored and processed by the neural population to encode and retrieve the associative memory. To investigate the nature of this population coding, we developed an optical and computational dissection method, demonstrating how associative learning drives reorganization of the neural network in the dorso-medial prefrontal cortex and generates novel circuits for associative memory and signal transformation.

DOI： 10.1038/s41467-023-41547-5

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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.

DOI： 10.1016/j.celrep.2023.112149

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Optica Publishing Group  

Raman microscopy is an emerging tool for molecular imaging and analysis of living samples. Use of Raman microscopy in life sciences is, however, still limited because of its slow measurement speed for spectral imaging and analysis. We developed a multiline-illumination Raman microscope to achieve ultrafast Raman spectral imaging. A spectrophotometer equipped with a periodic array of confocal slits detects Raman spectra from a sample irradiated by multiple line illuminations. A comb-like Raman hyperspectral image is formed on a two-dimensional detector in the spectrophotometer, and a hyperspectral Raman image is acquired by scanning the sample with multiline illumination array. By irradiating a sample with 21 simultaneous illumination lines, we achieved high-throughput Raman hyperspectral imaging of mouse brain tissue, acquiring 1108800 spectra in 11.4 min. We also measured mouse kidney and liver tissue as well as conducted label-free live-cell molecular imaging. The ultrafast Raman hyperspectral imaging enabled by the presented technique will expand the possible applications of Raman microscopy in biological and medical fields.

DOI： 10.1364/BOE.480611

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Other Link： https://opg.optica.org/viewmedia.cfm?URI=boe-14-3-1015&seq=0

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PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS).

DOI： 10.1016/j.jconrel.2022.12.050

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BACKGROUND: Accumulating evidence has implicated the role of neuroinflammation in the pathology of autism spectrum disorder (ASD), a neurodevelopmental disorder. OBJECTIVES: To investigate the expression of prostaglandin EP3 (EP3) receptor mRNA in the brain of ASD mouse model. METHODS: Pregnant mice were injected with valproic acid (VPA) 500 mg/kg intraperitoneally at 12.5 d gestation. The offspring were tested at the age of 5-6 weeks old for their social interaction behavior. Each mouse was assessed for prostaglandin EP3 receptor expression in the prefrontal cortical, hippocampal and cerebellar areas one day after the behavioral test. RESULTS: Compared to the naive, mice born to dams treated with VPA demonstrated a significantly shorter duration of sniffing behavior, a model of social interaction. Results further showed that the expression of EP3 receptor mRNA was significantly lower in all three brain regions of the mice born to VPA-treated dams. CONCLUSION: The present study provides further evidence of the relevance of the arachidonic acid cascade as an essential part of neuroinflammation in the pathology of ASD.

DOI： 10.2174/2211536612666230427152647

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Feeding behavior is adaptively regulated by external and internal environment, such that feeding is suppressed when animals experience pain, sickness, or fear. While the lateral parabrachial nucleus (lPB) plays key roles in nociception and stress, neuronal pathways involved in feeding suppression induced by fear are not fully explored. Here, we investigate the parasubthalamic nucleus (PSTN), located in the lateral hypothalamus and critically involved in feeding behaviors, as a target of lPB projection neurons. Optogenetic activation of lPB-PSTN terminals in male mice promote avoidance behaviors, aversive learning, and suppressed feeding. Inactivation of the PSTN and lPB-PSTN pathway reduces fear-induced feeding suppression. Activation of PSTN neurons expressing pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide enriched in the PSTN, is sufficient for inducing avoidance behaviors and feeding suppression. Blockade of PACAP receptors impaires aversive learning induced by lPB-PSTN photomanipulation. These findings indicate that lPB-PSTN pathway plays a pivotal role in fear-induced feeding suppression.

DOI： 10.1038/s41467-022-35634-2

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1186/s13041-022-00987-8

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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.

DOI： 10.1186/s13041-022-00915-w

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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.

DOI： 10.1038/s41398-021-01676-1

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In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.

DOI： 10.1038/s41598-021-95930-7

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In the mammalian cerebral neocortex, different regions have different cytoarchitecture, neuronal birthdates, and functions. In most regions, neuronal migratory profiles are speculated similar based on observations using thymidine analogs. Few reports have investigated regional migratory differences from mitosis at the ventricular surface. In this study, we applied FlashTag technology, in which dyes are injected intraventricularly, to describe migratory profiles. We revealed a mediolateral regional difference in the migratory profiles of neurons that is dependent on developmental stage; for example, neurons labeled at embryonic day 12.5-15.5 reached their destination earlier dorsomedially than dorsolaterally, even where there were underlying ventricular surfaces, reflecting sojourning below the subplate. This difference was hardly recapitulated by thymidine analogs, which visualize neurogenic gradients, suggesting a biological significance different from the neurogenic gradient. These observations advance our understanding of cortical development and the power of FlashTag in studying migration and are thus resources for future neurodevelopmental studies.

DOI： 10.1016/j.isci.2021.102277

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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 (POGZWT/Q1038R mice), showed ASD-like social behavioral deficits. Here, we have explored whether oxytocin (OXT) administration improves impaired social behavior in POGZWT/Q1038R mice and found that intranasal oxytocin administration effectively restored the impaired social behavior in POGZWT/Q1038R mice. We also found that the expression level of the oxytocin receptor gene (OXTR) was low in POGZWT/Q1038R mice. However, we did not detect significant changes in the number of OXT-expressing neurons between the paraventricular nucleus of POGZWT/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.

DOI： 10.1186/s13041-021-00769-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Pharmaceutical Society of Japan  

<p>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.</p>

DOI： 10.1248/bpb.b20-01006

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We previously showed that mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit attenuated light-induced phase shift. To explore the underlying mechanisms, we performed gene expression analysis of laser capture microdissected suprachiasmatic nuclei (SCNs) and found that lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is involved in the impaired response to light stimulation in the late subjective night in PACAP-deficient mice. L-PGDS-deficient mice also showed impaired light-induced phase advance, but normal phase delay and nonvisual light responses. Then, we examined the receptors involved in the response and observed that mice deficient for type 2 PGD2 receptor DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) show impaired light-induced phase advance. Concordant results were observed using the selective DP2/CRTH2 antagonist CAY10471. These results indicate that L-PGDS is involved in a mechanism of light-induced phase advance via DP2/CRTH2 signaling.

DOI： 10.1038/s42003-020-01281-w

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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.

DOI： 10.1016/j.pbb.2020.172876

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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.

DOI： 10.1038/s41467-020-14697-z

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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.

DOI： 10.1016/j.bbrc.2019.09.048

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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.

DOI： 10.1038/s41386-019-0441-5

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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.

DOI： 10.1093/ijnp/pyz041

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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.

DOI： 10.1523/JNEUROSCI.2468-18.2019

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Growing evidence suggests pivotal roles for epigenetic mechanisms in both animal models of and individuals with autism spectrum disorders (ASD). Neuron-restrictive silencer factor (NRSF) binds to neuron-restrictive silencing elements in neuronal genes and recruits co-repressors, such as mSin3, to epigenetically inhibit neuronal gene expression. Because dysregulation of NRSF is related to ASD, here we examined the effects of mS-11, a chemically optimized mimetic of the mSin3-binding helix in NRSF, on the behavioral and morphological abnormalities found in a mouse model of valproic acid (VPA)-induced ASD. Chronic treatment with mS-11 improved prenatal VPA-induced deficits in social interaction. Additionally, we found that NRSF mRNA expression was greater in the somatosensory cortex of VPA-exposed mice than of controls. Agreeing with these behavioral findings, mice that were prenatally exposed to VPA showed lower dendritic spine density in the somatosensory cortex, which was reversed by chronic treatment with mS-11. These findings suggest that mS-11 has the potential for improving ASD-related symptoms through inhibition of mSin3-NRSF binding.

DOI： 10.1016/j.pbb.2018.11.003

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Pharmacological Society  

<p>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.</p>

DOI： 10.1254/fpj.153.278

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DOI： 10.1063/1.5039567

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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.

DOI： 10.1371/journal.pone.0196946

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-VCH Verlag  

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.

DOI： 10.15252/embr.201744860

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We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart.

DOI： 10.1016/j.bbrc.2017.11.116

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Schizophrenia is a chronic psychiatric disorderwith complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair ofmonozygotic twin caseswith treatment-resistant schizophrenia, inwhich one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.schres.2016.10.012

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Authorship：Lead author,　Corresponding author   Language：English  

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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. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.10.089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

MicroRNAs (miRNAs) participate in a variety of functions in the brain. Understanding the in vivo localization of miRNAs is an important step for uncovering their roles in brain function. However, the in situ detection of low-abundance miRNAs in brain tissues remains difficult and requires extensive optimization of in situ hybridization (ISH) protocols in individual laboratories. Thus, detailed information regarding experimental conditions would serve as a useful reference for researchers in this field. Here, we investigated and summarized the effects of adjusting a series of critical steps, including tissue fixation, probe accessibility and hybridization stringency, to standardize the currently used miRNA ISH procedures. As a result, we successfully detected several low-abundance miRNAs by ISH using the following experimental conditions: (1) use of fresh brain tissues, (2) digestion of brain samples with proteinase K, (3) LNA-probe hybridization at a temperature 37 degrees C below the melting temperature of the RNA, (4) performance of high-stringency wash steps using 50% formamide in 1 x standard saline citrate (SSC) buffer. RT-PCR of the punched-out tissues using TaqMan (TM) primers confirmed the ISH results. Finally, double-fluorescence ISH successfully demonstrated the colocalization of miRNAs and mRNAs. Thus, the detailed information regarding the miRNA ISH procedures used in this study may help to resolve the technical hurdles observed in the in vivo localization of miRNAs, and the elucidation of the specific roles of miRNAs.

DOI： 10.3389/fnmol.2016.00126

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), which is a second receptor for prostaglandin (PG) D-2, is involved in inflammatory responses in peripheral tissue; however, its role in cognitive function remains unclear. Here, we demonstrate that CRTH2 is involved in cognitive function using a well-established animal model of cognitive dysfunction induced by MK-801, an N-methyl-D-aspartate receptor antagonist. Genetic deletion and pharmacological inhibition of CRTH2 suppressed MK-801-induced cognitive dysfunction. Pharmacological inhibition of cyclooxygenase-1, a rate-limiting enzyme in PG synthesis, also suppressed MK-801-induced cognitive dysfunction. Moreover, an MK-801-induced increase in c-Fos expression in the paraventricular nucleus (PVN) was abolished in the CRTH2-deficient mice. Together, these results suggest that PGD(2)-CRTH2 signaling is involved in both MK-801-induced cognitive dysfunction and neuronal activity regulation in the PVN. Furthermore, genetic association studies suggest that CRTH2 is weakly associated with cognitive function in humans. Our study provides evidence that PGD(2)-CRTH2 signaling is involved in cognitive function and may represent a potential therapeutic target for cognitive dysfunction in patients with psychiatric disorders. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbr.2016.07.050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

JMJD2B is a histone demethylase enzyme that regulates gene expression through demethylation of H3K9me3. Although mutations of JMJD2B have been suggested to be responsible for neurodevelopmental disorders, the function of JMJD2B in the central nervous system (CNS) remains to be elucidated. Here we show that JMJD2B has a critical role in the development of the CNS. We observed JMJD2B expression, which was especially strong in the hippocampus, throughout the CNS from embryonic periods through adulthood. We generated neuron-specific JMJD2B-deficient mice using the cre-loxP system. We found an increase in total spine number, but a decrease in mature spines, in the CA1 region of the hippocampus. JMJD2B-deficient mice exhibited hyperactive behavior, sustained hyperactivity in a novel environment, deficits in working memory and spontaneous epileptic-like seizures. Together these observations indicate that JMJD2B mutant mice display symptoms reminiscent of neurodevelopmental disorders. Our findings provide evidence for the involvement of histone demethylation in the formation of functional neural networks during development.

DOI： 10.1038/tp.2016.31

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Autism spectrum disorder (ASD) is a complex group of clinically heterogeneous neurodevelopmental disorders with unclear etiology and pathogenesis. Genetic studies have identified numerous candidate genetic variants, including de novo mutated ASD-associated genes; however, the function of these de novo mutated genes remains unclear despite extensive bioinformatics resources. Accordingly, it is not easy to assign priorities to numerous candidate ASD-associated genes for further biological analysis. Here we developed a convenient system for identifying an experimental evidence-based annotation of candidate ASD-associated genes. We performed trio-based whole-exome sequencing in 30 sporadic cases of ASD and identified 37 genes with de novo single-nucleotide variations (SNVs). Among them, 5 of those 37 genes, POGZ, PLEKHA4, PCNX, PRKD2 and HERC1, have been previously reported as genes with de novo SNVs in ASD; and consultation with in silico databases showed that only HERC1 might be involved in neural function. To examine whether the identified gene products are involved in neural functions, we performed small hairpin RNA-based assays using neuroblastoma cell lines to assess neurite development. Knockdown of 8 out of the 14 examined genes significantly decreased neurite development (P&lt;0.05, one-way analysis of variance), which was significantly higher than the number expected from gene ontology databases (P= 0.010, Fisher's exact test). Our screening system may be valuable for identifying the neural functions of candidate ASD-associated genes for further analysis and a substantial portion of these genes with de novo SNVs might have roles in neuronal systems, although further detailed analysis might eliminate false positive genes from identified candidate ASD genes.

DOI： 10.1038/jhg.2015.141

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Aims Leucine-rich alpha 2-glycoprotein (LRG) is considered as a biomarker of the clinical activities of chronic inflammatory diseases, including heart failure. However, its pathophysiological roles in cardiac remodelling after myocardial infarction (MI) remain to be clarified. In this study, we have addressed functional roles of LRG in cardiac remodelling after MI.
Methods and results MI was generated by ligating the left coronary artery in mice. Real-time reverse transcription (RT)-PCR and immunoblot analyses revealed that the expressions of LRG transcript and protein were up-regulated in post-infarct myocardium. LRG protein was produced by heart-infiltrating myeloid cells, such as macrophages and neutrophils. To elucidate functional roles of LRG in cardiac remodelling, we generated MI in wild-type (WT) and LRG-deficient (LRG(-/-)) mice and found that LRG gene ablation aggravated myocardial fibrosis with cardiac dysfunction after MI. Immunohistochemical analyses with anti-CD31 antibody revealed that capillary density decreased at border zone in LRG(-/-) mice compared with WT mice. Consistently, the expression of apelin receptor was reduced in LRG(-/-) mice, implying that the impaired angiogenic activity is associated with adverse cardiac remodelling in LRG(-/-) mice. Moreover, LRG gene ablation suppressed the activation of smad1/5/8, a pro-angiogenic signalling pathway. Finally, the transplantation of WT bone marrow cells into LRG(-/-) mice attenuated cardiac fibrosis with functional improvement after MI, accompanied by restoration of capillary density compared with the bone marrow transplantation from LRG(-/-) mice.
Conclusion LRG, produced by heart-infiltrating myeloid cells, suppresses adverse cardiac remodelling after MI as a novel cardioprotective factor. LRG signalling could be a therapeutic target against cardiovascular diseases.

DOI： 10.1093/cvr/cvv273

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

Dopamine (DA) has been suggested to modulate functions of glial cells including microglial cells. To reveal the regulatory role of DA in microglial function, in the present study, we investigated the effect of DA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial cell line BV-2. Pretreatment with DA for 24 h concentration-dependently attenuated LPS-induced NO production in BV-2 cells. The inhibitory effect of DA on LPS-induced NO production was not inhibited by SCH-23390 and sulpiride, D-1-like and D-2-like DA receptor antagonists, respectively. In addition, pretreatment with (-)-(6aR, 12bR)-4,6,6a, 7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY 208-243) and bromocriptine, D-1-like and D-2-like DA receptor agonists, respectively, did not affect the LPS-induced NO production. N-Acetylcysteine, which inhibits DA oxidation, completely inhibited the effect of DA. Tyrosinase, which catalyzes the oxidation of DA to DA quionone (DAQ), accelerated the inhibitory effect of DA on LPS-induced NO production. These results suggest that DA attenuates LPS-induced NO production through the formation of DAQ in BV-2 cells. (C) 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.jphs.2015.11.002

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DOI： 10.1186/s40303-016-0016-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

In the last couple of decades, the spatial resolution in optical microscopy has increased to unprecedented levels by exploiting the fluorescence properties of the probe. At about the same time, Raman imaging techniques have emerged as a way to image inherent chemical information in a sample without using fluorescent probes. However, in many applications, the achievable resolution is limited to about half the wavelength of excitation light. Here we report the use of structured illumination to increase the spatial resolution of label-free spontaneous Raman microscopy, generating highly detailed spatial contrast from the ensemble of molecular information in the sample. Using structured line illumination in slit-scanning Raman microscopy, we demonstrate a marked improvement in spatial resolution and show the applicability to a range of samples, including both biological and inorganic chemical component mapping. This technique is expected to contribute towards greater understanding of chemical component distributions in organic and inorganic materials.

DOI： 10.1038/ncomms10095

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

In diabetes mellitus, pituitary adenylate cyclase-activating polypeptide (PACAP) has insulinotropic and glucose-lowering properties. We previously demonstrated that transgenic mice overexpressing PACAP in pancreatic β-cells (PACAP-Tg) show attenuated pancreatic islet hyperplasia and hyperinsulinemia in type 2 diabetic models. To explore the underlying mechanisms, here we crossed PACAP-Tg mice with lethal yellow agouti (KKAy) diabetic mice, and performed gene chip analysis of laser capture microdissected pancreatic islets from four F1 offspring genotypes (wild-type, PACAP-Tg, KKAy, and PACAP-Tg:KKAy). We identified 1371 probes with &gt
16-fold differences between at least one pair of genotypes, and classified the probes into five clusters with characteristic expression patterns. Gene ontology enrichment analysis showed that genes involved in the terms ribosome and intracellular organelles such as ribonucleoprotein complex, mitochondrion, and chromosome organization were significantly enriched in clusters characterized by up-regulated genes in PACAP-Tg:KKAy mice compared with KKAy mice. These results may provide insight into the mechanisms of diabetes that accompany islet hyperplasia and amelioration by PACAP.

DOI： 10.1016/j.bbrep.2015.06.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed. (C) 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2015.03.062

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We examined the pancreatic function of p13 encoded by 1110001J03Rik, whose expression is decreased in pancreatic islets in high-fat-fed diabetic mice, by generating transgenic mice overexpressing p13 (p13-Tg) in pancreatic beta-cells. p13-Tg mice showed normal basal glucose metabolism; however, under high-fat feeding, these animals showed augmented glucose-induced first-phase and total insulin secretion, improved glucose disposal, greater islet area and increased mitotic insulin-positive cells. In addition, high-fat diet-induced 4-hydroxynonenal immunoreactivity, a reliable marker and causative agent of lipid peroxidative stress, was significantly decreased in p13-Tg mouse islets. These results indicate that p13 is a novel pancreatic factor exerting multiple beneficial effects against type 2 diabetes. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2015.04.074

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Depression is a complex neuropsychiatric disorder with an unclear molecular etiology. Inflammatory cytokines and molecular intermediates (including prostaglandins) are suggested to be involved in depression; however, the roles of prostaglandins and their respective receptors are largely unknown in depression. Using genetic and pharmacological approaches, we show here that chemoattractant receptorhomologous molecule expressed on T helper type 2 cells (CRTH2), a second receptor for prostaglandin D2 (PGD2), mediates depression-related behavior in mice. CRTH2-deficient (CRTH2(-/-)) mice showed antidepressant-like activity in a chronic corticosterone treatment-induced depression. Consistent with this observation, the pharmacological inhibition of CRTH2 via the clinically available drug ramatroban also rescued abnormal social interaction and depression-related behavior in well-established models, including chronic corticosterone-, lipopolysaccharide-, and tumor-induced pathologically relevant depression models. Importantly, chronic stress via corticosterone treatment increased mRNA levels in PGD2-producing enzymes, such as cyclooxygenase-2 and lipocalin-type PGD2 synthase, in the brain. Furthermore, the activity of the hippocampal noradrenergic system but not the dopaminergic or serotonergic systems was increased in CRTH2(-/-) mice. Together with the observation that untreated CRTH2(-/-) mice showed antidepressant-like activity in the forced swim test, these results provide evidence that central CRTH2-mediated signaling is critically involved in depression-related behavior. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbr.2015.02.013

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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.

DOI： 10.1371/journal.pone.0121707

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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.

DOI： 10.1371/journal.pone.0120526

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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. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2015.02.073

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock-in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet-1Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate-based screening and identified BMP type I receptor kinase inhibitors LDN-193189 and DMH1 as activators of luciferase. LDN-193189 induced ES cells to express the genes encoding Pet-1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF-beta receptor inhibitor SB-431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN-193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF-beta target gene Lefty, whereas the opposite effect was observed with SB-431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF-b receptor signaling are implicated in serotonergic differentiation.

DOI： 10.1111/jnc.12999

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Accumulating evidence from human genetic studies implicates the pituitary adenylate cyclase-activating polypeptide (PACAP) gene as a risk factor for psychiatric disorders, including schizophrenia and stress-related diseases. Mice with homozygous disruption of the PACAP gene display profound behavioral and neurological abnormalities that are ameliorated with the atypical antipsychotic and dopamine D-2 and serotonin (5-HT)(2) antagonist risperidone and the 5-HT2 receptor antagonist ritanserin; however, the underlying mechanisms remain unknown. Here, we investigated if PACAP heterozygous mutant (PACAP+/2) mice, which appear behaviorally normal, are vulnerable to aversive stimuli. PACAP+/2 mice were administered a 5-HT2 receptor agonist, (6)-2,5-dimethoxy-4-iodoamphetamine (DOI), a hallucinogenic drug, and their responses were compared with the littermate wild-type mice. After DOI injection, PACAP+/2 mice showed increased head-twitch responses, while their behavior was normal after saline. DOI induced deficits in sensorimotor gating, as determined by prepulse inhibition, specifically in PACAP+/2 mice. However, other 5-HT2 receptor-dependent responses, such as corticosterone release and hypothermia, were similarly observed in PACAP+/2 and wild-type mice. c-Fos expression analysis, performed in various brain regions, revealed that the DOI-induced increase in the number of c-Fos-positive cells was more pronounced in 5-HT(2)A receptor-negative cells in the somatosensory cortex in PACAP+/2 mice compared with wild-type mice. These results indicate that PACAP+/2 mice exhibit specific vulnerability to DOI-induced deficits in cortical sensory function, such as exaggerated head-twitch responses and sensorimotor gating deficits. Our findings provide insight into the neural mechanisms underlying impaired behavioral responses in which 5-HT2 receptors are implicated.

DOI： 10.1371/journal.pone.0089153

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) is a second prostaglandin D-2 receptor involved in mediating the allergic response; however, its central function is not yet known. Here, we demonstrate that central CRTH2 mediates emotional impairment. Lipopolysaccharide (LPS)-induced decreases in social interaction and novel exploratory behavior were observed in wild-type (CRTH2(+/+)) mice but not CRTH2-deficient (CRTH2(-/-)) mice, but both genotypes showed hypolocomotion and anorexia following LPS injection. Tumor (colon 26) inoculation, a more pathologically relevant model, induced decreases in social interaction and novel exploratory behavior in CRTH2(+/+), but not CRTH2(-/-) mice. In addition, the CRTH2 antagonists including clinically available ramatroban reversed impaired social interaction and novel exploratory behavior after either LPS or tumor inoculation in CRTH2(+/+) mice. Finally, LPS-induced c-Fos expression in the hypothalamic paraventricular nucleus (PVN) and central amygdala (CeA) was selectively abolished in CRTH2(-/-) mice. These results show that CRTH2 participates in LPS-induced emotional changes and activation in the PVN and CeA. Our study provides the first evidence that central CRTH2 regulates specific emotional behaviors, and that CRTH2 antagonism has potential as a therapeutic target for behavioral symptoms associated with tumors and infectious diseases.

DOI： 10.1523/JNEUROSCI.1407-13.2014

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Targeted mutation of STAT3 in myeloid cells dramatically altered the relative abundance of specific immune cell types within tumors, with no impact on tumor incidence. Although the immune system may provide early protection against cancer, tumors may exploit the healing arm of the immune system to enhance their growth and metastasis. For example, myeloid derived suppressor cells (MDSCs) are thought to promote tumor growth by several mechanisms, including the suppression of T cell activity. It has been suggested that STAT3 activation in myeloid cells modulates multiple aspects of MDSC physiology, including their expansion and activity. Whereas most animal studies investigating tumor immunology have used tumor implants, we used transgenic mice (Smo*) that spontaneously develop medulloblastoma brain tumors to investigate the temporal accumulation of MDSCs within tumors and how myeloid STAT3 disruption affects MDSC and other immune cell types. We found distinct populations of MDSC in medulloblastoma tumors, with a high prevalence of CD11b(+)Ly6G(+)Ly6C(low/-) cells, described previously by others as G-MDSCs. These were found early in tumor development, in premalignant lesions located on the surface of the cerebellum of 28-day-old mice. In fully developed tumors, pSTAT3 was found in the majority of these cells. Conditional STAT3 gene disruption in myeloid cells resulted in an enhanced proinflammatory phenotype of macrophages in Smo* mice. Moreover, a significant reduction in the abundance of G-MDSCs and Tregs was observed within tumors along with an increased presence of CD4(+) and CD8(+) cells. Despite these alterations in immune cells induced by myeloid STAT3 disruption, we found no effect on tumor incidence in Smo* mice with this deletion.

DOI： 10.1189/jlb.1012531

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The recruitment of mural cells such as pericytes to patent vessels with an endothelial lumen is a key factor for the maturation of blood vessels and the prevention of hemorrhage in pathological angiogenesis. To date, our understanding of the specific trigger underlying the transition from cell growth to the maturation phase remains incomplete. Since rapid endothelial cell growth causes pericyte loss, we hypothesized that suppression of endothelial growth factors would both promote pericyte recruitment, in addition to inhibiting pathological angiogenesis. Here, we demonstrate that targeted knockdown of apelin in endothelial cells using siRNA induced the expression of monocyte chemoattractant protein-1 (MCP-1) through activation of Smad3, via suppression of the PI3K/Akt pathway. The conditioned medium of endothelial cells treated with apelin siRNA enhanced the migration of vascular smooth muscle cells, through MCP-1 and its receptor pathway. Moreover, in vivo delivery of siRNA targeting apelin, which causes exuberant endothelial cell proliferation and pathological angiogenesis through its receptor APJ, led to increased pericyte coverage and suppressed pathological angiogenesis in an oxygen-induced retinopathy model. These data demonstrate that apelin is not only a potent endothelial growth factor, but also restricts pericyte recruitment, establishing a new connection between endothelial cell proliferation signaling and a trigger of mural recruitment. © 2013 The Author(s).

DOI： 10.1007/s10456-013-9349-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ASSOC RESEARCH VISION OPHTHALMOLOGY INC  

PURPOSE. To investigate the role of the apelin-APJ system in the development of choroidal neovascularization (CNV).
METHODS. Experimental CNV was induced by laser photocoagulation in wild-type (WT), apelin-deficient (apelin-KO), and apelin receptor (APJ)-deficient (APJ-KO) mice. The gene expression levels of angiogenic or inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction. APJ expression in CNV lesions was examined by immunohistochemistry. The sizes of the CNV lesions in the three mouse models were measured and compared histologically using isolectin B4 staining. Macrophage recruitment was measured by flow cytometric analysis. Proliferation of endothelial cells was determined using the alamar Blue assay.
RESULTS. Laser photocoagulation significantly increased expression of apelin and APJ in the retina-retinal pigment epithelium (RPE) complex. APJ immunoreactive cells were found in the CNV lesions and colocalized with platelet endothelial cell adhesion molecule-1, an endothelial cell marker. The sizes of the CNV lesions in apelin-KO or APJ-KO mice decreased significantly compared with those in the WT mice. Macrophages in the RPE complex of the apelin-KO mice, in which gene expression of the inflammatory factors was almost equal to that in WT mice, were recruited as a result of laser photocoagulation to the same degree as in WT mice. In addition, apelin small and interfering RNA (siRNA) suppressed proliferation of endothelial cells independently of vascular endothelial growth factor (VEGF) receptor 2 signaling, while VEGF increased expression of apelin and APJ in human umbilical vein endothelial cells.
CONCLUSIONS. The results suggested that the apelin-APJ system contributes to CNV development partially independent of the VEGF pathway.

DOI： 10.1167/iovs.13-11611

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Language：Japanese   Publisher：5  

DOI： 10.1254/fpj.139.198

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Other Link： https://jlc.jst.go.jp/DN/JALC/10000262803?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with D-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30 min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by D-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2 mg/mL. None of the individual components, for example, D-octaarginine, had any influence on CF uptake, demonstrating that only D-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120 min. Confocal laser scanning microphotographs of cells incubated with D-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that D-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejpb.2012.01.008

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Language：Japanese   Publisher：(公財)日本眼科学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H2O2-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.

DOI： 10.1371/journal.pone.0023968

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Language：Japanese   Publisher：PHARMACEUTICAL SOC JAPAN  

Progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis mainly induced by vascular endothelial growth factor (VEGF). Anti-angiogenic therapy using anti-VEGF antibodies is effective in treating diabetic retinopathy, even though its efficacy is not long-lasting. Since many factors are involved in angiogenesis, it is reasonable to seek new therapeutic target molecules in pathological retinal angiogenesis. We have found that apelin/APJ system is involved in not only physiological but also pathological retinal angiogenesis using a mouse model of oxygen-induced retinopathy (OIR). Oxygen-induced vessel loss in the retinas of OIR model leads to a significant increase in the capillary density accompanied by abnormal vessel growth, similar to aneurysms, which are hardly detected in the retinas of control mice. Compared with age-matched control mice, retinal apelin expression was dramatically increased during retinal angiogenesis in OIR model. Immunostaining for APJ, apelin receptor, in retinal from OIR model revealed that APJ was localized in proliferating endothelial cells in the retinal vascular plexus. Retinal angiogenesis in the OIR model was rarely observed in apelin deficient mice, although temporal expression pattern of VEGF was similar to that of wild-type OIR model. In addition, clinical study showed that vitreous concentrations of apelin were significantly higher in the proliferative diabetic retinopathy group than in the control group. Taken together, these findings clearly suggest that apelin/APJ system may be a crucial factor for pathological retinal angiogenesis. Inhibition of this system could offer new therapeutic opportunities against ischemic retinopathy.

DOI： 10.1248/yakushi.131.1201

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

The present study investigated the function of caspase-4 in endoplasmic reticulum (ER) stress-induced apoptosis in human neuronal cell line SH-SY5Y. Tunicamycin, which is known to induce ER stress, activated both caspase-9 and caspase-4, and the activation of caspase-4 preceded that of caspase-9. The caspase-4 inhibitor LEVD-CHO suppressed both the apoptosis and caspase-9 activation. In addition, human recombinant active caspase-4 cleaved wild type and D330A mutant substituted Asp-330 for alanine of human recombinant procaspase-9, but did not cleave D315A mutant substituted Asp-315 for alanine. These results suggest that caspase-4 directly activates caspase-9 by the processing of procaspase-9 at Asp-315 in ER stress induced neuronal apoptosis.

DOI： 10.1254/jphs.10217SC

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Background: Neuregulin-1 (NRG1) is one of the susceptibility genes for schizophrenia and implicated in the neurotrophic regulation of GABAergic and dopaminergic neurons, myelination, and NMDA receptor function. Postmortem studies often indicate a pathologic association of increased NRG1 expression or signaling with this illness. However, the psychobehavioral implication of NRG1 signaling has mainly been investigated using hypomorphic mutant mice for individual NRG1 splice variants.
Methodology/Principal Findings: To assess the behavioral impact of hyper NRG1 signaling, we generated and analyzed two independent mouse transgenic (Tg) lines carrying the transgene of green fluorescent protein (GFP)-tagged type-1 NRG1 cDNA. The promoter of elongation-factor 1 alpha gene drove ubiquitous expression of GFP-tagged NRG1 in the whole brain. As compared to control littermates, both heterozygous NRG1-Tg lines showed increased locomotor activity, a nonsignificant trend toward decreasing prepulse inhibition, and decreased context-dependent fear learning but exhibited normal levels of tone-dependent learning. In addition, social interaction scores in both Tg lines were reduced in an isolation-induced resident-intruder test. There were also phenotypic increases in a GABAergic marker (parvalbumin) as well as in myelination markers (myelin basic protein and 2&apos;,3&apos;-cyclic nucleotide 3&apos;-phosphodiesterase) in their frontal cortex, indicating the authenticity of NRG1 hyper-signaling, although there were marked decreases in tyrosine hydroxylase levels and dopamine content in the hippocampus.
Conclusions: These findings suggest that aberrant hyper-signals of NRG1 also disrupt various cognitive and behavioral processes. Thus, neuropathological implication of hyper NRG1 signaling in psychiatric diseases should be evaluated with further experimentation.

DOI： 10.1371/journal.pone.0014185

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Objective-To investigate the role of endogenous apelin in pathological retinal angiogenesis.
Methods and Results-The progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis, mainly induced by vascular endothelial growth factor (VEGF) and erythropoietin. Although antiangiogenic therapies using anti-VEGF drugs are effective in treating retinal neovascularization, they show a transient efficacy and cause general adverse effects. New therapeutic target molecules are needed to resolve these issues. It was recently demonstrated that the apelin/APJ system, a newly deorphanized G protein-coupled receptor system, is involved in physiological retinal vascularization. Retinal angiography and mRNA expression were examined during hypoxia-induced retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Compared with age-matched control mice, retinal apelin expression was dramatically increased during the hypoxic phase in oxygen-induced retinopathy model mice. APJ was colocalized in proliferative cells, which were probably endothelial cells of the ectopic vessels in the vitreous body. Apelin deficiency hardly induced hypoxia-induced retinal angiogenesis despite the upregulation of VEGF and erythropoietin mRNA in oxygen-induced retinopathy model mice. Apelin small and interfering RNA suppressed the proliferation of endothelial cells independent of the VEGF/VEGF receptor 2 signaling pathway.
Conclusion-These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis. (Arterioscler Thromb Vasc Biol. 2010; 30: 2182-2187.)

DOI： 10.1161/ATVBAHA.110.209775

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

The present study examined the effect of the nitric oxide (NO) donor NOC18 on lipopolysaccharide (LPS)-induced NO production to investigate a regulation mechanism of NO production by microglial cells. LPS increased the levels of NO and inducible NO synthase (iNOS) protein in BV-2 murine microglial cells in a concentration-dependent manner. Pretreatment with NOC18 for 24 h concentration-dependently attenuated the LPS-induced iNOS protein expression and NO production. The inhibitory effect of NOC18 on LPS-induced NO production was partially blocked by LY83583, a soluble guanylate cyclase inhibitor. Pretreatment with dibutyryl guanosine-3',5'-cyclic monophosphate (DBcGMP), a cell-permeable cGMP analogue, for 24 h attenuated partially LPS-induced iNOS protein expression and NO production. Furthermore, the effects of LPS on iNOS and NO production were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125, and LPS-induced phosphorylation of JNK and c-Jun was inhibited by NOC18 and DBcGMP. These results suggest that NO production by microglial cells is controlled by a negative feedback mechanism via the NO/cGMP signaling pathway.

DOI： 10.1254/jphs.10060FP

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Authorship：Lead author,　Corresponding author   Language：English  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Objective -Apelin is an endogenous ligand for the G protein -coupled receptor, APJ, and participates in multiple physiological processes. To identify the roles of endogenous apelin, we investigated the phenotype of apelin-deficient (apelin-KO) mice.
Methods and Results -Apelin-KO mice showed impaired retinal vascularization and ocular development, which were analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and the mouse corneal micropocket assay. Apelin-KO mice showed significantly impaired retinal vascularization in the early postnatal period. Retinal apelin/ APJ mRNAs were transiently upregulated during the first 2 postnatal weeks but were undetectable in adults. There were no differences in VEGF or FGF2 mRNA expression, or in the morphology and localization of GFAP-positive astrocytes, in the apelin-KO retinas at P5. The corneal pocket assay showed that angiogenic responses to VEGF and FGF2 were remarkably decreased in apelin-KO mice. The reduced responses to VEGF and FGF2 in apelin-KO mice were partially restored by apelin, but apelin alone did not induce angiogenesis.
Conclusions -Our results suggest that spatiotemporally regulated apelin/ APJ signaling participates in retinal vascularization in a cooperative manner with VEGF or FGF2, and contributes to normal ocular development.

DOI： 10.1161/ATVBAHA.108.163402

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DOI： 10.1254/fpj.130.281

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Other Link： https://jlc.jst.go.jp/DN/JALC/00300745410?from=CiNii

Language：English   Publisher：電気学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：HUMANA PRESS INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：HUMANA PRESS INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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DOI： 10.1248/yakushi.131.1201

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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DOI： 10.1016/j.neures.2010.07.2446

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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DOI： 10.1016/j.neures.2010.07.1538

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：Japanese   Presentation type：Oral presentation (general)  

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