L-type amino acid transporter 1 (LAT1) is a major essential amino acid transporter in cancer. Leucine is one of the LAT1 substrates and acts as a signaling molecule to regulate cell growth and proliferation by stimulating mTORC1 pathway. Recently, several groups, include us, reported that inhibition of LAT1 suppresses the growth of cancer cells. The inhibition of LAT1 is a promising procedure for cancer therapeutics. Many researchers have been developing LAT1 inhibitors, but all of them inhibits LAT1 in a competitive manner. Due to abundant amounts of amino acids in living organisms, the competitive inhibition seems not to be the most practical way to control the function of the amino acid transporter in vivo. Thus, we have developed a series of non-competitive inhibitors of LAT1, called OKY compounds.

In this study, we found that a classical LAT1 competitive inhibitor, BCH, and one of the OKY compounds suppressed mTORC1 pathway in the same manner, and both compounds even made similar effects on comprehensive phosphoproteomes in vitro. These results indicate that the inhibition of LAT1 causes the same effect on cellular signaling in both competitive and non-competitive manners.

LAT1 (SLC7A9) is a large-neutral amino acid transporter that forms heterodimer with CD98hc to function at the plasma membrane. LAT1 is a target for cancer diagnosis and therapy due to its important roles on cancer cell growth. PET imaging using FDG is a common cancer diagnosis. Yet, high accumulation of FDG in inflammatory lesions causes false positive results. In contrast, we and others reported that LAT1 selective PET tracers including ¹⁸F-FAMT (3-fluoro-L-a-methyl-tyrosine) were highly accumulated in tumors but not in inflammatory lesions.

Here, we demonstrated that FAMT was taken up by tumors but not by inflammatory lesions in animal models indicating no LAT1 function in inflammatory lesions, despite similar LAT1 expression in tumors and inflammatory lesions. LAT1 formed complex with CD98hc at the plasma membrane in tumors. However, LAT1 retained as monomer inside the cells of inflammatory lesions while CD98hc existed as dimer. Membrane proteome showed other SLC7 transporters in the inflammatory lesions whereas LAT1 dominated in tumor. Taken all together, in inflammatory lesions unlike tumors, LAT1 exists as a monomer inside of the cells and has no function at the plasma membrane, and likelihood CD98hc forms complex with SLC7 family beside LAT1. Our results suggest the different roles of LAT1 in tumors and inflammatory lesions.

Short chain fatty acids, including acetate, propionate and butyrate, have been shown to regulate various metabolic processes such as energy and lipid metabolism. Of these short chain fatty acids, butyrate has been identified as a ligand for GRP41/43, both of which are know no stimulate GLP-1 production from L cells in the intestine. Thus, the butyrate blood levels would correlate with GLP-1 production and one of the determinants of the butyrate level is its uptake/release by the liver. Human liver-specific organic anion transporter 7 (OAT7), expressed at the hepatic sinusoidal membrane, has been functionally characterized as an exchange transporter of butyrate with sulfate conjugated steroids.

The purpose of this study is to clarify the regulation of butyrate transport function. As a first step we attempted to identify OAT7 interacting proteins by yeast two-hybrid and immunoprecipitation followed by mass spectrometry in human liver cell lines, HepG2 and Huh7. One of the proteins identified by yeast two-hybrid, PDZK1/NHERF3, was likely to regulate OAT7 function. We are currently seeking other regulatory proteins for OAT7 by proteomic analysis of the immunoprecipitant.

Iguratimod (IGU) is a disease-modifying antirheumatic drug, whose activity is mainly ascribed to the inhibition of NF-kB pathway. Interestingly, we have noticed that the serum uric acid (UA) levels were decreased in 22% of rheumatoid arthritis patients taking IGU. Since there have been no reports of IGU affecting UA, we decided to explore how IGU lowers the serum UA level.

We first examined whether IGU inhibits the xanthine oxidase (XO) activity. Xanthine and XO were incubated with the drug and the production of UA was measured by absorbance at 290 nm. UA production was not inhibited by IGU. Next, we examined whether IGU or its metabolites inhibit the UA transporters. As M3 and M4 are the major metabolites in the urine, we examined the inhibitory activities of IGU and these metabolites against the major UA transporters, GLUT9 and URAT1, in the xenopus oocytes expression system. IGU and M4 inhibited the URAT1, and their IC₅₀s were 12.61 ± 0.97 μM and 120.07 ± 26.69 μM, respectively. However, the estimated maximal concentrations of these compounds in the proximal tubule were 4 - 7 times lower than the IC₅₀.

In conclusion, UA lowering effect of IGU is not likely to be explained by its or its metabolite's direct interaction with XO, URAT1, or GLUT9.

Endothelin (ET)-1 is involved in various diseases, including cancer, hypertension, atherosclerosis, diabetes, and fibrotic diseases, although ET-1 is originally identified as endothelium-derived vasocontractile peptide. ET receptors belong to the class A of G protein-coupled receptor, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). ET_AR and ET_BR generally exhibit the opposite responses, although many exceptions exist. Here, we attempted to identify ET_AR or ET_BR specific binding proteins to understand difference of ET_AR- and ET_BR-mediated responses upon ET-1 stimulation. We found that GRP78 exhibited a stronger binding affinity toward ET_BR than ET_AR. Overexpression of GRP78 promotes ET_BR-mediated ERK activation. In addition, the silencing of GRP78 suppressed ET_BR-mediated ERK activation. On the other hand, ET_BR can localize GRP78 to cell periphery. Our results suggest that interaction of ET_BR with GRP78 affects the ERK activation and GRP78 localization.

Since charged molecule cannot permeate cell membrane, urate movement across the epithelial cell layer has to be transcellularly by carrier or endocytosis/exocytosis, or paracellularly. We have reported that the paracellular route is the major urate transport pathway across the blood-placental barrier. In this study, the mechanism of urate paracellular transport was investigated in several epithelial cell lines. Very little urate passed through MDCK and LLC-PK1 cell layers. Interestingly, one Caco-2 cell line was urate non-permeable while another Caco-2 cell line was found to be urate-permeable. Urate paracellular movement across the Caco-2 cell layer was partially inhibited by DIDS, which inhibits chloride transport.

Detection and quantification of claudin proteins that are important for paracellular transport of ions were performed by LC/MS. Claudin 1, 3, 4, 6, 7 and 12 were detected in urate-permeable cell lines, BeWo and Caco-2 cells. However, overexpression of these claudins in MDCK cells did not increase urate paracellular transport.

In conclusion, overexpression of single claudin was not sufficient to make urate-non-permeable cell become urate permeable.

Purpose: To identify novel time-dependent (TD) inhibitors of uptake transporters in vitro.

Methods: HEK293 cells overexpressing uptake transporters OATP1B1/1B3, OAT1/3, OCT1/2, and MATE1/2-K were used to determine IC₅₀ values of corresponding inhibitors with or without 3 hours of preincubation. A total of 64 transporter-inhibitor combinations were analyzed. A shift in IC50 greater than 2.5-fold (i.e., IC₅₀ with preincubation ≤ 0.4 x IC₅₀ without preincubation) was considered relevant. In addition, transwell permeability of the inhibitors across a low efflux MDCK cell (MDCK-LE) monolayer was measured.

Results: TD inhibition was observed, albeit with different frequencies, across all classes of uptake transporters investigated. The proportion of inhibitors tested positive with at least one member of a cognate transporter pair was 3/5 for OATP1B1/1B3, 1/10 for OAT1/3, 6/9 for OCT1/2, and 1/8 for MATE1/2-K. In particular, ledipasvir, an antiviral previously not recognized as an OCT inhibitor, was shown to potently suppress both OCT1 and 2 upon preincubation (IC₅₀ with preincubation: 0.15 µM and 74.3 µM, respectively). MDCK-LE permeability of the inhibitors ranged between 0.012 and 16.9*10^-6 cm/s, and compounds with low to medium P_app (≤ 5*10^-6 cm/s) were more likely to show TD behavior, as such compounds were involved in 10/15, or 66.7%, of observed cases of TD inhibition. However, the association between MDCK-LE permeability and TD effect was not statistically significant. Among inhibitors that were non-substrates of their respective transporters, the magnitude of IC₅₀ shift correlated positively with cLogP (Spearman's r = 0.43, P=0.008) and molecular weight (r = 0.67, P<0.0001).

Conclusion: Since TD behavior was seen not only in OATPs but also in OATs, OCTs, and MATEs, the phenomenon of TD transporter inhibition seems to extend beyond OATPs. Non-substrate inhibitors with high hydrophobicity and high molecular weight were more liable to exhibiting a TD effect, while TD inhibition was less typical of high MDCK-LE permeability compounds.

Sansoninto (SAT) is prescribed for annoyance and insomnia. We previously demonstrated SAT attenuates aggressive behavior in social isolation-reared (SI) mice. However the mechanism by which SAT attenuates aggression is still unknown. Arginine vasopressin (AVP) plays a critical role in the regulation of aggression in mammals. AVP is regulated by 5-HTergic and DAergic systems. In this study, the effect of SAT on expression of 5-HTergic and DAergic system-related genes and AVP was examined using SI mice.

SAT was treated for 2 weeks. Group-reared (GR) mice were used as control. AVP level in bed nucleus of the stria terminalis was measured by EIA. Expression of 5-HTergic and DAergic system-related genes were analyzed by qPCR.

SI mice showed higher AVP level than GR mice. SAT-treated SI mice showed lower AVP level as much as GR mice. Catechol-O-methyltransferase (COMT) and 5-HT_3a receptor mRNA level was higher in hypothalamus and amygdala of SI mice compared with GR mice. SAT-treated SI mice showed lower level of COMT and 5-HT_3a receptor mRNA in hypothalamus, not in amygdala, than SI mice.

These results suggest that SAT reduces the increased AVP level via regulation of hypothalamic 5-HTergic and DAergic systems.

Nyoshinsan is one of the kampo medicine used in the treatment for menopausal disorder. We have already reported that ovariectomized (OVX) rats were seen in the decreases of voluntary activity at the dark time and serotonin level in amygdala (Behav. Brain. Res. 227(1)1-6(2012)). In this study, we examined effects of Nyosihnsan on a voluntary activity in OVX mice. The female ICR mice of 9-week old were OVX or sham operation. Either Nyoshinsan (750 mg/kg/day, p.o.) or water were administered to each group for 8 weeks (6 times/week) starting from 8 weeks after OVX. The voluntary activity of mice was evaluated by using an activity sensor (model NS-AS01 neuroscience, Inc.) at dark time (19:00-7:00). Moreover, RNA expression level of tryptophan hydroxylase (TPH) was measured in hippocampus and prefrontal cortex by Real-time PCR. In the result, Nyoshinsan significantly prevented decreased voluntary activity at dark periods induced by OVX. In addition Nyoshinsan suppressed down-regulation of TPH mRNA expression level induced by OVX. These results revealed that these OVX induced despair-like behaviors were improved by administration of Nyoshinsan. Moreover, it was also thought that the voluntary activity mediated by serotonin level in hippocampus.

Human iPSC-derived neurons are expected to be applied to toxicity evaluations in nonclinical studies. Microelectrode array (MEA), measurement system of the electrophysiological activity, are suitable to evaluate the seizure liability of drugs. We have previously reported the electrophysiological responses to several convulsive compounds using MEA in cultured hiPSC-derived neurons. However, the identification of analytical parameters to detecting seizure liability remains an important issue. We identify the analytical parameters enabling the separation of drug-induced responses between convulsants and negative control drugs, and the separation among the mechanism of action (MoA) in convulsants by using principal component analysis. Furthermore, we have succeeded in the prediction of MoA in PTZ and Linopirdine from MEA data using the identified analytical parameters. These our analysis method will be effective for detecting seizure liability and predicting the MoA of new drugs.

Multi-electrode array (MEA) assays using human induced pluripotent stem cell (hiPSC)-derived neurons are expected to predict the convulsion toxicity of new drugs. In the reliability of the evaluation system, it is important to compare the response of hiPSC-neuron with the response in the living brain. In this study, we compared the responses to typical convulsants in cultured hiPSC-derived cortical neuronal network compared with mouse acute hippocampal slice. 4-Aminopyridine (4-AP), Pilocarpine, Picrotoxin(PTX), Pentylenetetrazol (PTZ), and negative control acetaminophen, DMSO were tested in both samples using MEA system. The change of number of burst, inter burst interval (IBI) and CV of IBI in negative control and DMSO administration were almost same both samples. Although the number of burst increased both sample in convulsants administration, dose-dependency were difference between hiPSC-derived neurons and acute slices. However, CV of IBI were decreased dose-dependently both samples. In three convulsants, it was found that the hiPS neuron and the acute slice exhibit partially similar firing patterns.

Micro-electrode array (MEA) assay using human iPSC-derived neurons are expected to one of in vitro assays to predict the toxicity and predict the mechanism of action of drugs. MEA subteam of NeuTox Committee in Health and Environmental Science Institute (HESI) have started the pilot study for the prediction of seizure liability of drugs. In this study, we aimed to develop an analytical method enabling the evaluation of toxicity of convulsants using deep learning. Human iPSC-derived cortical neurons and astrocytes were cultured on 24-wells MEA plate for extracellular recording using MED64 Presto. HESI twelve compounds were tested at 5 concentrations for each compound (n>6). We firstly had artificial intelligence (AI) learned the data of convulsants and the data of non-convulsants. Next this AI predicted the Toxicity of the data not used for learning. The toxicity probability of unlearned sample data was 90% or more, and the toxicity probability of the unlearned convulsants was also 80% or more. In addition, the negative probability of non-convulsants was more than 80%. These results indicated that this AI analysis method is useful for predicting the convulsion toxicity using hiPSC-derived neurons.

Organophosphates (OPs) inhibit cholinesterase and hyperactivate the acetylcholinergic nervous system in the brain, causing motor excitement (e.g., tremor and seizures). However, the mechanism underlying the motor excitement by OPs remains unknown. Here, we performed behavioral and immunohistochemical studies in mice to investigate the tremorgenic mechanism of paraoxon, an active metabolite of parathion. Treating animals with paraoxon (0.15-0.6 mg/kg, i.p.) elicited kinetic tremor in a dose-dependent manner. Expressional analysis of Fos protein, a biomarker of neural excitation, revealed that a tremorgenic dose of paraoxon (0.6 mg/kg) significantly and region-specifically elevated Fos expression in the dorsolateral striatum (dlST) and the inferior olive (IO) among 48 brain regions examined. Moderate to slight increases in Fos expression was also observed in the cerebral cortex, hippocampus, nucleus accumbens, medial striatum, globus pallidus, medial habenula, and solitary nucleus, while these changes were not statistically significant. Paraoxon-induced tremor was inhibited by the nicotinic acetylcholine (nACh) receptor antagonist mecamylamine (MEC), but not affected by the muscarinic acetylcholine receptor antagonist trihexyphenidyl (THP). In addition, paraoxon-induced Fos expression in the dlST and IO was also antagonized by MEC, but not by THP. The present results suggest that OPs elicit kinetic tremor primarily by activating dlST and IO neurons via nACh receptors.

[Background] We studied whether rebamipide would exert healing effects in an acetic acid-induced model of oral stomatitis in animals either untreated or pretreated with 5-FU. [Methods] The oral stomatitis model was prepared by anesthetizing male Golden Syrian hamsters. The center of the cheek pouch was then exteriorized and sandwiched between ring forceps 5 mm in inside diameter, followed by submucosal injection of 25 µL of 10% acetic acid solution through the ring forceps. [Results] In animals un-pretreated with 5-FU, rebamipide at 3 mg/mL was shown to significantly inhibit acetic acid-induced oral stomatitis in comparison with animals that did not receive rebamipide. The area of oral injury in 5-FU-pretreated animals was significantly greater than that in animals not pretreated with 5-FU. The recovery time in 5-FU-pretreated group was longer than that in the 5-FU-un-pretreated group. In 5-FU-pretreated animals, the area of oral injury was significantly reduced in those that received 3 mg/mL rebamipide in comparison with those that did not receive rebamipide. [Conclusion] The present findings suggest that rebamipide has healing effects on acetic acid-induced oral stomatitis in animals both pretreated and un-pretreated with 5-FU.

Extracting teeth of patients treated with bisphosphonates (BPs) occasionally induces the necrosis of jaw (BRONJ), but the cause of disease is still unclear. I found out that the intracellular BP of osteoblastic cells was gradually accumulated in lysosomes. In the present study, I investigated the mechanism of osteoblastic cell death induced by BPs. MC3T3-E1 cells were used as osteoblatic cells. The uptake of BP into cells was observed by fluorescent pamidronate. Apoptosis was evaluated by PSVue 480 and CellEvent caspase 3/7. Formation of autophagosome and autolysosome was observed using DAPGreen and DALGreen respectively. Autophagosome and autolysosome were frequently observed in the cytosol of normal MC3T3-E1 cells. On the other hands, autolysosomes of cells treated with BPs were gradually accumulated around nuclei. The mitochondria of BPs-treated cells were decreased in both response and quantity. Moreover, BPs shrank nuclei of cells. After a while phosphatidylserine (early apoptotic marker) exposed on cell membranes was detected, and finally the activation of caspase 3 was observed. Mitochondria in cells treated with BPs were not co-localized with autolysosomes. These results suggest that BPs may accumulate autolysosome around nuclei of osteoblastic cells, and induce apoptosis due to inhibiting autophagic flux.

Unsaturated carbonyl compounds such as acrolein (ACR) and methyl vinyl ketone (MVK) are major cytotoxic factors in the gas phase extract of cigarette smoke. ACR and MVK induce cell membrane damage and cell death through protein kinase C and NADPH oxidases. We have previously reported that several antioxidants such as reduced glutathione (GSH) and N-acetylcysteine (NAC) can suppress ACR- and MVK-induced cell membrane damage and cell death, although the molecular mechanism has remained to be clarified. In this study, we have elucidated the mechanism for suppression of ACR- and MVK-induced cell injury by antioxidants. The molecules with thiol group such as GSH, NAC, and cysteine effectively suppressed cell membrane damage and cell death induced by cigarette smoke gas phase, ACR, and MVK. The results of HPLC and MS showed that GSH and NAC directly reacted with ACR and MVK. Cysteine and cysteine derivatives suppressed ACR-induced protein carbonylation. Current results suggest that GSH, NAC, and cysteine directly reacted with ACR and MVK, and suppressed unsaturated carbonyl compounds-induced cell damage by preventing protein carbonylation.

Toxicogenomics project is a Japanese government and pharmaceutical companies joint project to gather the gene expression data after exposure to about 150 compounds to rats in vivo for up to 28 days, rats and humans in vitro. The data are available for public use on the internet as "Open TG-GATES". Analysing the microarray data from "Open TG-GATES", genes fall in the following criteria were selected 1) increase more than 2 times, 2) the expression changes occur within 7 days from the initial dose, 3) those changes occur with several carcinogenic agents, 4) not or minimally expressed in the normal liver, and 5) related to proliferation or apoptosis. Several genes fell in the criteria and we focused on one of them, p75-NTR associated cell death executor (NADE) gene. NADE is known to bind to some partner proteins in the cell and in most cases, cells die by apoptosis. We further investigated whether NADE is a promising early carcinogenic marker using carcinogen exposed rats. Sprague-Dawley rats were treated with diethylnitrosamine for up to 8 weeks followed by up to another 11 weeks of no treatment. The liver was excised to obtain RNA and protein samples and paraffin embedded tissue sections. The NADE expression pattern correlates with the development and growth of cancerous cells. It is likely that the NADE plays a role in chemically induced carcinogenesis in the rat liver and it can be a good early gene marker of carcinogen.

Purpose: This study was aimed to develop a DMN-induced liver fibrosis model in rats and to evaluate effects of hMSC on the model.

Methods: DMN was given i.p. to SD rats 3 times weekly for 4 weeks. hMSC was injected at the day after the last DMN treatment. Blood was collected once a week to perform blood biochemistry test. Liver was removed from week 3 to 6 to observe the content of fibrosis and to perform histopathological examination.

Results: DMN treated rats showed a progressive increase in plasma ASAT, ALAT, γGT, Tbil and hyaluronan from week 1 to 4, a significant decrease in plasma Alb and a significant increase in plasma ALP, a significant increase in fibrosis area ratio and hydroxyproline of liver compared to the non-treatment rats. Histopathological examination indicated DMN induced inflammation and liver fibrosis from 3^rd week after start of DMN treatment. Administration of hMSC did not affect the model rats under the present conditions.

Conclusion: A rat liver fibrosis model was developed under the present study conditions and the model can be reproduced consistently within a relatively short period of time and can be used to assess the drug efficacy of potential anti-fibrotic agents.

Fast muscles (white muscles) are innervated by fast-type motor neurons (MNs); slow muscles (red muscles) are by slow-type MNs. VAChT-Cre is a Cre-driver mouse line that can direct DNA recombination in postnatal slow-type motor neurons (Misawa et al., genesis, 54, 568-572, 2016). To selectively eliminate slow-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which diphtheria toxin A (DTA) was expressed after the loxP-site excision. The VAChT-Cre;NSE-DTA mice (delta SlowMN mice) were born normally but showed progressive body weight loss, tremor and reduced life-span (average life, ca. 40 weeks). Muscular atrophy was evident in red muscles including the soleus and diaphragm, however white muscles were normal. Complex fiber-type transitions were observed in each muscles. The delta SlowMN mice showed hunched back posture (kyphosis) possibly by reduced trunk muscles. The tremor was kinetic in nature and most conspicuous in head and neck. The observed abnormalities were largely different from those observed in mutant SOD1-expressed ALS model mice in which fast-type motor neurons are known to be preferentially affected. The delta SlowMN mice could be a novel MN disease model characterized in proximal muscle atrophy, tremor and bulbar involvement.

Idiopathic pulmonary fibrosis (IPF) is a refractory disease that progresses from alveolar damage and inflammation to interstitial fibrosis. In this study, we confirmed the progress of the pathological condition of pulmonary fibrosis model in mice and investigated the effects of IPF therapeutic agent nintedanib ethanesulfonate (ofev).

Bleomycin was intravenously administered to 9-10 weeks old male ICR mice for 5 days. To confirm the progress of the pathological condition, the mice were euthanized after 28, 42 and 56 days of initial administration of bleomycin, lungs were collected and weighed. The content of lung hydroxyproline (HP) and fibrosis area were measured, and histopathological examination were performed. Ofev was administered to mice by gavage at 3 and 10 mg/kg once a day for 42 consecutive days.

Increases in relative lung weight and lung HP content were noted in mice given bleomycin for 5 consecutive days intravenously. The formation of fibrotic lesions was confirmed and increases in the fibrosis regions were observed over time, histopathologically.

In addition, orally administration of ofev at 10 mg/kg/day to mice for 42 days, suppression of increase in lung relative weight and lung HP content was confirmed.

p13 is mitochondrial protein originally identified as one of the proteins down-regulated in pancreatic islets of diabetic mice. Previously, we generated transgenic mice overexpressing p13 in pancreatic beta cells and demonstrated that overexpressed p13 exerts multiple beneficial effects against type 2 diabetes, such as the increase in islets size and insulin secretion. Here, we performed further histological analysis using pancreatic tissue slices of p13-knockout mice in order to investigate the roles of endogenous p13 in pancreatic morphogenesis and function. Although there was no significant difference in islet number, the average islet size was decreased by 42% in p13-knockout mice compared with wild-type mice. Notably, very small islets (<0.001 mm²) were observed specifically in p13-knockout mice. The present results suggest that p13 plays a critical role in islets morphogenesis.

Reactive oxygen species (ROS) is produced in immune cells during immune responses and is necessary for host defense and inflammation. Furthermore, ROS acts as signals for gene expression and required for T cell proliferation and activation. While low levels of ROS play important roles in cell activation, high levels of ROS induce significant damage to cells. To monitor redox state in living cells we generated transgenic mice expressing a green fluorescent protein (roGFP) whose fluorescence varies with redox state (J Invest Dermatol. 34, 1701-1709, 2014). CRO mice, measuring the redox state of whole cells and MRO mice, measuring the redox state of mitochondria were generated. Immune cells were isolated from CRO and MRO mice, and treated with hydrogen peroxide (oxidized state) or DTT (reduced state) to evaluate the maximum oxidation and reduction value in immune cells. Next, splenocytes isolated from CRO and MRO mice were stained with cell surface markers, and the redox state in various types of immune cells was analyzed by flow cytometry. We found that T cells were more oxidized than B cells in both CRO and MRO. This system should be a powerful and convenient tool for analyzing redox state in various types of immune cells.

Although accumulating evidence suggests that serotonin (5-HT) controls emotional behaviors and cognition via acting on its receptors, it is not clear that the receptor subtype responsible for the specific function. Fourteen pharmacologically distinct 5-HT receptor subtypes have been identified, but some of them lack any selective ligands because of their similarity. It is expensive and impractical to obtain knock-out mice for all the subtypes. Therefore we attempted to knock out 5-HT receptor genes by injecting virus vectors expressing sgRNA to adult Cas9 knock-in mice. This approach would provide easier and low-cost knock out of 5-HT receptor genes in specific cell types and brain regions. First, we targeted 5-HT1A receptors in the dorsal raphe nucleus (DRN) because the phenotype of 5-HT1A knock-out mice has been clarified. A 5-HT1A agonist, 8-OH-DPAT induced hypothermia is attenuated by the injection of viral vector expressing sgRNA for 5-HT1A gene into DRN of Cas9 knock-in mice. DNA sequencing and mismatch cleavage assay detected the target gene editing. Immunohistochemical analysis revealed that most of the virally induced Cas9 were expressed in GAD-positive GABAergic neurons.

Osteoclasts are derived from the monocyte-macrophage lineage of the bone marrow and are bone-resorbing cells essential for bone homeostasis. Osteoclasts contain more mitochondria and require more oxygen to support the energy demands associated with bone resorption. However, local oxygen distribution within bone marrow has not been completely understood. Intravital two-photon microscopy is a powerful tool for investigating biological processes in live animals. In this study, we performed two-photon phosphorescence lifetime imaging to characterize the oxygen tension in osteoclasts.

　Two-photon-enhanced phosphorescent probe, iridium complex (BTPDM1), was injected into knock-in mice in which EGFP is expressed in osteoclast lineage cells. The emissive triplet state of the BTPDM1 is sensitive to local oxygen tension. By measuring the phosphorescence from EGFP positive cells, oxygen tension in osteoclasts was determined. Furthermore, we quantitatively analyzed changes in oxygen tension in osteoclasts by measuring the phosphorescence decay time in EGFP positive cells. Thus, we have succeeded in detecting oxygen tension in vivo. This method is applicable to various cell types for determining local oxygen tension inside bone marrow.

Molecular target anticancer drugs have less adverse effects than conventional compounds but often provide patients with unfavorable events. Development of an appropriate administration protocol that alleviate the adverse effects and maximize the desirable effects requires monitoring of drug concentrations in individuals. This strategy is currently inaccessible, owing to no convenient method usable at a clinical site. Furthermore, as for recent drugs whose therapeutic windows remain unevaluated, it is difficult to control the adverse effects. To address these shortcomings, we describe a rapid and simple procedure for determination of the concentrations in blood samples using lenvatinib, a multi-kinase inhibitor, as a test reagent. This method stems from electrochemical measurement with diamond sensor that induces more stable reaction than classical materials. When guinea-pig plasma mixed with lenvatinib was examined, the sensor could detect a clinically relevant concentration of ≥ 1 µM. Time necessary for all the processes including sample's pretreatment did not exceed 10 minutes. This procedure may contribute to advances in personalized medicine.

Imatinib is a classical molecular targeted anticancer agent that specifically suppresses a hyperactivated tyrosine kinase and used for treatment of the patients with chronic myeloid leukemia. The application has been recently expanded to gastrointestinal stromal tumor. This drug is initially administered to all the patient at the same dose without normalization by parameters of the individuals such as the body surface area and weight and the variation in metabolism. Therefore, it is difficult to expect the effects of the therapy. Optimization of the administration protocol for individual patients requires immediate determination of plasma level of the compounds at clinical site. To address this issue, we show a simple procedure with an electrochemical approach. The sensor consists of boron-doped diamond electrode, a state-of-the-art material that elicits more stable reaction than classical materials. We examined guinea-pig plasma containing imatinib at different concentrations. With the procedure, each measurement was completed in 35 sec. This method was sensitive to the drug of 0.3 to 10 µM, the range included in a therapeutic window. The methodology described here may contribute to advances in pharmacotherapies for cancer.

Protein phosphorylation is a major and essential post-translational modification in eukaryotic cells that plays a critical role in various cellular processes. While recent advances in mass spectrometry based proteomics allowed us to identify approximately 200,000 phosphorylation sites, it is not fully understood which sites are phosphorylated by a specific kinase and which extracellular stimuli regulate the protein phosphorylation via intracellular signaling cascades. Recently, we have developed an in vitro approach termed the kinase-interacting substrate screening (KISS) method and an in vivo approach termed kinase-oriented substrate screening (KIOSS) method. Using KIOSS method, we analyzed the phosphorylation signals downstream of dopamine in mouse striatal slices, and found that about 100 proteins including ion channels and transcription factors were phosphorylated probably by PKA or MAPK. Here, we present an on-line database system which provides the phosphorylation signals identified by our KISS and KIOSS methods as well as those previously reported in the literature. The database system and its web portal, named KANPHOS (Kinase-Associated PHOspho-Signaling), were built based on the Next Generation XooNIps. We also demonstrate how to retrieve proteins and pathways in striatal medium-sized spiny neurons modulated by extracellular dopaminergic stimulation.

Fluorescence labeling of proteins is a key technique in live-cell imaging. We have developed a turn-on fluorescence labeling technique named DeQODE (De-Quenching of organic dye emission) tag technology. In this technology, a specially designed quenched fluorescent probe called QODE (Quenched organic dye emission) probe is used. The QODE probe consists of a fluorescent moiety and a quenching moiety. The DeQODE tag is the single chain antibody (scFv) that binds the quenching moiety to turn on fluorescence emission of the QODE probe. We here report a new QODE probe employing arylazopyrazoles moiety (AAP) as a quencher, and silicon rhodamine as a fluorophore. To produce the DeQODE tag for the new QODE probe, we obtained scFv clones against AAP. Two clones were found to express in a mammalian cell line 293T. The cells expressing these clones showed strong fluorescence in the presence of the QODE probe. Washing out of the free QODE probe was unnecessary for high-contrast imaging. This property is advantageous for application for in vivo imaging and high content drug screening.

An important advance in the RNA interference (RNAi) field was the discovery that plasmid vector-mediated expression of short hairpin RNA (shRNA) can substitute for synthetic small interference RNA (siRNA) s in vitro and in vivo. But the constitutive and/or ubiquitous knockdown of target gene by this method has still limited the utility, especially if the inhibition of target gene leads to lethality, which prevents in vivo functional analysis. To overcome these limitations, the time (or period)- and cell (or tissue)- specific regulation of shRNA expression should have been required as the double-conditional mice. Conventional protocols for such an artificial regulation of gene expression in vivo are based on the tetracycline-controlled reversible system for the time- and the Cre-loxP system for cell-specificities respectively. The double-conditional transgenic mice are expected by mating their two lines of genetic engineered mice at last. Therefore, we have designed the "All-in-One" type single double-conditional shRNA expression vectors having both systems within a single vector format to make the transgenic mice with double-conditional RNAi without much effort.

Positron Emission Tomography (PET) is a non-invasive molecular imaging technique for clinical diagnostic with the detection of pathological lesions. Small radioactive molecules such as fluorodeoxyglucose have been mainly used as PET tracers. However, they have some challenges in the aspect of their specificity. Recently, small protein ligands (6-7 kDa), affibody molecules, are attracting increasing interest as an innovative PET tracer owing to their promised high specificity and affinity. In this study, we propose a novel radiolabeling method for protein with F-18, termed TAG-encoded cell-free protein synthesis system with F-18 labeled amino acid, to use affibody molecules as PET tracers. Furthermore, we show the successful preparation of radiolabeled affibody molecules targeted to HER2 and PD-L1 with this method. A small animal PET imaging with F-18 labeled affibodies surely demonstrated the accumulation of tracers in tumors expressing HER2 or PD-L1. This technique will be a powerful tool for the evaluation of potential candidates for PET tracers in preclinical settings because of its facile radiolabeling of protein just by adding the template DNA plasmids.

G-protein-coupled receptors (GPCRs) may form homomeric or heteromeric complexes and cooperatively mediate intracellular responses. Previously, we showed modulation of type 1 metabotropic glutamate receptor (mGluR1) function by metabotropic gamma-aminobutyric acid receptor (GABA_BR) in cerebellar Purkinje cells. The activity of mGluR1 is mediated by a G_q protein, and has a crucial role in synaptic plasticity and motor learning. GABA_BR inhibits neuronal activity through G_i protein, which regulates the release of neurotransmitters and the activity of ion channels. In this report, we investigated in greater detail the relationship of these GPCRs using non-neuronal cells. Using live cell imaging and biochemical analysis, we showed that mGluR1 and GABA_BR form complexes at the cell surface. Moreover, using cAMP homogenous luminescence assay and calcium imaging, we found that mGluR1 and GABA_BR regulate their signal transduction of each other. These findings provide a new insight into neuronal GPCR signaling and demonstrate a novel regulatory mechanism of synaptic transmission. This interaction would be involved in several important physiological and pathophysiological functions related to mGluR1 and GABA_BR, such as cerebellar motor learning and its dysfunction.

Renin-angiotensin system is believed to have important roles in blood pressure regulation. Baro and chemoreceptor afferents project to the nucleus tractus solitarius (NTS) neurons. The expression of angiotensin II type 1 (AT₁) and 2 (AT₂) receptors in the NTS is confirmed. However the physiological roles of angiotensin II in NTS are not fully understood. We have previously reported that angiotensin II increased the frequency of spontaneous EPSCs in 33 % of the cells through the activation of AT₁ receptors and decreased it in 39 % of the cells through the activation of AT₂ receptors. In this report we aimed to reveal the effects of angiotensin II on tractus solitarius evoked EPSCs (eEPSCs) in the NTS by using a slice patch-clamp technique.

 Angiotensin II decreased the amplitude of eEPSCs in 56 % of the neurons and had no effect in 44 % of the cells. Under the presence of AT₁ receptor blocker (ARB) losartan (10 μM), angiotensin II increased the eEPSCs in 37 % of the cells and decreased it in 15 % of the cells. The other neurons (48 %) showed no responses.

These results suggest that the activation of AT₁ receptors induces opposite effects on spontaneous and synchronous release of glutamate.

The molecular basis with which we acquire and maintain sociality has been still unknown. Autism spectrum disorder (ASD) is defined by social communication deficits, indicating the molecular basis of sociality should be damaged in this condition. Many genes associated with ASD encode proteins involved in synapse formation or maintenance, especially synaptic adhesion molecules (SAMs). On the other hand, sex hormones may be involved in ASD onset, but its molecular basis remains uncertain. Here, we show a novel interaction between sex hormones and SAMs. We focused on Octodon Degus as a model animal due to their highly organized sociality. Degus are medium-sized diurnal rodents, and communicate with each other by using more than 20 vocal repertoires. We investigated amino-acid sequences of several SAMs expressed in degus brains and compared with those of humans. Interestingly, a particular SAM of degus shared more than 90% homology with human sequence. We analyzed the binding of the SAM pairs and revealed that a sex hormone disrupted their binding. We also found that one of these SAMs directly binds to the sex hormone. Therefore, we show a possible molecular mechanism of sex hormones affecting ASD and sociality formation.

Primary cilia are microtubule-based organelles mediating sensory and neuroendocrine signaling. The importance of cilia function is underscored by ciliopathies often presenting clinical manifestations such as obesity. Many neurons possess primary cilia that are enriched for certain G protein-coupled receptors, including melanin-concentrating hormone (MCH) receptor 1 (MCHR1). The MCH system is known to mediate distinct aspects of energy balance and vital behavior. Although short cilia have been observed in genetic obese mice, a possible correlation between MCHR1-positive neuronal cilia length and energy metabolism has not been characterized. Here, we established a novel protocol to detect ciliary receptors in rat hippocampal slice culture. Ciliary MCHR1 were abundantly located in the CA1 and CA3 but not in DG. The features in each region were not uniform; the length of ciliary MCHR1 in CA1 were significantly longer than that in the CA3. Then, by using our culture system, we provide the first evidence that endogenous MCHR1-positive cilia length in neuron is significantly reduced by MCH at nanomolar order, and this appears to selectively occur in CA1 neurons. Future work will investigate the molecular mechanism of cilia shortening in responses to MCH.

Neurotransmitter release is confined to a specialized area of the presynaptic plasma membrane known as the active zone which consists of a large number of synaptic proteins including Munc13. The composition and in situ arrangement of the active zone proteins remain unclear. In this study, we developed an optimized immunostaining method to visualize active zone proteins at synapses in the hippocampus, and analyzed their nanoscale spatial distribution by multi-color and three-dimensional super-resolution imaging. We found that active zone proteins form discrete nanoscale supramolecular assemblies in an ordered arrangement as we have previously found for Munc13 proteins. The distance of individual supramolecular assemblies to Munc13 assembly, which marks the synaptic vesicle release site, varied among active zone proteins. Interestingly, the composition and distribution of active zone proteins varied among types of synapses, e.g. Schaffer collateral synapses, perforant path synapses, and mossy fiber synapses. Our results provide insight into supramolecular structure responsible for universality and diversity of synaptic functions.

Generation of reactive oxygen species (ROS) causes lipids, proteins and DNA damage, resulting in neuronal damage and neruoinflammation. Ratanasampil (RNSP), a traditional Tibetan medicine, clinically used for the mild-to moderate AD patients living at high altitude. In vivo, RNSP improved the learning and memory in an AD mouse model (Tg2576). However, mechanism underlying the effects of RNSP is unknown. In SH-SY5Y cells, RNSP significantly ameliorated the H₂O₂ –induced cytotoxicity. Furthermore, RNSP significantly reduced the H₂O₂-induced 8-oxo-2′-deoxyguanosine and attenuated the phosphorylation of p38 and ERK 1/2. In MG6 microglia, RNSP significantly ameliorated the cytotoxicity induced by hypoxia-reoxygenation. Furthermore, RNSP significantly suppressed the H6/R24-induced pro-inflammatory cytokines, ROS, DNA damage and phosphorylation of IκBα. These observations suggest that RNSP suppressed the H₂O₂-induced neuronal death through downregulation of p38-ERK activation and regulated the H/R-induced neuroinflammation through inhibition of oxidative stress and the activation of NFκB in MG6 cells. Therefore, RNSP may be beneficial for preventing oxidative stress-induced neuronal death and neuroinflammation.

Radiation therapy (RT) is effective method to remove brain tumors. RT is also applied to pediatric patients, although comorbid adverse events, such as intellectual or cognitive disfunction, is severe problem. To understand the molecular events that occurs after RT will be promising to form pharmaceutical method to ameliorate these disfunctions. We examined the X-ray sensitivity of primary neuronal culture of embryonic rat (embryonic days of 16.5 – 18.5 days) cortex by employing trypan blue exclusion test. We found that the death fraction of cells after irradiation increased. We also performed immunofluorescence staining to detect a K⁺-Cl^- co-transporter KCC2, which plays an important role in mediating intracellular Cl^- concentration ([Cl^-]_i), and found that perimembrane KCC2 signals declined, suggesting that decline of KCC2 would result in decrease of [Cl^-]_i, followed by hyperexcitability. We then testified whether afterward oxytocin administration restore declined KCC2 signals in X-ray irradiated cells and found that 10nM of oxytocin administration restored KCC2. In addition, we performed γ-ray irradiation to head of 10-week-old mice. It was found that KCC2 decreased in mice cortex which were irradiated with 3 Gy γ-ray irradiation by using western blot analysis. Collectively thought from these results, we assume that KCC2 is useful to analyze the effect of irradiation and to examine the afterward restoration by drug administration.

Hydrophilic antioxidant ergothioneine (ERGO) is not synthesized in mammals, but ingested from daily life in humans. Oral administration of ERGO exhibits several beneficial effects in the brain in experimental animals. ERGO promotes neuronal differentiation of neural progenitor cells in primary culture, and involvement of the activation of mTOR pathway has been proposed (Cell Signal 53, 269, 2018), although its directly interacting proteins have not yet been clarified. The aim of the present study is to perform comprehensive study to clarify the pathways involved in the pharmacological activity of ERGO. After repeated oral dose of ERGO or vehicle alone in normal mice, hippocampal dentate which is important in neurogenesis was isolated, and membrane proteome analysis using LC-MS/MS was conducted. Accordingly, 3,337 proteins were identified, and we found change in expression of proteins associated with mitochondria and synapse formation. Mouse neural stem cells were primarily cultured for 6 days, followed by incubation with ERGO. Gene product of neuronal marker b-III tubulin was increased by ERGO, confirming neuronal differentiation. Effect of ERGO on mitochondrial proteins is now under investigation.

Gelatin can be enzymatically hydrolyzed to yield collagen hydrolysates potentially applicable in the food industry. When collagen hydrolysates are ingested, several di- and tri-peptides (collagen peptide, CP) with various physiological activities are detected in human blood. We investigated whether CPs exert trophic effects on the differentiation of primary cultured cerebellar granule cells (CGC), using the MTT assay. Addition of specific tripeptides (TP-X) contained in the collagen hydrolysate in differentiating CGC cultures prevented extensive neuronal degeneration, which was observed in growth media containing low potassium (15 mM, K15). When CGC was cultured in K15, the cell viability was 64.4% relative to high potassium (25 mM) at 7 days in vitro. Under this condition, TP-X (10 μM) increased cell survival up to 76.6%. The effect was similar to the neuroprotective effect resulting from supplementation with 100 ng/mL brain-derived neurotrophic factor (76.5%) or 100 μM N-methyl-D-aspartate (97.2%). Currently, the mechanism underlying TP-X-mediated neuronal survival is unclear. Various functions of CP in skin, cartilage, and bone have been reported previously, and the present study further suggests a new possibility of improvement of cranial nerve function upon treatment with CP.

Caffeine (1,3,7-trimethylxanthine) consumption reduces the incidence of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We have previously shown that not only caffeine but also uric acid, which is a final metabolite of caffeine, increase the intracellular glutathione (GSH) levels in the hippocampus that is due to promotion of cysteine uptake into neurons (Neuroscience 2011). In our recent study, paraxanthine (1,7-dimethylxanthine), a major metabolite of caffeine also increased cysteine uptake in mouse hippocampus slices. In this presentation, we focused on the effect of caffeine metabolites on the cysteine uptake and the GSH levels in mouse hippocampal neurons. We analyzed the cysteine and GSH levels in hippocampal neurons after an intraperitoneal injection of 10 mg/kg caffeine, uric acid or paraxanthine into C57BL/6 mice. The cysteine and GSH contents of the tissues were quantitated by HPLC-fluorescence detection and the GSH levels in neuronal cells in hippocampal slices were detected by CMFDA staining. We show that caffeine and its metabolites promote cysteine uptake leading to the increased GSH levels in hippocampal neurons.

Accumulation of amyloid-β (Aβ) around cerebral blood vessels is found in more than 80% of Alzheimer's disease (AD) patients, and peripheral Aβ can accumulate in brain triggering degeneration. Recently, periodontitis has been reported positively link to AD, however, the mechanism of peripheral Aβ transport into brain is unclear. we hypothesized that periodontitis may involve in peripheral Aβ transport into brain. In the present study, we aim to examine the expression of Receptor for advanced glycation end products (RAGE) on brain endothelial cells after infection with Porphyromonas gingivalis (P.g.), the major pathogenic bacteria of periodontitis, because RAGE is mediated in transporting peripheral Aβ into brain. The mRNA level and immunofluorescent signal of of RAGE were significantly increased in hCMEC/D3 cells after P.g exposure. The expression of RAGE on CD31-positve endothelial cells were significantly increased in the P.g infected mice compared to control mice. Moreover, Aβ were detected on CD31-positve endothelial cells surrounding cerebral blood vessels in the P.g infected mice. These observations suggested that increased RAGE expression in endothelial cells is involved in Aβ influx into brain after P.g infection.

Glutamate excitotoxicity via NMDA receptors is associated with retinal ganglion cell (RGC) death in retinal diseases, such as glaucoma and diabetic retinopathy. We have previously reported that the apelin receptor is expressed in the RGCs and intravitreal injection of apelin inhibits RGC death induced by NMDA in mice. In the present study, we investigated whether systemic administration of an apelin receptor agonist protects the RGCs from NMDA-induced excitotoxicity. The apelin agonist ML233 (5 mg/kg) was administered intraperitoneally at 1 h before intravitreal injection of NMDA (10 nmol) in mice. The effect of ML233 on RGC death was assessed by immunohistochemistry with anti-Brn-3a and anti-calretinin antibodies. ML233 significantly prevented the decrease of the number of Brn-3a and calretinin-positive RGCs at 24 h after NMDA injection. Moreover, ML233 markedly suppressed NMDA-induced cell death of calretinin-positive amacrine cells, which are exquisitely sensitive to glutamate excitotoxicity in the retina. Our results suggest that systemic administration of apelin receptor agonists prevents retinal neuronal death induced by excitotoxicity via NMDA receptors.

In the hippocampal CA1 area, characteristic oscillations in local field potentials (LFPs) have been related to important functions for memory encoding and consolidation. Synchronous or sequential activity of neurons during oscillations in LFPs is thought to encode information, but the relationships between LFPs and activity of multiple pyramidal neurons have not fully been investigated. In this study, we examined population dynamics of subthreshold membrane potentials, which underlie the firing activity, using multiple whole-cell recordings of up to four CA1 pyramidal neurons simultaneously with recordings of CA1 LFPs from anesthetized mice. In particular, we compared theta frequency-band (3-10 Hz) oscillations between LFPs (i.e., type 2 theta) and membrane potentials of multiple neurons under urethane anesthesia. We found weak but significant correlations of event timings and frequencies of theta oscillations between LFPs and subthreshold membrane potentials. Our results provide an insight into our understanding of how subthreshold dynamics of each cell is incorporated in collective ensemble activity.

In the striatum, dopamine modulates these functions via cAMP/PKA signal-mediated mechanisms. Recent studies revealed that structural organization and cortical innervation are different among subregions of the striatum. Therefore, we investigated dopamine signaling in subregions of the striatum. Mouse striatal slices were divided into seven subregions: (1) rostral part, (2-1) intermediate medial part, (2-2) intermediate lateral part, (2-3) intermediate most lateral part, (3) caudal part, (4) most caudal part, (5) nucleus accumbens. Each slice was treated with a D1 receptor agonist, SKF81297 (1 μM) and activity of cAMP/PKA signal was evaluated with the phosphorylation of DARPP-32, GluA1. The stimulatory effects of SKF81297 on the phosphorylation were the lowest in the subregion (3) in the rostrocaudal axis and in the subregion (2-3) in the mediolateral axis. Treatment of slices with a PDE10A inhibitor, papaverine, or SKF81297 plus a muscarinic receptor antagonist, atropine, increased the phosphorylation in subregions where the effect of SKF81297 on the phosphorylation was low. Thus, dopamine D1 receptor/cAMP/PKA signaling is differentially regulated in each subregion of the striatum, and the differences are mediated by PDE10 and/or muscarinic receptor.

Dietary habits are important factors affecting the development of emotion. We have shown that long-term powdered food (PF) feeding in mice increases locomotor activity and social interaction time (SI). Although the increased SI indicates low anxiety, the elevated plus maze test (EPM) shows not only anxiety-related behavior but also impulsive behavior. In this study, we investigated whether the PF feeding causes changes in anxiety-related behavior. Mice fed a PF for 17 weeks were compared with mice fed a standard food. The % of open arm time (OAT) and total number of arm entries were increased in PF-fed mice in the EPM. Moreover, we examined the effects of methylphenidate (MP), dopamine transporter (DAT) and noradrenaline transporter (NAT) inhibitor, atomoxetine (AT), selective NAT inhibitor, GBR12909 (GB), selective DAT inhibitor, and PD168077 (PD), selective D4 receptor agonist, on the changes of EPM in PF-fed mice. MP and AT are clinically used to treat ADHD symptoms. The OAT in PF-fed mice was decreased by MP, AT and PD, but not GB. These results suggest that the PF feeding may cause low anxiety or impulsivity, possibly via NA and DA systems and increase the risk for onset of ADHD-like behaviors.

Pharmacological studies have suggested that serotonin 5-HT_2C receptor is involved in locomotor activity, anxiety, and fear memory. However, the results of locomotor activity and anxiety in 5-HT_2C receptor knockout mice are mixed, and the effects of 5-HT_2C receptor knockout on fear memory have not yet been addressed. In the present study, we reconciled these inconsistent results by analyzing behavioral data in details. We revealed that the higher locomotor activity in 5-HT_2C receptor knockout mice is observed only in the late phase of the test. Moreover, we found that 5-HT_2C receptor knockout mice display a hesitating attitude, staying in the center area and risk assessment behavior, in the elevated plus maze test. This phenotype might explain the inconsistency of previous studies. In the contextual fear conditioning test, 5-HT_2C receptor knockout mice tended to show rapid within-session extinction of fear, but not between-session extinction, compared to the wild type mice.

Establishment of dyskinesia, one of the symptoms (side effects) induced by levodopa was assessed in a unilateral 6-OHDA injection rat model. Since it has become clear that serotonin system has an important role in this rat model (levodopa-induced dyskinesia [LID] rats), a serotonergic agonist for inhibiting dyskinesia-like symptom was also assessed.

AIMs scores (locomotive, limb, axial, orolingual, and total) were high in the LID rats. Repeatedly administered tandospirone for 14 days also decreased AIMs scores (limb, axial, orolingual, and total) significantly; the reactivity was dose-dependent. The following findings were also noted in the LID rats: decreases in the contents of DA, DOPAC, HVA, 5-HT, and glutamate and DOPAC/DA and glutamate/GABA ratios in the striatum; decreases in DOPAC/DA and HVA/DA ratios, and an increase in glutamate/GABA ratio in the hypothalamus. Changes in the contents and ratios in the hypothalamus were improved by 14-day repeated administration of tandospirone.

As described above, tandospirone decreased AIMs scores without affecting rotational behavior induced by L-dopa administration in LID rats and inhibited development of side effects. The possibility is suggested that the inhibiting action occurs through at least the hypothalamus.

It has been speculated that serotonin release in the forebrain is involved in model-based decision making. However, there is so far no direct evidence proving this hypothesis because there had been no method that selectively controls serotonergic activity. To resolve this problem, we developed transgenic mice expressing Archaerhodopsin T (ArchT) only in central serotonergic neurons. A lithium devaluation task was used to assess model-based decision making. In this paradigm, a mouse is first trained to poke its nose to illuminated holes to get a food pellet, and then the food is devalued by pairing it with lithium-induced illness. If the mouse associates the devaluation with nose-poking by mental simulation though the mouse has never experienced these two events simultaneously, the mice will refrain from poking its nose to holes (i.e. model based-decision making). Our results indicated that optogenetic silencing of serotonergic neurons in the dorsal raphe nucleus, but not the median raphe nucleus, impaired model-based decision making. Thus it is likely that serotonergic activity in the dorsal raphe nucleus has a pivotal role in model-based decision making.

Social isolation is suggested to be a detrimental for the confusional states and abnormal interaction with cognitive impairment after the isoflurane plus surgery. However, the underlying mechanisms of these states remain unclear. After 2 hours exposure of isoflurane with abdominal surgery followed by social isolation for 24 h (ISO+SI-24h), the spontaneous alternations in Y-maze in male mice (7-10 weeks old) was significantly decreased, indicating that the spatial working memory was impaired by ISO+SI-24h. In general, only raring the 7-days of SI without the surgery, mice exhibit normal behaviors. However, the exposure of isoflurane with abdominal surgery in mice followed by raring 7 days of SI enhanced the mounting and sniffing behaviors against intruder mice in the home cage. In addition, the protein level of hippocampal dopamine D₂ receptors, but not prefrontal cortex was significantly decreased in mice with isoflurane plus surgery and SI. Since D₂ receptor is important for the cognitive function and psychosocial, these results imply the possibility that decrease in D₂ receptor contribute to the postoperative abnormal social interaction and cognitive dysfunction.

Among voltage-gated Ca²⁺ channels (VGCCs), low VGCCs/T-type Ca²⁺ channels (T-channels) regulate neuronal excitation and spontaneous neurotransmitter release, while high VGCCs are essential for evoked neurotransmitter release. It is still largely open to question how VGCCs contribute to CNS actions of psychostimulants, such as amphetamine and methamphetamine (MA). Interestingly, it has been reported that genetic deletion of Ca_v3.2 T-channels reduces amphetamine-induced hyperlocomotion (HL) in mice. Here, we examined effects of a selective T-channel blocker, TTA-A2, on MA-induced HL and brain Fos expression in C57BL/6J mice, as compared to pregabalin, a high VGCC α2δ inhibitor. TTA-A2, administered i.p. at 1 mg/kg, strongly suppressed MA-induced HL. In contrast, i.p. pregabalin at 1, 10 or 30 mg/kg had no such effect, although it exhibited slight suppressive effect at 3 mg/kg. MA caused cFos expression in specific brain areas including the prefrontal cortex, striatum, paraventricular hypothalamic nucleus, and hippocampal dentate gyrus and CA3 region, which were almost abolished by TTA-A2. Together, T-channels appear to play a critical role in MA-induced neuronal and behavioral excitation in C57BL/6J mice.