Publications by authors named "Kazuki Nagayasu"

67 Publications

Secreted PLA-III is a possible therapeutic target to treat neuropathic pain.

Biochem Biophys Res Commun 2021 Jul 5;568:167-173. Epub 2021 Jul 5.

Department of Molecular Pharmacology, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto, 606-8501, Japan; Department of Pharmacology and Therapeutic Innovation, Nagasaki University Institute of Biomedical Sciences, 852-8521, Japan; Laboratory for the Study of Pain, Research Institute for Production Development, Kyoto, 606-0805, Japan. Electronic address:

Lysophosphatidic acid (LPA) plays a critical role in developing and maintaining chronic pain in various animal models. Previous studies have reported that cytosolic and calcium-independent phospholipase A (PLA) is involved in the LPA receptor-mediated amplification of LPA production in the spinal dorsal horn (SDH) after nerve injury, while the involvement of secreted PLA (sPLA) remains unclear. The present study revealed that only sPLA -III among 11 species of PLA showed a significant upregulation of gene expression in the SDH. Intraspinal injection of adeno-associated virus-miRNA targeting sPLA-III prevented hyperalgesia and unique hypoalgesia in mice treated with partial sciatic nerve ligation. In addition, intrathecal treatment with antisense oligodeoxynucleotide or siRNA targeting sPLA-III significantly reversed the established thermal hyperalgesia. In the high-throughput screening of sPLA-III inhibitors from the chemical library, we identified two hit compounds. Through in vitro characterization of PLA inhibitor profiles and in vivo assessment of the anti-hyperalgesic effects of known PLA inhibitors as well as hit compounds, sPLA-III was found to be a novel therapeutic target molecule for the treatment of Neuropathic pain.
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http://dx.doi.org/10.1016/j.bbrc.2021.06.058DOI Listing
July 2021

MrgprB4 in trigeminal neurons expressing TRPA1 modulates unpleasant sensations.

J Pharmacol Sci 2021 Aug 28;146(4):200-205. Epub 2021 Apr 28.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.

Gentle touch such as stroking of the skin produces a pleasant feeling, which is detected by a rare subset of sensory neurons that express Mas-related G protein-coupled receptor B4 (MrgprB4) in mice. We examined small populations of MrgprB4-positive neurons in the trigeminal ganglion and the dorsal root ganglion, and most of these were sensitive to transient receptor potential ankyrin 1 (TRPA1) agonist but not TRPV1, TRPM8, or TRPV4 agonists. Deficiency of MrgprB4 did not affect noxious pain or itch behaviors in the hairless plantar and hairy cheek. Although behavior related to acetone-induced cold sensing in the hind paw was not changed, unpleasant sensory behaviors in response to acetone application or sucrose splash to the cheek were significantly enhanced in Mrgprb4-knockout mice as well as in TRPA1-knockout mice. These results suggest that MrgprB4 in the trigeminal neurons produces pleasant sensations in cooperation with TRPA1, rather than noxious or cold sensations. Pleasant sensations may modulate unpleasant sensations on the cheek via MrgprB4.
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http://dx.doi.org/10.1016/j.jphs.2021.04.006DOI Listing
August 2021

Striatal TRPV1 activation by acetaminophen ameliorates dopamine D2 receptor antagonist-induced orofacial dyskinesia.

JCI Insight 2021 May 24;6(10). Epub 2021 May 24.

Department of Molecular Pharmacology, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.

Antipsychotics often cause tardive dyskinesia, an adverse symptom of involuntary hyperkinetic movements. Analysis of the US Food and Drug Administration Adverse Event Reporting System and JMDC insurance claims revealed that acetaminophen prevented the dyskinesia induced by dopamine D2 receptor antagonists. In vivo experiments further showed that a 21-day treatment with haloperidol increased the number of vacuous chewing movements (VCMs) in rats, an effect that was inhibited by oral acetaminophen treatment or intracerebroventricular injection of N-(4-hydroxyphenyl)-arachidonylamide (AM404), an acetaminophen metabolite that acts as an activator of the transient receptor potential vanilloid 1 (TRPV1). In mice, haloperidol-induced VCMs were also mitigated by treatment with AM404 applied to the dorsal striatum, an effect not seen in TRPV1-deficient mice. Acetaminophen prevented the haloperidol-induced decrease in the number of c-Fos+preproenkephalin+ striatal neurons in wild-type mice but not in TRPV1-deficient mice. Finally, chemogenetic stimulation of indirect pathway medium spiny neurons in the dorsal striatum decreased haloperidol-induced VCMs. These results suggest that acetaminophen activates the indirect pathway neurons by activating TRPV1 channels via AM404.
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http://dx.doi.org/10.1172/jci.insight.145632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262333PMC
May 2021

Intranasal oxytocin administration ameliorates social behavioral deficits in a POGZ mouse model of autism spectrum disorder.

Mol Brain 2021 03 16;14(1):56. Epub 2021 Mar 16.

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterized by core symptoms of impaired social behavior and communication. Recent studies have suggested that the oxytocin system, which regulates social behavior in mammals, is potentially involved in ASD. Mouse models of ASD provide a useful system for understanding the associations between an impaired oxytocin system and social behavior deficits. However, limited studies have shown the involvement of the oxytocin system in the behavioral phenotypes in mouse models of ASD. We have previously demonstrated that a mouse model that carries the ASD patient-derived de novo mutation in the pogo transposable element derived with zinc finger domain (POGZ mice), showed ASD-like social behavioral deficits. Here, we have explored whether oxytocin (OXT) administration improves impaired social behavior in POGZ mice and found that intranasal oxytocin administration effectively restored the impaired social behavior in POGZ mice. We also found that the expression level of the oxytocin receptor gene (OXTR) was low in POGZ mice. However, we did not detect significant changes in the number of OXT-expressing neurons between the paraventricular nucleus of POGZ 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.
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http://dx.doi.org/10.1186/s13041-021-00769-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962304PMC
March 2021

Pronociceptive Roles of Schwann Cell-Derived Galectin-3 in Taxane-Induced Peripheral Neuropathy.

Cancer Res 2021 Apr 19;81(8):2207-2219. Epub 2021 Feb 19.

Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Sakyo-ku, Kyoto, Japan.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency ( mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell-derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN. SIGNIFICANCE: These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2799DOI Listing
April 2021

Transient Receptor Potential Melastatin 3 Is Functionally Expressed in Oligodendrocyte Precursor Cells and Is Upregulated in Ischemic Demyelinated Lesions.

Biol Pharm Bull 2021 ;44(2):181-187

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University.

Oligodendrocyte precursor cells (OPCs) are glial cells that differentiate into oligodendrocytes and myelinate axons. The number of OPCs is reportedly increased in brain lesions in some demyelinating diseases and during ischemia; however, these cells also secrete cytokines and elicit both protective and deleterious effects in response to brain injury. The mechanism regulating the behaviors of OPCs in physiological and pathological conditions must be elucidated to control these cells and to treat demyelinating diseases. Here, we focused on transient receptor potential melastatin 3 (TRPM3), a Ca-permeable channel that is activated by the neurosteroid pregnenolone sulfate (PS) and body temperature. Trpm3/Pdgfra OPCs were detected in the cerebral cortex (CTX) and corpus callosum (CC) of P4 and adult rats by in situ hybridization. Trpm3 expression was detected in primary cultured rat OPCs and was increased by treatment with tumor necrosis factor α (TNFα). Application of PS (30-100 µM) increased the Ca concentration in OPCs and this effect was inhibited by co-treatment with the TRP channel blocker Gd (100 µM) or the TRPM3 inhibitor isosakuranetin (10 µM). Stimulation of TRPM3 with PS (50 µM) did not affect the differentiation or migration of OPCs. The number of Trpm3 OPCs was markedly increased in demyelinated lesions in an endothelin-1 (ET-1)-induced ischemic rat model. In conclusion, TRPM3 is functionally expressed in OPCs in vivo and in vitro and is upregulated in inflammatory conditions such as ischemic insults and TNFα treatment, implying that TRPM3 is involved in the regulation of specific behaviors of OPCs in pathological conditions.
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http://dx.doi.org/10.1248/bpb.b20-00510DOI Listing
January 2021

Prediction of pharmacological activities from chemical structures with graph convolutional neural networks.

Sci Rep 2021 01 12;11(1):525. Epub 2021 Jan 12.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Many therapeutic drugs are compounds that can be represented by simple chemical structures, which contain important determinants of affinity at the site of action. Recently, graph convolutional neural network (GCN) models have exhibited excellent results in classifying the activity of such compounds. For models that make quantitative predictions of activity, more complex information has been utilized, such as the three-dimensional structures of compounds and the amino acid sequences of their respective target proteins. As another approach, we hypothesized that if sufficient experimental data were available and there were enough nodes in hidden layers, a simple compound representation would quantitatively predict activity with satisfactory accuracy. In this study, we report that GCN models constructed solely from the two-dimensional structural information of compounds demonstrated a high degree of activity predictability against 127 diverse targets from the ChEMBL database. Using the information entropy as a metric, we also show that the structural diversity had less effect on the prediction performance. Finally, we report that virtual screening using the constructed model identified a new serotonin transporter inhibitor with activity comparable to that of a marketed drug in vitro and exhibited antidepressant effects in behavioural studies.
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http://dx.doi.org/10.1038/s41598-020-80113-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803991PMC
January 2021

Transient receptor potential vanilloid 4 agonist GSK1016790A improves neurological outcomes after intracerebral hemorrhage in mice.

Biochem Biophys Res Commun 2020 08 16;529(3):590-595. Epub 2020 Jul 16.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.

Intracerebral hemorrhage (ICH) is one of the most severe subtypes of stroke with high morbidity and mortality. Although a lot of drug discovery studies have been conducted, the drugs with satisfactory therapeutic effects for motor paralysis after ICH have yet to reach clinical application. Transient receptor potential vanilloid 4 (TRPV4), a Ca-permeable cation channel and activated by hypoosmolarity and warm temperature, is expressed in various cell types. The present study investigated whether TRPV4 would participate in the brain damage in a mouse model of ICH. ICH was induced by intrastriatal treatment of collagenase. Administration of GSK1016790A, a selective TRPV4 agonist, attenuated neurological and motor deficits. The inhibitory effects of the TRPV4 agonist in collagenase-injected WT mice were completely disappeared in TRPV4-KO mice. The TRPV4 agonist did not alter brain injury volume and brain edema at 1 and 3 days after ICH induction. The TRPV4 agonist did not show any differences with respect to the increased number of Iba1-positive microglia/macrophages, GFAP-positive astrocytes, and Gr1-positive neutrophils at 1 and 3 days after ICH induction. Quantitative RT-PCR experiments revealed that the TRPV4 agonist significantly upregulated the expression level of c-fos, a marker of neuronal activity, while the agonist gave no effects on the expression level of cytokines/chemokines at 1 day after ICH induction, These results suggest that stimulation of TRPV4 would ameliorate ICH-induced brain injury, presumably by increased neuronal activity and TRPV4 provides a novel therapeutic target for the treatment for ICH.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.103DOI Listing
August 2020

The Role of Dorsal Raphe Serotonin Neurons in the Balance between Reward and Aversion.

Int J Mol Sci 2020 Mar 21;21(6). Epub 2020 Mar 21.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.

Background: Reward processing is fundamental for animals to survive and reproduce. Many studies have shown the importance of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons in this process, but the strongly correlative link between the activity of DRN 5-HT neurons and rewarding/aversive potency is under debate. Our primary objective was to reveal this link using two different strategies to transduce DRN 5-HT neurons.

Methods: For transduction of 5-HT neurons in wildtype mice, adeno-associated virus (AAV) bearing the mouse tryptophan hydroxylase 2 (TPH2) gene promoter was used. For transduction in Tph2-tTA transgenic mice, AAVs bearing the tTA-dependent TetO enhancer were used. To manipulate the activity of 5-HT neurons, optogenetic actuators (CheRiff, eArchT) were expressed by AAVs. For measurement of rewarding/aversive potency, we performed a nose-poke self-stimulation test and conditioned place preference (CPP) test.

Results: We found that stimulation of DRN 5-HT neurons and their projections to the ventral tegmental area (VTA) increased the number of nose-pokes in self-stimulation test and CPP scores in both targeting methods. Concomitantly, CPP scores were decreased by inhibition of DRN 5-HT neurons and their projections to VTA.

Conclusion: Our findings indicate that the activity of DRN 5-HT neurons projecting to the VTA is a key modulator of balance between reward and aversion.
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http://dx.doi.org/10.3390/ijms21062160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139834PMC
March 2020

Pathogenic POGZ mutation causes impaired cortical development and reversible autism-like phenotypes.

Nat Commun 2020 02 26;11(1):859. Epub 2020 Feb 26.

Technology and Developmental Team for Mouse Phenotype Analysis, RIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.

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.
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http://dx.doi.org/10.1038/s41467-020-14697-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044294PMC
February 2020

Acute restraint stress augments the rewarding memory of cocaine through activation of α adrenoceptors in the medial prefrontal cortex of mice.

Neuropharmacology 2020 04 6;166:107968. Epub 2020 Feb 6.

Laboratory of Molecular Pharmacology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, 920-1192, Japan. Electronic address:

Stress augments the rewarding memory of cocaine, which plays a critical role in inducing cocaine craving. However, the neurobiological mechanisms underlying the enhancing effect of stress remain unclear. Here, we show that noradrenaline (NA) transmission in the medial prefrontal cortex (mPFC) mediates stress-induced enhancement of cocaine craving. When mice were exposed to acute restraint stress immediately before the posttest session of the cocaine-induced conditioned place preference (CPP) paradigm, the CPP score was significantly higher than that in non-stressed mice. Because extracellular NA levels have been reported to be increased in the mPFC during stress exposure, we assessed the effects of NA on mPFC layer 5 pyramidal cell activity. Whole-cell recordings revealed that NA application induces depolarization and a concomitant increase in spontaneous excitatory postsynaptic currents (sEPSCs). The NA effects were inhibited by terazosin, but not by yohimbine or timolol, and the sEPSC increase was not observed in the presence of tetrodotoxin, suggesting the involvement of postsynaptic α, but not α or β, adrenoceptors in the NA effects. Additionally, intra-mPFC injection of terazosin before stress exposure attenuated the stress-induced increase in cocaine CPP. Intra-mPFC injection of phenylephrine, an α adrenoceptor agonist, before the posttest session without stress exposure significantly enhanced cocaine CPP. Furthermore, chemogenetic suppression of mPFC pyramidal cells with inhibitory DREADD (designer receptors exclusively activated by designer drugs) also suppressed the stress-induced CPP enhancement. These findings suggest that the stress-induced increase in NA transmission activates mPFC pyramidal cells via α adrenoceptor stimulation, leading to enhancement of cocaine craving-related behavior.
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http://dx.doi.org/10.1016/j.neuropharm.2020.107968DOI Listing
April 2020

Myelin Oligodendrocyte Glycoprotein 35-55 (MOG 35-55)-induced Experimental Autoimmune Encephalomyelitis: A Model of Chronic Multiple Sclerosis.

Bio Protoc 2019 Dec 20;9(24):e3453. Epub 2019 Dec 20.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Multiple sclerosis (MS) is the common demyelinating disease of human central nervous system. Among mouse models available to study MS, including the cuprizone application and lysolecithin-injection models, experimental autoimmune encephalomyelitis (EAE) model is widely used so that chronic EAE model of C57BL/6J can reflect the autoimmune pathogenesis of MS well. Here we introduce the EAE model based on C57BL/6J mice, which is generated by injection of myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) as an antigen. After immunization with complete Freund's adjuvant, clinical signs and changes in body weight are observed one or two weeks later. The EAE model will continue to be useful for development of therapeutics for MS.
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http://dx.doi.org/10.21769/BioProtoc.3453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853963PMC
December 2019

Autism-associated protein kinase D2 regulates embryonic cortical neuron development.

Biochem Biophys Res Commun 2019 11 18;519(3):626-632. Epub 2019 Sep 18.

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan; Department of Pharmacology, Graduate School of Dentistry, Osaka University, Suita, Osaka, 565-0871, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.048DOI Listing
November 2019

Identification of neuron-type specific promoters in monkey genome and their functional validation in mice.

Biochem Biophys Res Commun 2019 10 23;518(4):619-624. Epub 2019 Aug 23.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan. Electronic address:

Viral gene delivery is one of the most versatile techniques for elucidating the mechanisms underlying brain dysfunction, such as neuropsychiatric disorders. Due to the complexity of the brain, expression of genetic tools, such as channelrhodopsin and calcium sensors, often has to be restricted to a specified cell type within a circuit implicated in these disorders. Only a handful of promoters targeting neuronal subtypes are currently used for viral gene delivery. Here, we isolated conserved promoter regions of several subtype-specific genes from the macaque genome and investigated their functionality in the mouse brain when used within lentiviral vectors (LVVs). Immunohistochemical analysis revealed that transgene expression induced by the promoter sequences for somatostatin (SST), cholecystokinin (CCK), parvalbumin (PV), serotonin transporter (SERT), vesicular acetylcholine transporter (vAChT), substance P (SP) and proenkephalin (PENK) was largely colocalized with specific markers for the targeted neuronal populations. Moreover, by combining these results with in silico predictions of transcription factor binding to the isolated sequences, we identified transcription factors possibly underlying cell-type specificity. These findings lay a foundation for the expansion of the current toolbox of promoters suitable for elucidating these neuronal phenotypes.
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http://dx.doi.org/10.1016/j.bbrc.2019.08.101DOI Listing
October 2019

Psychiatric-disorder-related behavioral phenotypes and cortical hyperactivity in a mouse model of 3q29 deletion syndrome.

Neuropsychopharmacology 2019 11 19;44(12):2125-2135. Epub 2019 Jun 19.

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1038/s41386-019-0441-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887869PMC
November 2019

CRISPR/Cas9-mediated in vivo gene editing reveals that neuronal 5-HT receptors in the dorsal raphe nucleus contribute to body temperature regulation in mice.

Brain Res 2019 09 10;1719:243-252. Epub 2019 Jun 10.

Department of Neuropharmacology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, N15 W7 Kita-ku, Sapporo 060-8638, Japan.

Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT receptor agonist-induced hypothermia was attenuated and antidepressant effect of a selective serotonin reuptake inhibitor (SSRI) was enhanced by microinjection of the viral vector expressing sgHtr1a into the DRN of Cre-dependent Cas9 knock-in mice. These results suggest that this in vivo CRISPR/Cas9-mediated 5-HT receptor gene knockout strategy provides a reliable and low-cost method for elucidating 5-HT receptor functions in specific cell types and brain regions. Further, we demonstrate that the neuronal 5-HT receptor in the DRN regulates body temperature and antidepressant effect of SSRI.
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http://dx.doi.org/10.1016/j.brainres.2019.06.009DOI Listing
September 2019

Depletion of microglia ameliorates white matter injury and cognitive impairment in a mouse chronic cerebral hypoperfusion model.

Biochem Biophys Res Commun 2019 07 13;514(4):1040-1044. Epub 2019 May 13.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Microglia are immune cells in the central nervous system (CNS) and essential for homeostasis that are important for both neuroprotection and neurotoxicity, and are activated in a variety of CNS diseases. Microglia aggravate cognitive impairment induced by chronic cerebral hypoperfusion, but their precise roles under these conditions remain unknown. Here, we used PLX3397, a colony-stimulating factor 1 receptor inhibitor, to deplete microglia in mice with chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis (BCAS). Cognitive impairment induced 28 days after BCAS was significantly improved in mice fed a diet containing PLX3397. In PLX3397-fed mice, microglia were depleted and white matter injury induced by BCAS was suppressed. In addition, the expression of proinflammatory cytokines, interleukin 6 and tumor necrosis factor alpha, was suppressed in PLX3397-fed mice. Taken together, these findings suggest that microglia play destructive roles in the development of cognitive impairment and white matter injury induced by chronic cerebral hypoperfusion. Thus, microglia represent a potential therapeutic target for chronic cerebral hypoperfusion-related diseases.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.055DOI Listing
July 2019

[The biological basis and application of lentiviral vector and adeno-associated viral vector in pharmacological research].

Authors:
Kazuki Nagayasu

Nihon Yakurigaku Zasshi 2019 ;153(5):204-209

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University.

Viral vectors, including lentiviral vectors and adeno-associated viral vectors, have been used as a delivery tool for transduction of neuronal and glial cells with a variety of genetic tools in vitro and in vivo. Although viral vector technologies are essential for application of genetic tools especially in vivo, less attention has been paid to the biological basis of these technologies than to genetic tools delivered. Here we would like to summarize the biological basis of lentiviral vectors and adeno-associated viral vectors and briefly introduce the recent advances from the perspective of the application of these viral vectors to pharmacological research.
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http://dx.doi.org/10.1254/fpj.153.204DOI Listing
August 2019

Pathophysiological Role of TRPM2 in Age-Related Cognitive Impairment in Mice.

Neuroscience 2019 06 15;408:204-213. Epub 2019 Apr 15.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.

Aging causes various functional changes, including cognitive impairment and inflammatory responses in the brain. Transient receptor potential melastatin 2 (TRPM2), a Ca-permeable channel expressed abundantly in immune cells, exacerbates inflammatory responses. Previously, we reported that TRPM2 on resident microglia plays a critical role in exacerbating inflammation, white matter injury, and cognitive impairment during chronic cerebral hypoperfusion; however, the physiological or pathophysiological role of TRPM2 during age-associated inflammatory responses remains unclear. Therefore, we examined the effects of TRPM2 deletion in young (2-3 months) and older (12-24 months) mice. Compared with young wild-type (WT) mice, middle-aged (12-16 months) WT mice showed working and cognitive memory dysfunction and aged (20-24 months) WT mice exhibited impaired spatial memory. However, these characteristics were not seen in TRPM2 knockout (TRPM2-KO) mice. Consistent with the finding of cognitive impairment, aged WT mice exhibited white matter injury and hippocampal damage and an increase in the number of Iba1-positive cells and amounts of pro-inflammatory cytokines in the brain; these characteristics were not seen in TRPM2-KO mice. These findings suggest that TRPM2 plays a critical role in exacerbating inflammatory responses and cognitive dysfunction during aging.
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http://dx.doi.org/10.1016/j.neuroscience.2019.04.012DOI Listing
June 2019

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation.

J Neurosci 2019 05 18;39(22):4208-4220. Epub 2019 Mar 18.

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan,

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 ( ) mice versus WT mice. Golgi staining of hippocampal CA1 neurons revealed a reduced spine densities and atypical morphologies in the male mice. Furthermore, viral miR-132 overexpression reversed the reduction in hippocampal spinal density in the male 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. 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 and 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.
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http://dx.doi.org/10.1523/JNEUROSCI.2468-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538856PMC
May 2019

An Adenosine A Receptor Antagonist Improves Multiple Symptoms of Repeated Quinpirole-Induced Psychosis.

eNeuro 2019 Jan-Feb;6(1). Epub 2019 Feb 27.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder characterized by the repeated rise of concerns (obsessions) and repetitive unwanted behavior (compulsions). Although selective serotonin reuptake inhibitors (SSRIs) is the first-choice drug, response rates to SSRI treatment vary between symptom dimensions. In this study, to find a therapeutic target for SSRI-resilient OCD symptoms, we evaluated treatment responses of quinpirole (QNP) sensitization-induced OCD-related behaviors in mice. SSRI administration rescued the cognitive inflexibility, as well as hyperactivity in the lateral orbitofrontal cortex (lOFC), while no improvement was observed for the repetitive behavior. D receptor signaling in the central striatum (CS) was involved in SSRI-resistant repetitive behavior. An adenosine A antagonist, istradefylline, which rescued abnormal excitatory synaptic function in the CS indirect pathway medium spiny neurons (MSNs) of sensitized mice, alleviated both of the QNP-induced abnormal behaviors with only short-term administration. These results provide a new insight into therapeutic strategies for SSRI-resistant OCD symptoms and indicate the potential of A antagonists as a rapid-acting anti-OCD drug.
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http://dx.doi.org/10.1523/ENEURO.0366-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397953PMC
April 2019

Glutamatergic neurons in the medial prefrontal cortex mediate the formation and retrieval of cocaine-associated memories in mice.

Addict Biol 2020 01 7;25(1):e12723. Epub 2019 Feb 7.

Laboratory of Molecular Pharmacology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan.

In drug addiction, environmental stimuli previously associated with cocaine use readily elicit cocaine-associated memories, which persist long after abstinence and trigger cocaine craving and consumption. Although previous studies suggest that the medial prefrontal cortex (mPFC) is involved in the expression of cocaine-addictive behaviors, it remains unclear whether excitatory and inhibitory neurons in the mPFC are causally related to the formation and retrieval of cocaine-associated memories. To address this issue, we used the designer receptors exclusively activated by designer drugs (DREADD) technology combined with a cocaine-induced conditioned place preference (CPP) paradigm. We suppressed mPFC neuronal activity in a cell-type- and timing-dependent manner. C57BL/6J wild-type mice received bilateral intra-mPFC infusion of an adeno-associated virus (AAV) expressing inhibitory DREADD (hM4Di) under the control of CaMKII promotor to selectively suppress mPFC pyramidal neurons. GAD67-Cre mice received bilateral intra-mPFC infusion of a Cre-dependent AAV expressing hM4Di to specifically silence GABAergic neurons. Chemogenetic suppression of mPFC pyramidal neurons significantly attenuated both the acquisition and expression of cocaine CPP, while suppression of mPFC GABAergic neurons affected neither the acquisition nor expression of cocaine CPP. Moreover, chemogenetic inhibition of mPFC glutamatergic neurons did not affect the acquisition and expression of lithium chloride-induced conditioned place aversion. These results suggest that the activation of glutamatergic, but not GABAergic, neurons in the mPFC mediates both the formation and retrieval of cocaine-associated memories.
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http://dx.doi.org/10.1111/adb.12723DOI Listing
January 2020

Manipulation of dorsal raphe serotonergic neurons modulates active coping to inescapable stress and anxiety-related behaviors in mice and rats.

Neuropsychopharmacology 2019 03 30;44(4):721-732. Epub 2018 Oct 30.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Major depression and anxiety disorders are a social and economic burden worldwide. Serotonergic signaling has been implicated in the pathophysiology of these disorders and thus has been a crucial target for pharmacotherapy. However, the precise mechanisms underlying these disorders are still unclear. Here, we used species-optimized lentiviral vectors that were capable of efficient and specific transduction of serotonergic neurons in mice and rats for elucidation of serotonergic roles in anxiety-like behaviors and active coping behavior in both species. Immunohistochemical analyses revealed that lentiviral vectors with an upstream sequence of tryptophan hydroxylase 2 gene efficiently transduced serotonergic neurons with a specificity of approximately 95% in both mice and rats. Electrophysiological recordings showed that these lentiviral vectors induced sufficient expression of optogenetic tools for precise control of serotonergic neurons. Using these vectors, we demonstrate that acute activation of serotonergic neurons in the dorsal raphe nucleus increases active coping with inescapable stress in rats and mice in a time-locked manner, and that acute inhibition of these neurons increases anxiety-like behaviors specifically in rats. These findings further our understanding of the pathophysiological role of dorsal raphe serotonergic neurons in different species and the role of these neurons as therapeutic targets in major depression and anxiety disorders.
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http://dx.doi.org/10.1038/s41386-018-0254-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372597PMC
March 2019

TRPM2 confers susceptibility to social stress but is essential for behavioral flexibility.

Brain Res 2019 02 28;1704:68-77. Epub 2018 Sep 28.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.

Transient receptor potential melastatin 2 (TRPM2) is a Ca-permeable, nonselective cation channel and a member of the TRP channel superfamily that acts as a sensor of intracellular redox states. TRPM2 is widely distributed in many tissues and highly expressed in the brain, but the physiological roles of TRPM2 in the central nervous system remain unclear. In this study, TRPM2-deficient mice were examined in a series of behavioral tests. TRPM2-deficient mice did not significantly differ from wild-type littermates in muscle strength, light/dark transition test, rotarod, elevated plus maze, social interaction, prepulse inhibition, Y-maze, forced swim test, cued and contextual fear conditioning, and tail suspension test. In the Barnes circular maze, TRPM2-deficient mice learned the fixed escape box position at similar extent to wild-type littermates, suggesting normal reference memory. However, performance of the first reversal trial and probe test were significantly impaired in TRPM2-deficient mice. In the T-maze delayed alternation task, TRPM2 deficiency significantly reduced choice accuracy. These results indicate that TRPM2-deficient mice shows behavioral inflexibility. Meanwhile, social avoidance induced by repeated social defeat stress was significantly attenuated in TRPM2-deficient mice, suggesting that TRPM2 deficiency confers stress resiliency. Our findings indicate that TRPM2 plays an essential role in maintaining behavioral flexibility but it increases susceptibility to stress.
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http://dx.doi.org/10.1016/j.brainres.2018.09.031DOI Listing
February 2019

TRPM2 Exacerbates Central Nervous System Inflammation in Experimental Autoimmune Encephalomyelitis by Increasing Production of CXCL2 Chemokines.

J Neurosci 2018 09 10;38(39):8484-8495. Epub 2018 Sep 10.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the CNS characterized by demyelination and axonal injury. Current therapies that mainly target lymphocytes do not fully meet clinical need due to the risk of severe side effects and lack of efficacy against progressive MS. Evidence suggests that MS is associated with CNS inflammation, although the underlying molecular mechanism is poorly understood. Transient receptor potential melastatin 2 (TRPM2), a Ca-permeable nonselective cation channel, is expressed at high levels in the brain and by immune cells, including monocyte lineage cells. Here, we show that TRPM2 plays a pathological role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Knockout (KO) or pharmacological inhibition of TRPM2 inhibited progression of EAE and TRPM2-KO mice showed lower activation of Iba1-immunopositive monocyte lineage cells and neutrophil infiltration of the CNS than WT mice. Moreover, CXCL2 production in TRPM2-KO mice was significantly reduced at day 14, although the severity of EAE was the same as that in WT mice at that time point. In addition, we used BM chimeric mice to show that TRPM2 expressed by CNS-infiltrating macrophages contributes to progression of EAE. Because CXCL2 induces migration of neutrophils, these results indicate that reduced expression of CXCL2 in the CNS suppresses neutrophil infiltration and slows progression of EAE in TRPM2-KO mice. Together, the results suggest that TRPM2 plays an important role in progression of EAE pathology and shed light on its putative role as a therapeutic target for MS. Current therapies for multiple sclerosis (MS), which mainly target lymphocytes, carry the risk of severe side effects and lack efficacy against the progressive form of the disease. Here, we found that the transient receptor potential melastatin 2 (TRPM2) channel, which is abundantly expressed in CNS-infiltrating macrophages, plays a crucial role in development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE progression was suppressed by Knockout (KO) or pharmacological inhibition of TRPM2; this was attributed to a reduction in CXCL2 chemokine production by CNS-infiltrating macrophages in TRPM2-KO mice, resulting in suppression of neutrophil infiltration into the CNS. These results reveal an important role of TRPM2 in the pathogenesis of EAE and shed light on its potential as a therapeutic target.
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http://dx.doi.org/10.1523/JNEUROSCI.2203-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596171PMC
September 2018

The impact of mouse strain-specific spatial and temporal immune responses on the progression of neuropathic pain.

Brain Behav Immun 2018 11 29;74:121-132. Epub 2018 Aug 29.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.

The present study was designed to investigate the correlation between the spatial and temporal aspects of immune responses and genetic heterogeneity in the progression of peripheral neuropathic pain. To address this issue, we first screened four inbred mouse strains (C57BL/6J, C3H/He, DBA/2, and A/J mice) to identify high- and low-responder strains to mechanical hypersensitivity induced by partial sciatic nerve ligation (pSNL). Among these strains, the C57BL/6J strain showed the highest vulnerability to pSNL-induced mechanical hypersensitivity, whereas the C3H/HeSlc strain was most resistant. C3H/HeSlc mice exhibited a significant increase in CD206-immunoreactivity (anti-inflammatory macrophages) in the dorsal root ganglia (DRG) at 3 and 7 days, and lower Iba1-immunoreactivity (microglia) in the spinal cord from 3 to 14 days after pSNL than C57BL/6J mice. These phenomena might be associated with a decrease in the production of inflammatory factors (interleukin-1β, interleukin-6, and CX3CL1) in the DRG and the poor responsiveness of spinal microglia (i.e. microglial production of IL1β, CCL2, and TNFα) against CX3CL1 in C3H/HeSlc mice. Behavioral experiments using bone marrow (BM) chimeric mice derived by crossing C3H/HeSlc and C57BL/6J strains showed that the strength of mechanical hypersensitivity 3 days following pSNL was inversely correlated with the increase in the ratio of anti-inflammatory/pro-inflammatory DRG macrophages, which was based on the BM-derived hematopoietic cells from donor mice. By contrast, the intensity of Iba1-immunoreactivity (microglia) in the spinal cord was dependent on the phenotypes of recipient mice, but not affected by the phenotypes of BM-derived donor hematopoietic cells. These findings suggest that the strain-specific aspects of DRG macrophages and spinal microglia might be related to the early and late phases of pSNL-induced mechanical hypersensitivity, respectively. This study presents a greater understanding of the differences in neuropathic pain among genetically heterogeneous inbred mouse strains, and provides further insights into the spatial and temporal roles of the immune system in the pathogenesis of neuropathic pain.
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http://dx.doi.org/10.1016/j.bbi.2018.08.013DOI Listing
November 2018

TRPA1 sensitization during diabetic vascular impairment contributes to cold hypersensitivity in a mouse model of painful diabetic peripheral neuropathy.

Mol Pain 2018 Jan-Dec;14:1744806918789812. Epub 2018 Jul 3.

1 Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.

Background Diabetic peripheral neuropathy is a common long-term complication of diabetes. Accumulating evidence suggests that vascular impairment plays important roles in the pathogenesis of diabetic peripheral neuropathy, while the mechanism remains unclear. We recently reported that transient receptor potential ankyrin 1 (TRPA1) is sensitized by hypoxia, which can contribute to cold hypersensitivity. In this study, we investigated the involvement of TRPA1 and vascular impairment in painful diabetic peripheral neuropathy using streptozotocin-induced diabetic model mice. Results Streptozotocin-induced diabetic model mice showed mechanical and cold hypersensitivity with a peak at two weeks after the streptozotocin administration, which were likely to be paralleled with the decrease in the skin blood flow of the hindpaw. Streptozotocin-induced cold hypersensitivity was significantly inhibited by an antagonist HC-030031 (100 mg/kg) or deficiency for TRPA1, whereas mechanical hypersensitivity was unaltered. Consistent with these results, the nocifensive behaviors evoked by an intraplantar injection of the TRPA1 agonist allyl isothiocyanate (AITC) were enhanced two weeks after the streptozotocin administration. Both streptozotocin-induced cold hypersensitivity and the enhanced AITC-evoked nocifensive behaviors were significantly inhibited by a vasodilator, tadalafil (10 mg/kg), with recovery of the decreased skin blood flow. Similarly, in a mouse model of hindlimb ischemia induced by the ligation of the external iliac artery, AITC-evoked nocifensive behaviors were significantly enhanced three and seven days after the ischemic operation, whereas mechanical hypersensitivity was unaltered in TRPA1-knockout mice. However, no difference was observed between wild-type and TRPA1-knockout mice in the hyposensitivity for current or mechanical stimulation or the deceased density of intraepidermal nerve fibers eight weeks after the streptozotocin administration. Conclusion These results suggest that TRPA1 sensitization during diabetic vascular impairment causes cold, but not mechanical, hypersensitivity in the early painful phase of diabetic peripheral neuropathy. However, TRPA1 may play little or no role in the progression of diabetic peripheral neuropathy.
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http://dx.doi.org/10.1177/1744806918789812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055098PMC
December 2018

Activation of GABAergic Neurons in the Nucleus Accumbens Mediates the Expression of Cocaine-Associated Memory.

Biol Pharm Bull 2018 ;41(7):1084-1088

Laboratory of Molecular Pharmacology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University.

Cocaine-associated environmental cues elicit craving and relapse to cocaine use by recalling the rewarding memory of cocaine. However, the neuronal mechanisms underlying the expression of cocaine-associated memory are not fully understood. Here, we investigated the possible contribution of γ-aminobutyrate (GABA)ergic neurons in the nucleus accumbens (NAc), a key brain region associated with the rewarding and reinforcing effects of cocaine, to the expression of cocaine-associated memory using the conditioned place preference (CPP) paradigm combined with designer receptors exclusively activated by designer drugs (DREADD) technology. The inhibitory DREADD hM4Di was selectively expressed in NAc GABAergic neurons of vesicular GABA transporter-Cre (vGAT-Cre) mice by infusing adeno-associated virus (AAV) vectors. Ex vivo electrophysiological recordings revealed that bath application of clozapine-N-oxide (CNO) significantly hyperpolarized membrane potentials and reduced the number of spikes induced by depolarizing current injections in hM4Di-positive NAc neurons. Additionally, systemic CNO injections into cocaine-conditioned mice 30 min before posttest session significantly reduced CPP scores compared to saline-injected mice. These results indicate that chemogenetic inhibition of NAc GABAergic neurons attenuated the expression of cocaine CPP, suggesting that NAc GABAergic neuronal activation is required for the environmental context-induced expression of cocaine-associated memory.
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http://dx.doi.org/10.1248/bpb.b18-00221DOI Listing
October 2018

Neurotropin inhibits neuronal activity through potentiation of sustained K currents in primary cultured DRG neurons.

J Pharmacol Sci 2018 Jul 31;137(3):313-316. Epub 2018 May 31.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Neurotropin (NTP) is a Japanese analgesic agent for treating neuropathic pain; however, its method of action remains unclear. This study examined the effects of NTP on the activity of small dorsal root ganglion (DRG) neurons using whole-cell patch clamp recordings. After 3 days of treatment, NTP decreased current injection-induced firing activity of cultured DRG neurons by raising the current threshold for action potential generation. Additionally, NTP increased the sustained component of voltage-gated potassium (K) channel currents without affecting other K currents. These results suggest that NTP inhibits the firing activity of DRG neurons through augmentation of sustained K current.
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http://dx.doi.org/10.1016/j.jphs.2018.05.005DOI Listing
July 2018

TRPV4 is functionally expressed in oligodendrocyte precursor cells and increases their proliferation.

Pflugers Arch 2018 05 22;470(5):705-716. Epub 2018 Mar 22.

Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Oligodendrocytes, which differentiate from oligodendrocyte precursor cells (OPCs), ensheath axons with myelin, play an essential role in rapid conduction of action potentials and metabolically support neurons. Elucidation of the mechanisms underlying the proliferation, migration, differentiation, and survival of OPCs is considered indispensable for determining the causes of central nervous system diseases. However, the relationship between these functions of OPCs and their intracellular Ca signaling has not been fully elucidated. Here, we investigated the function of transient receptor potential vanilloid 4 (TRPV4), a Ca-permeable channel that responds to hypo-osmolarity, mild temperature, mechanical stimulation, and endogenous arachidonic acid metabolites, in OPCs. Trpv4 mRNA was detected in OPCs in vivo and in primary cultured rat OPCs. In Ca imaging experiments, treatment with the selective TRPV4 agonist GSK1016790A induced sustained elevation of the intracellular Ca concentration in OPCs in a concentration-dependent manner, which was almost completely suppressed by co-treatment with the selective TRPV4 antagonist HC067047. Stimulation of TRPV4 by GSK1016790A augmented OPC proliferation, which was abolished by co-treatment with HC067047, the intracellular Ca chelator BAPTA-AM, and the protein kinase C inhibitor bisindolylmaleimide II. By contrast, GSK1016790A did not significantly affect the migration or differentiation of OPCs. Taken together, these results suggest that TRPV4 is functionally expressed in OPCs and increases the proliferation of these cells without affecting their ability to differentiate into oligodendrocytes.
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http://dx.doi.org/10.1007/s00424-018-2130-3DOI Listing
May 2018