Publications by authors named "Hisashi Shirakawa"

82 Publications

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

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

Synthesis and biological evaluation of radioiodinated 3-phenylcoumarin derivatives targeting myelin in multiple sclerosis.

Bioorg Med Chem Lett 2020 12 22;30(24):127562. Epub 2020 Sep 22.

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

Myelin is a lipid multilayer involved in the rate of nerve transmission, and its loss is a pathological feature of multiple sclerosis in brains. Since in vivo imaging of myelin may be useful for drug development, early diagnosis, and monitoring the disease stage, we designed, synthesized, and evaluated eight novel radioiodinated 3-phenylcoumarin derivatives as imaging probes targeting myelin. In the biodistribution study using normal mice, all compounds displayed sufficient brain uptake, ranging from 2.5 to 5.0% ID/g, at 2 min postinjection. On ex vivo autoradiography, [I]18 and [I]21, which have a dimethylamino group, showed high binding affinity for myelin in the normal mouse brain. In addition, the radioactivity accumulation of [I]21 in the white matter of the spinal cord in the experimental autoimmune encephalomyelitis mice was lower than that in naive mice. These results suggest that [I]21 shows potential as a single photon emission computed tomography probe targeting myelin.
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http://dx.doi.org/10.1016/j.bmcl.2020.127562DOI Listing
December 2020

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

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

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

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

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

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

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

Physiological and Pathophysiological Roles of Transient Receptor Potential Channels in Microglia-Related CNS Inflammatory Diseases.

Biol Pharm Bull 2018 ;41(8):1152-1157

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

Central nervous system (CNS) inflammation is a potential therapeutic target for neurodegenerative diseases. In recent years, a number of studies have focused on the links between neurodegenerative diseases and CNS glial cells, especially microglia. Microglia are the main resident immune cells in the CNS and represent approximately 10-15% of all CNS cells. Microglia play an important role in maintaining brain homeostasis at rest by surveying the environment, and engulfing apoptotic cells and debris in the healthy brain. However, under certain pathological conditions, microglia can generate neurotoxic factors, such as pro-inflammatory cytokines and molecules like nitric oxide (NO), which lead to CNS inflammatory diseases. In this review, we discuss the evidence that regulation of microglial ion channels may modulate CNS inflammation and subsequent tissue damage in neurological disorders. In particular, we discuss the role of transient receptor potential (TRP) channels in microglia in both acute and chronic inflammatory conditions, and describe the physiological and pathophysiological roles of TRP channels in CNS inflammatory pathways. Additionally, we describe the benefits of stimulation/inhibition of TRP channels in animal models of microglia-related CNS inflammatory diseases.
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http://dx.doi.org/10.1248/bpb.b18-00319DOI Listing
October 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

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

TRPM2 Channel Aggravates CNS Inflammation and Cognitive Impairment via Activation of Microglia in Chronic Cerebral Hypoperfusion.

J Neurosci 2018 04 9;38(14):3520-3533. Epub 2018 Mar 9.

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

Chronic cerebral hypoperfusion is a characteristic seen in widespread CNS diseases, including neurodegenerative and mental disorders, and is commonly accompanied by cognitive impairment. Recently, several studies demonstrated that chronic cerebral hypoperfusion can induce the excessive inflammatory responses that precede neuronal dysfunction; however, the precise mechanism of cognitive impairment due to chronic cerebral hypoperfusion remains unknown. Transient receptor potential melastatin 2 (TRPM2) is a Ca-permeable channel that is abundantly expressed in immune cells and is involved in aggravation of inflammatory responses. Therefore, we investigated the pathophysiological role of TRPM2 in a mouse chronic cerebral hypoperfusion model with bilateral common carotid artery stenosis (BCAS). When male mice were subjected to BCAS, cognitive dysfunction and white matter injury at day 28 were significantly improved in TRPM2 knock-out (TRPM2-KO) mice compared with wild-type (WT) mice, whereas hippocampal damage was not observed. There were no differences in blood-brain barrier breakdown and HO production between the two genotypes at 14 and 28 d after BCAS. Cytokine production was significantly suppressed in BCAS-operated TRPM2-KO mice compared with WT mice at day 28. In addition, the number of Iba1-positive cells gradually decreased from day 14. Moreover, daily treatment with minocycline significantly improved cognitive perturbation. Surgical techniques using bone marrow chimeric mice revealed that activated Iba1-positive cells in white matter could be brain-resident microglia, not peripheral macrophages. Together, these findings suggest that microglia contribute to the aggravation of cognitive impairment by chronic cerebral hypoperfusion, and that TRPM2 may be a potential target for chronic cerebral hypoperfusion-related disorders. Chronic cerebral hypoperfusion is manifested in a wide variety of CNS diseases, including neurodegenerative and mental disorders that are accompanied by cognitive impairment; however, the underlying mechanisms require clarification. Here, we used a chronic cerebral hypoperfusion mouse model to investigate whether TRPM2, a Ca-permeable cation channel highly expressed in immune cells, plays a destructive role in the development of chronic cerebral hypoperfusion-induced cognitive impairment, and propose a new hypothesis in which TRPM2-mediated activation of microglia, not macrophages, specifically contributes to the pathology through the aggravation of inflammatory responses. These findings shed light on the understanding of the mechanisms of chronic cerebral hypoperfusion-related inflammation, and are expected to provide a novel therapeutic molecule for cognitive impairment in CNS diseases.
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http://dx.doi.org/10.1523/JNEUROSCI.2451-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596050PMC
April 2018

Ketamine-Induced Prefrontal Serotonin Release Is Mediated by Cholinergic Neurons in the Pedunculopontine Tegmental Nucleus.

Int J Neuropsychopharmacol 2018 03;21(3):305-310

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

Background: Ketamine rapidly elicits antidepressive effects in humans and mice in which serotonergic activity is involved. Although α4β2 nicotinic acetylcholine receptor (α4β2 nAChR) in the dorsal raphe nucleus plays a key role in the ketamine-induced prefrontal serotonin release, the source of cholinergic afferents, and its role is unclear.

Methods: Prefrontal serotonin levels after ketamine injection were measured by microdialysis in rats. Electrolytic lesion of pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus was made with constant direct current.

Results: Bilateral lesion of the pedunculopontine tegmental nucleus, but not laterodorsal tegmental nucleus, attenuated prefrontal serotonin release induced by systemic ketamine. Intra-pedunculopontine tegmental nucleus, but not intra-laterodorsal tegmental nucleus ketamine perfusion, increased prefrontal serotonin release. This increase was attenuated by intra-dorsal raphe nucleus injection of dihydro-β-erythroidine, an α4β2 nAChR antagonist, or NBQX, an AMPA receptor antagonist.

Conclusions: These results suggest the ketamine-induced serotonin release in medial prefrontal cortex is mediated by cholinergic neurons projecting from pedunculopontine tegmental nucleus to dorsal raphe nucleus via α4β2 nAChRs.
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http://dx.doi.org/10.1093/ijnp/pyy007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838842PMC
March 2018

Pathophysiological Role of Transient Receptor Potential Ankyrin 1 in a Mouse Long-Lasting Cystitis Model Induced by an Intravesical Injection of Hydrogen Peroxide.

Front Physiol 2017 7;8:877. Epub 2017 Nov 7.

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

Chronic inflammatory bladder disorders, such as interstitial cystitis/bladder pain syndrome, are associated with poor quality of life. The exact pathological processes remain unclear, but accumulating evidence suggests that reactive oxidative species (ROS) are involved in urinary bladder disorders. Transient receptor potential ankyrin 1 (TRPA1), the most sensitive TRP channel to ROS, was shown to be responsible for urinary bladder abnormalities and hyperalgesia in an acute cystitis model. However, the roles of TRPA1 in chronic inflammatory bladder are not fully understood. We previously established a novel mouse cystitis model induced by intravesical injection of hydrogen peroxide (HO), resulting in long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes. In the present study, we investigated the pathophysiological role of TRPA1 in the HO-induced long-lasting cystitis mouse model. Under anesthesia, 1.5% HO solution was introduced transurethrally into the bladder of female wild-type (WT) and TRPA1-knockout mice and maintained for 30 min. This increased the number of voids in WT mice at 1 and 7 days after injection, but reduced the number in TRPA1-knockout mice at 1 day but not 7 days after injection. Spontaneous locomotor activities (increase in freezing time and decrease in distance moved) were reduced at 3 h after injection in WT mice, whereas the spontaneous visceral pain-related behaviors were attenuated in TRPA1-knockout mice. Furthermore, upregulation of mRNA in the spinal cord at 1 day after injection was observed in WT but not TRPA1-knockout mice. However, there was no difference in histopathological changes in the urinary bladder, such as edematous thickening in the submucosa, between WT and TRPA1-knockout mice at 1 or 7 days after injection. Finally, mRNA levels in the L5-S1 dorsal root ganglion were not altered, but levels in the urinary bladder were drastically increased at 1 and 7 days after injection. Taken together, these results suggest that TRPA1 contributes to acute bladder hyperactivity such as frequent urination and bladder pain, but does not appear to play a major role in the pathological processes of long-lasting cystitis.
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http://dx.doi.org/10.3389/fphys.2017.00877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715365PMC
November 2017

Distinct Mechanism of Cysteine Oxidation-Dependent Activation and Cold Sensitization of Human Transient Receptor Potential Ankyrin 1 Channel by High and Low Oxaliplatin.

Front Physiol 2017 1;8:878. Epub 2017 Nov 1.

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

Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1) is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD), which causes sensitization of TRPA1 to reactive oxygen species (ROS). Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1) with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1 mM) induced outward-rectifier whole-cell currents and increased the intracellular Ca concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser)-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala)-expressing cells showed similar whole-cell currents and Ca influx. By contrast, a lower concentration of oxaliplatin (100 μM) did not increase the intracellular Ca concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser)-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower concentration induces cold-induced cysteine oxidation-dependent opening of hTRPA1 via PHD inhibition.
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http://dx.doi.org/10.3389/fphys.2017.00878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672011PMC
November 2017

Colchicine alleviates acute postoperative pain but delays wound repair in mice: roles of neutrophils and macrophages.

Mol Pain 2017 Jan-Dec;13:1744806917743680

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

Background: Acute postoperative pain is induced by most incisional surgeries and usually resolves with wound repair. However, many patients experience moderate to severe pain despite receiving currently available postoperative pain relief. Accumulating evidence suggests that inflammatory cells, neutrophils, and macrophages infiltrating the wound site contribute to the acute inflammation, pain, and subsequent wound repair. Colchicine is commonly used to relieve pain in gout by inhibiting the infiltration of granulocytes and other motile cells. In this study, we examined the effects of colchicine on acute postoperative pain and wound repair by correlating the infiltration of neutrophils and macrophages in a mouse model of postoperative pain induced by plantar incision. Furthermore, these effects of colchicine were compared with clodronate liposomes, which selectively deplete circulating macrophages.

Results: Plantar incision induced mechanical hypersensitivity in the ipsilateral hind paw that peaked one day and lasted for three days after the surgery. Treatment with colchicine significantly attenuated the early infiltration of Gr1-positive cells (neutrophils) around the incision site and mechanical hypersensitivity, which was accompanied with inhibition of the subsequent infiltration of Iba1-positive cells (macrophages) and macrophage polarization toward the proinflammatory M1 phenotype. By contrast, an intravenous injection of clodronate liposomes significantly inhibited the infiltration of macrophages around the incision site but had little effect on the infiltration of neutrophils or mechanical hypersensitivity. Importantly, colchicine treatment significantly delayed wound closure after the incisional surgery, whereas clodronate liposome administration had no effect on wound closure.

Conclusion: These results suggest that colchicine can alleviate acute postoperative pain and also enhance the risk of delayed wound repair, which are associated with the suppression of neutrophil and subsequent proinflammatory M1 macrophage infiltration around the incision site, while the involvement of macrophages may be limited.
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http://dx.doi.org/10.1177/1744806917743680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5692123PMC
September 2018

Chronic antidepressant potentiates spontaneous activity of dorsal raphe serotonergic neurons by decreasing GABA receptor-mediated inhibition of L-type calcium channels.

Sci Rep 2017 10 19;7(1):13609. Epub 2017 Oct 19.

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

Spontaneous activity of serotonergic neurons of the dorsal raphe nucleus (DRN) regulates mood and motivational state. Potentiation of serotonergic function is one of the therapeutic strategies for treatment of various psychiatric disorders, such as major depression, panic disorder and obsessive-compulsive disorder. However, the control mechanisms of the serotonergic firing activity are still unknown. In this study, we examined the control mechanisms for serotonergic spontaneous activity and effects of chronic antidepressant administration on these mechanisms by using modified ex vivo electrophysiological recording methods. Serotonergic neurons remained firing even in the absence of glutamatergic and GABAergic ionotropic inputs, while blockade of L-type voltage dependent Ca channels (VDCCs) in serotonergic neurons decreased spontaneous firing activity. L-type VDCCs in serotonergic neurons received gamma-aminobutyric acid B (GABA) receptor-mediated inhibition, which maintained serotonergic slow spontaneous firing activity. Chronic administration of an antidepressant, citalopram, disinhibited the serotonergic spontaneous firing activity by weakening the GABA receptor-mediated inhibition of L-type VDCCs in serotonergic neurons. Our results provide a new mechanism underlying the spontaneous serotonergic activity and new insights into the mechanism of action of antidepressants.
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http://dx.doi.org/10.1038/s41598-017-13599-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648823PMC
October 2017

Sphingosine-1-phosphate induces Ca signaling and CXCL1 release via TRPC6 channel in astrocytes.

Glia 2017 06 16;65(6):1005-1016. Epub 2017 Mar 16.

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

A biologically active lipid, sphingosine-1-phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein-coupled receptors, some of which generates Ca mobilization. In astrocytes, S1P is reported to evoke Ca signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca -permeable cation channels expressed in astrocytes and involved in Ca influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P-induced cellular responses. In Ca imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca in astrocytes, followed by sustained elevation. The sustained Ca response was markedly suppressed by S1P receptor antagonist JTE013, S1P receptor antagonist CAY10444, or non-selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca signaling in astrocytes via G -coupled receptors S1P and S1P , followed by Ca influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1.
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http://dx.doi.org/10.1002/glia.23141DOI Listing
June 2017

A rat long-lasting cystitis model induced by intravesical injection of hydrogen peroxide.

Physiol Rep 2017 Feb 27;5(4). Epub 2017 Feb 27.

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

Novel longer lasting inflammatory bladder animal models are needed to better understand the pathophysiology of chronic cystitis. We previously developed a relatively long-lasting mouse cystitis model by intravesical injection of hydrogen peroxide (HO). To further evaluate its pathophysiology, in this study, we established and analyzed a rat cystitis model. Under anesthesia, 1.5% HO solution was introduced transurethrally into the bladder of female rats, and kept for 30 min. The HO injection significantly increased the number of micturition events up to day 14 and decreased urine volume per micturition, with the smallest volumes on day 3, compared with the vehicle-treated group. Cystometric analysis on day 7 revealed that intercontraction intervals were significantly shortened without affecting the baseline, threshold, or maximum pressures. Intravesical resiniferatoxin-evoked nociceptive behaviors, such as freezing, were significantly enhanced on days 7 and 14. Furthermore, histopathology revealed hemorrhage, edema, infiltration of neutrophils into the lamina propria, and urothelial denudation in the early phase (day 1). These damages were gradually repaired, while hyperplasia of the urothelium, vascularization, increases in fibroblast counts, and infiltration of mast cells and eosinophils were observed through the later phase (days 7 and 14). These results suggest that intravesical HO injection induces relatively long-lasting cystitis with enhanced bladder activity and pain sensation in rats. This approach thus provides a novel rat long-lasting cystitis model that allows us to analyze detailed symptoms and pathophysiology of HO-induced cystitis model than the mouse model and may be used to investigate the pathophysiology and treatment of chronic bladder hypersensitive disorders, such as bladder pain syndrome/interstitial cystitis.
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http://dx.doi.org/10.14814/phy2.13127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328770PMC
February 2017

Sequential PET estimation of cerebral oxygen metabolism with spontaneous respiration of O-gas in mice with bilateral common carotid artery stenosis.

J Cereb Blood Flow Metab 2017 Oct 9;37(10):3334-3343. Epub 2017 Feb 9.

1 Department of Investigative Radiology, National Cerebral and Cardiovascular Center Research Institute, Japan.

Positron emission tomography with O-labeled gases (O-PET) is important for in vivo measurement of cerebral oxygen metabolism both in clinical and basic settings. However, there are currently no reports concerning O-PET in mice. Here, we developed an O-PET method applicable to mice with spontaneous respiration of O-gas without a tracheotomy catheter. Sequential O-PET was also performed in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) induced by placement of microcoils. O-gas with isoflurane was supplied to the nose of mouse with evacuation of excess O-gas surrounding the body. O-PET was performed on days 3, 7, 14, 21, and 28 after surgery. Cerebral blood flow (CBF), cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO) were calculated in whole brains. A significant decrease in CBF and compensatory increase in OEF in the BCAS group produced CMRO values comparable to that of the sham group at three days post-operation. Although CBF and OEF in the BCAS group gradually recovered over the first 28 days, the CMRO showed a gradual decrease to 68% of sham values at 28 days post-operation. In conclusion, we successfully developed a noninvasive O-PET method for mice.
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http://dx.doi.org/10.1177/0271678X17692815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624393PMC
October 2017