We have previously clarified the physiological roles of Epac2 (exchange protein directly activated by cAMP 2)-mediated signaling in insulin secretion induced by incretin and sulfonylureas. There have been three Epac2 isoforms identified to date: a full length Epac2A, Epac2B lacking N-terminus cAMP binding domain, and Epac2C, the shortest isoform predominantly expressed in the liver. To further investigate the cellular functions of Epac2 in pancreatic β-cell, we first intended to ablate both Epac2A and Epac2B in insulin-secreting MIN6-K8 cells by the CRISPR/Cas9 system (KO-1 cells). In the KO-1 cells, although protein expression of Epac2A was ablated, the expressions of multiple Epac2B-like isoforms were still detected. We then ablated all these isoforms and established the Epac2-null cell lines (KO-2 cells). While relatively weak activation of Rap1, a downstream molecule of Epac2, by cAMP was found in the KO-1 cells, no Rap1 activation was observed upon cAMP stimulation in the KO-2 cells. Interestingly, insulin secretion in response to Epac-selective cAMP analog and GLP-1, an incretin, was reduced in both KO-1 and KO-2 cells to the same extent, compared to the parental MIN6-K8 cells. These results indicate that Rap1 activation through newly identified Epac2B-like isoforms is not involved in insulin secretion, suggesting their roles mediating the cellular functions different from insulin secretion in pancreatic β-cells.

Increases in the cytosolic Ca²⁺ concentration ([Ca²⁺]c) in pancreatic β-cells mediate glucose-stimulated insulin secretion. It has been suggested that increases in the mitochondrial Ca²⁺ concentration ([Ca²⁺]m) are also involved in insulin secretion promoting mitochondrial ATP production. However, [Ca²⁺]m dynamics during glucose stimulation require further clarification. Using mitochondrial Ca²⁺ indicator CEPIA2mt, we here analyzed glucose-stimulated [Ca²⁺]m dynamics in a pancreatic β-cell line MIN6. During glucose stimulation, [Ca²⁺]c showed oscillatory changes with intervals of 2–3 min. [Ca²⁺]m, on the other hand, showed very subtle and unexpected changes: it decreased with an increase in [Ca²⁺]c and increased with a decrease in [Ca²⁺]c. However, upon shRNA-mediated knockdown of MICU1, a gatekeeper protein of mitochondrial calcium uniporter (MCU), [Ca²⁺]m increased in phase with [Ca²⁺]c oscillations having much greater amplitudes than those in control cells. Despite the remarkable increase in [Ca²⁺]m dynamics, glucose-stimulated [Ca²⁺]c dynamics remained almost the same. These results indicate that [Ca²⁺]m is tightly regulated by MICU1 during glucose stimulation, and that increases in [Ca²⁺]m above the baseline level seem not to be necessary for the generation of glucose-stimulated [Ca²⁺]c oscillations.

Basement membrane proteins play important roles for cells as adherent extracellular scaffolds and signal transduction factors via integrins. Laminin, a major component of the basement membrane, is comprised of three subunits, α, β, and γ chains. Among these chains, only laminin α chain is capable of signaling via integrins. Previously, we studied components of the basement membrane in rat anterior pituitary gland, and found that laminin α1, α3, and α5 chains are located in the basement membrane on the parenchymal cell side, but not in the endothelial cell side. However, the effect of laminin on endocrine cells in the gland is not clear. In order to elucidate this issue, dispersed rat anterior pituitary cells were cultured on laminins containing α1, α3, or α5 chains as ligands. Cultured cells adhered to laminin containing α3 or α5, and the morphology of these cells changed to flat shape. Double-immunostaining showed that endocrine cells express integrin β1 and α3. The antibody against integrin β1 blocked the morphological change of the cells. Furthermore, we found that adhesion to laminins containing α3 or α5 induced growth hormone release from rat anterior pituitary cells. These findings show that laminin α3 and α5 play a functional role as a signaling molecule to regulate endocrine cells in rat anterior pituitary gland.

It has been known that the increase of visceral fat is closely involved in abnormal glucose tolerance, hypertension, and hyperlipidemia, leading to the other diseases, such as atherosclerosis and type 2 diabetes. Recent reports have shown that serum copper concentration was increased in obese and/or diabetic patients. Here, we investigated the effects of copper chelator, cuprizone on high fat diet (HFD)-induced obesity in mice. We administered cuprizone (0.2%w) mixed in food pellets. Mice were divided in 4 groups, fed with (1) normal chow (NCD), (2) NCD with cuprizone (NCD+C), (3) HFD or (4) HFD with cuprizone (HFD+C) for 4 weeks, and then metabolic parameters were obtained. Serum copper levels were decreased in both cuprizone groups. Cuprizone significantly decreased the body weight in HFD+C without changes of food intake. Specifically in HFD+C, cuprizone decreased 60% of epididymal and inguinal fat weights, but did not change the weight of skeletal muscle, both soleus and gastrocnemius. Furthermore, we found that cuprizone ameliorated HFD-induced insulin resistance (ipGTT and ITT) with the modifications of gene expression pattern in liver, based on the comparison between NCD, HFD and HFD+C. These results suggest that cuprizone would be a candidate of leading compounds for anti-obesity agent.

[Aim of the study] Canola oil (CAN) ingestion is known to shorten the life-span of male SHRSP and the life-shortening is preceded by a decrease in plasma testosterone and an increase in aldosterone. The present study was conducted to clarify the target of the toxicity. [Methods] Male SHRSP were given an AIN-93G diet containing 10w/w% soybean oil (Control) or CAN and tap water ad libitum for 8 weeks. At the 8th week the animals were sacrificed and plasma concentrations of testosterone, aldosterone, LH and FSH were measured using ELISA kits. Testes were isolated and histopathologically examined. Gene expressions for ACE and ACE2 in the lung, kidney and testis were measured. [Results and discussion] CAN-induced decrease in plasma testosterone and increase in aldosterone were confirmed. Both, plasma LH and FSH were similar between the Control and CAN groups. Leydig cell counts in the testis were comparable between the two groups. The ratios of gene expression for ACEs to that for ACE (ACE2/ACE) in both, the testis and the kidney of the CAN group were significantly lower than those in the Control group, while ACE2/ACE in the lung of the two groups were similar.These results demonstrate that CAN ingestion does not affect gonadotropin secretion from the pituitary while suppresses the testicular function without pathomorphological changes nor Leydig cell count. The decreased ACE2/ACE in the testis and the kidney of the CAN group may affect the local renin-angiotensin-aldosterone system (RAAS). In this presentation the relationship among testosterone, aldosterone and RAAS in the CAN toxicity is discussed.

The pharmacokinetic and pharmacodynamic interactions between prescription drugs and epigallocatechin gallate (EGCG), a polyphenol and the main catechin in green tea. Many Japanese people consume green tea in beverages and foods. Functional foods that contain green tea or EGCG are claimed to reduce allergies and have inhibitory effects on lipid absorption. There are 5 prescription drugs with descriptions of possible interactions with green tea on the package inserts; however, none of them described specific interactions with EGCG. Studies have suggested the effects of EGCG on cytochrome P450 (CYP), organic anion transporting polypeptide (OATP), and biomolecules that affect the pharmacokinetics of various prescription drugs. In multiple databases in mid 2019, there are 34 drugs which showing OATP-mediated interactions with EGCG, and investigated interactions between some of these drugs and EGCG that caused inhibition of intracellular drug uptake in the literature mainly involving in vitro experiments, which increases the drug blood concentration and risk of associated side effects. The literature shows that the blood concentration of EGCG following green tea intake is at most 1 μM, and I could not find a manuscript on specific accumulation in tissues or organs. Based on the literature, since the pharmacokinetics of bortezomib, nadolol, warfarin, and many statins is affected by EGCG at concentrations close to the blood drug concentration range, there is a possibility that green tea consumption during drug administration could be restricted depending on evidence accumulation in the future.

The asymmetry between the hemispheres of the brain is particularly well known in humans. Language centers are unevenly distributed in the left hemisphere, and the right hemisphere is known to function primarily in spatial recognition. In addition, in animal species other than humans, reports on the asymmetry of neuronal structure due to changes in the expression level of glutamate receptor-expressing neurons in the mouse hippocampus, and the difference in symptom expression by behavioral experiments using unilateral hippocampal rats there is. However, since the dominant brain has traditionally been negative in rodents, it is not clear that there is a left-right difference in brain activity in rodents. In this study, we analyzed changes in rat brain activity during taste stimulation by sweeteners using functional MRI. However, the experimental results were asymmetric. Imaging device magnetic field inhomogeneities may be related to this asymmetry. Therefore, we tried to detect changes in brain activity in the same individual by changing the direction of the rat by 180 degrees during the MRI imaging experiment. As a result, in this experiment, it was shown that even if the direction of the rat is changed, there is no effect on the brain activity detection site, and there is a difference in the brain activity of the rat.

Coriandrum sativum (CS) has been used as folk remedies for Ebers papyrus, an ancient medical record in Egypt over 3000 years. It has been reported that CS shows the effects of the antioxidant and anti-inflammatory. In this study, we examined the protective role of CS leaf extract (CSLE) against the cytotoxicity induced by hydrogen peroxide (H₂O₂) on neurite outgrowth of PC12 cells.

PC12 cells seeded onto 12-well plate (2×10⁴cells/well) were cultured in DMEM medium containing FBS (-), which NGF (12.5 ng/mL) was also added at this time. After 24h, the cells were incubated for 3 days in serum free DMEM containing either CSLE (0.01μg/mL, 0.1μg/mL, 1μg/mL, 10μg/mL) or ascorbic acid (AA: 50μg/mL) with H₂O₂. On day1 and 3, morphometric analysis of the neurites and length was performed by Neurocyte Image Analyzer software. In addition, the expression levels of neurofilament-L (NF-L) were measured by real-time RT-PCR.

NGF-induced neurite outgrowth action was significantly suppressed by H₂O₂, and significant improvement was observed in the CSLE (0.01μg/mL, 0.1μg/mL), dose-dependently, and AA. The result of real-time RT-PCR, NF-L level was significantly increased by adding of CSLE and AA compared to H₂O₂ group.

These results demonstrate that CSLE has cytoprotective action against hydrogen peroxide-induced cell damage as well as AA.

Recently, we proposed the Japanese traditional foods as the potential anti-obesity food materials based on our animal and human studies. In this study, we evaluated effects of the commercially available Japanese traditional Nuru-Neba diet (JNN): contained with root kelp, wakame, agar, white cloud ear, shiitake, nameko, okra, mekabu, cut tororo, shimeji, on the abdominal fat in the high fat mice.

5-week-old male ICR mice were divided as follows: high-fat diet group (HFD group), high-fat diet and JNN group (HFD + JNN 60 mg, HFD + JNN 180 mg and HDF + JNN 300 mg). Each mouse was reared individually and then allowed to free access to diet of 2.7 g per day for six weeks. At the end of the treatment period, the visceral fat was collected. The cholesterol concentrations were determined from plasma and the expression levels of leptin in visceral fat were measured by real-time RT-PCR.

The visceral fat was significantly and dose-dependently decreased in the HFD + JNN group compared to the HFD group. The cholesterol in plasma was significantly decreased in the HFD + JNN 300 mg group compared to the HFD group. The expression level of leptin was significantly suppressed in the HFD + JNN. These results indicate that JNN can be helpful in weight control along with maintaining high-mucopolysaccharide of intestinal mucosa.

In this study, we cleared the different effects of commercially available Japanese Nuru-Neba diet (JNN) on the visceral fat in normal and high fat diet mice considering about changes in adiponectin and leptin levels.

5-week-old male ICR strain mice were divided as follows: normal diet group (ND group), ND + JNN (0.35g) group, high-fat diet group (HFD group), HFD + JNN group. Each mouse was reared individually and then allowed to free access to diet of 3.5 g per day for eight weeks. At the end of the treatment period, the visceral fat was collected. The expression levels of adiponectin and leptin in visceral fat were measured by real-time RT-PCR.

In ND+JNN group not HFD+JNN group, the significant increases of adiponectin levels were seen. The changes in body weight in ND+JNN group were same as seen in those of ND group. In visceral fat leptin levels, any changes could not be seen in ND+JNN group. However, in HFD group, more than 500 times of leptin levels were detected compared to those of ND group. In HFD+JNN group, the abnormal leptin levels induced by high fat diet was significantly suppressed.  

These results suggest that daily intake of NJJ activates adiponectin secretion under normal conditions and has an obesity preventive effect and that obesity is prevented by suppressing leptin resistance in the obese state.

The aging society leads to increase in diseases such as dementia. Alzheimer's disease (AD) is the most common form of dementia. It is known Coriandrum sativum (CS) impart an antioxidative effect. Therefore, it is hypothesized that CS can ameliorate the degenerative brain diseases by decreasing the oxidative stress caused by ageing. In this study, we examined whether CS seed extract (CSSE) could improve the memory impairment in SAMP8 mice or not.

10-week-old male SAMP8 mice were divided into two groups orally administrated with water (SAMP8(-)) or CSSE (SAMP8(+); 200mg/kg body weight/day). 10-week-old male ICR was used as normal control group also orally administrated with water.

The mean escape time of SAMP8(-) mice was significantly longer than that of ICR mice in Barnes maze test. However, SAMP8(+) mice showed the shorter mean escape time when compared with SAMP8(-) mice. The mRNA levels of neurofilament was significantly decreased in frontal lobe of SAMP8(-) mice, but significantly increased in SAMP8(+) mice. In addition, the mRNA levels of nNOS was significantly increased in in frontal lobe of SAMP8(-) mice, but significantly reduced in SAMP8(+) mice. It was indicated that continuous oral administration of CSSE for 12 weeks could ameliorate the aging-induced memory decline in the senescence-accelerated SAMP8 mice.

PHD finger protein 24 (Phf24), also known as Gαi-interacting protein (GINIP), was found to be absent in Noda epileptic rat (NER) (Behav. Genet., 47, 609, 2017). In this study, to explore the role of Phf24 in modulating CNS functions, we analyzed behavioral characteristics of Phf24-knockout (KO) rats, especially changes in seizure sensitivity, emotional responses and cognitive functions. Phf24-KO rats showed higher intensity and incidence of seizures induced by pentylenetetrazole (PTZ) and pilocarpine than F344 rats (control). Furthermore, PTZ-induced kindling was significantly facilitated in Phf24-KO rats. Anxiety-like behavior and cognitive function were analyzed by elevated plus maze and Morris water maze tests, respectively. Phf24-KO rats at old age exhibited reduced anxiety (impulsive) behaviors in the elevated plus-maze test compared with F344 (control) rats. In addition, Phf24-KO rats showed impaired learning behaviors in Morris water maze test. The memory retention ability was also disrupted in Phf24-KO rats. These results suggest that Phf24 negatively regulates epileptogenesis (seizure induction and development) and plays important roles in controlling emotion and cognition.

p13 is mitochondrial protein highly expressed in heart tissue. Recently, our unbiased compound screen has shown that some cardiotonic drugs change p13 mRNA expression in vitro, suggesting p13 may play a role in cardiac function. To reveal the role of endogenous p13 in cardiac function, here, we investigated histological changes, mitochondrial complex 1 activity, and mRNA expression levels of mitochondria-related genes in the heart of p13 knockout (p13-KO) mice. Although no apparent abnormalities were observed in the weight and histology, complex 1 activity was significantly reduced in the p13-KO heart. In addition, mRNA expression levels of apoptosis-related genes, such as Bcl-xL, were significantly reduced in the p13-KO heart. These results suggest that endogenous p13 may be involved in energy metabolism and apoptosis in heart tissue.

【Purpose】

Idiopathic pulmonary fibrosis (IPF) is a progressive and intractable lung disease characterized by the proliferation of fibroblasts and loss of pulmonary function. Although many bleomycin-induced pulmonary fibrosis has been studied as IPF model in rodent, IPF model in nonhuman primates has not been reported. In this study, we investigate a cynomolgus monkey model of bleomycin-induced pulmonary fibrosis by IPF related biomarker and pathology.

【Methods】

Two cynomolgus monkeys were injected transtrachealy with bleomycin (2mg/kg) once a week for the first 2 weeks. The blood and bronchoalveolar lavage fluid (BALF) were collected at 0, 1, 4, 7, 9, 14, 21, 28 days after the first bleomycin injection, and cytokine levels were measured. On day 29, lung hydroxyproline content was measured. The formalin fixed lungs were stained with HE or Masson's trichrome for microscopic observation.

【Result and Discussion】

After bleomycin injection, BALF IL-1β levels were significantly increased on day 1 and returned almost normal level on day 4. The BALF MCP-1 levels were gradually increased and reached peak from day 4 to day 9. The BALF TGF-beta1 levels reached the maximum on days 7 or 9. The serum TGF-beta1 levels showed almost the same tendency as the BALF levels. The lung hydroxyproline contents of bleomycin injected monkeys were increased about 1.4 times more than normal. Histological examination showed a significant interstitial fibrosis with destruction of the alveolar architecture and was similar to IPF of human. This study provided new model using nonhuman primate for drug development in IPF.

Object：Prenatal hypoxic stress (e.g., threatened abortion) is thought to be a risk factor of neurodevelopmental disease, such as autism spectrum disorder. However, a critical target molecular which was altered by hypoxic stress in the brain and its detail molecular mechanism is still unclear. To elucidate these problems, we firstly addressed by generating hypoxia rat model.

Method: Pregnant F344 rats were exposed low O₂ condition by using hypoxic chamber for 24 hours. After postnatal day 50, autism-like behaviors in rats were carried out by using social interaction test and novel objective recognition (NOR) test. In gene expression analysis, neuro2a cells were exposed 1% O₂ condition and collected for RT-PCR.

Result：Hypoxia rats didn't show any abnormalities in general behaviors. In behavioral test, hypoxia rats showed decrease social interaction time compared to F344 rats reared at normal condition (control rats). In addition, hypoxia rats showed impairment of memory function in NOR test. Furthermore, RT-PCR results showed decrease the autism-related protein Mecp gene, inhibitory postsynaptic protein gephyrin gene and GABA_A receptor β3 subunit gene in neuro2a cells treated hypoxic condition.

Conclusion: these results indicated that the prenatal hypoxic stress might cause decrease inhibitory synapse-related genes, which destroy the excitatory/inhibitory synaptic balance and showed autism-like behavior in rats.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder that manifests clinically as a hypermetabolic with skeletal muscle rigidity when MH-susceptible (MHS) individual is exposed to commonly used volatile anesthetics. At present, definitive diagnosis of MH before anesthesia requires the detection of the functional abnormality using biopsied muscle samples, a painful test for patients and requiring skillful diagnosticians. Recent reports showed that the frequency of MH episodes has increased though the MH crises are very rare case. The exact percentage of MHS is difficult to determine, but the prevalence of MH can be estimated up to 1:2750 due to the autosomal -dominant inheritance in humans. The noninvasive diagnosis such as the genetic screening of the MHS is asked for, because safer anesthesia is performed. Approximately 50 % of known cases of MH are caused by mutations in the gene locus of the ryanodine receptor type 1 (RyR1, calcium release channel) and the numbers of RyR1 mutation sites reported in the patients have been increased up to about 200, but only 48 have been formally shown to be causative; the remainder await confirmatory studies. So we have employed the fast and easy making procedures for these mutants to be expressed in stable cultured cells to be discussed in this paper.

Intracellular calcium ion (Ca²⁺i) is one of the most important cation that controls several cellular functions, and thus there were already huge number of literature about Ca²⁺i imaging in various cells. Although it is believed that Ca²⁺i increase is accompanied by extracellular Ca²⁺(Ca²⁺o) decrease, the kinetics of Ca²⁺o decrease remain unknown because of the limited imaging options. To overcome this limitation, we developed a Ca²⁺ image sensor (CIS) that is highly selective to Ca²⁺ but not to other cations within wide dynamic range (from 100 mM to 100 mM). We used CIS for the imaging of Ca²⁺o in acute hippocampal slices. Stimulation with glutamate (Glu) decreased Ca²⁺o to around 200 nM within 3 s, which returned to the baseline level (2 mM) with slow kinetics (5 min). Glu stimulates both neurons and glial cells to evoke Ca²⁺i elevation, thereby reducing Ca²⁺o. Thus, we tested NMDA to selectively stimulate NMDA receptor in neurons. NMDA mimicked Glu-evoked Ca²⁺o decrease. Interestingly, Ca²⁺o decrease was initiated at hippocampal CA1-2, before spreading along the pyramidal layers. Glutamate- and NMDA-evoked Ca²⁺o decreases were inhibited by a NMDA receptor antagonist D-APV, suggesting involvement of neuronal NMDA receptors in decrease in Ca²⁺o. So far, many scientists neglected Ca²⁺o, but the CIS would tell us its importance for understanding brain functions.

Multi-electrode (MEA) assays using human induced pluripotent stem cell (hiPSC)-derived neurons are expected to predict the toxicity and the pharmacological effects. If we can measure the release of neurotransmitter using this MEA, it is possible to evaluate the drug related to release of neurotransmitters, and it is expected to improve the accuracy of medicinal effects. In this study, we aimed to develop the carbon nanotube (CNT) MEA chip, which enables in both electrochemical measurement of DA release and conventional field potential measurement.

The CNT-MEA chip was fabricated by electro-plating method. Detection sensitivity to dopamine (DA) in the fabricated CNT-MEA chip was examined by electrochemical measurement method. The change of DA release to methamphetamine (MTH) were measured using cultured human iPSC-derived dopamine neurons on CNT-MEA.

As a result of the electrochemical measurement, an oxidation peak current was observed at 0.25 V, and the detection limit and linearity of DA was less than 5 nM. We have succeeded in real time detection of DA release using human iPS cell derived DA neurons, and detected the changes in the amount of DA release depending on MTH dose. Furthermore, in the human iPSC-derived DA neuron, a change of spike pattern at MTH administration was detected by conventional field potential measurement. CNT-MEA is expected as a new MEA measurement method that improves the accuracy of toxicity prediction and the pharmacological effects.

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 is 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). Since the redox state may change during in vitro analysis it should be necessary to fix the cells. Here we evaluate the fixation methods to analyze redox state in vitro. We compared aldehyde and organic solvent-based fixation methods. To fix redox state of roGFP protein N-ethylmaleimide which react thiol and modify cysteine residues in protein was used. Splenocytes were isolated from roGFP mice and treated with hydrogen peroxide (oxidized state) or DTT (reduced state) to induce the maximum oxidation and reduction status. Oxidized or reduced cells fixed with various fixation methods were accessed by flow cytometry. Organic solvents lead to a severe loss of fluorescence of roGFP protein. On the other hand, fixation with aldehyde and N-ethylmaleimide was useful to maintain fluorescence and redox status of roGFP. This system should be a powerful and convenient tool for analyzing redox state in various types of immune cells in vitro.

Ion channel proteins play important roles in controlling various cell functions by regulating the ion permeability of cell membranes in response to stimuli. Dysfunctions of ion channels cause severe diseases; therefore, ion channels are important drug targets. However, it is difficult to measure the detailed effects of drugs on ion channels efficiently, and drug discovery affecting ion channel proteins has been lacking as compared to that affecting other proteins such as enzymes. This study describes the development of a novel electrophysiological method that significantly increases the measurement efficiency for the ion channels. The method is based on the artificial lipid bilayer method. By contacting a gold electrode containing channels with a lipid-solution interface, the channels are incorporated into the membrane simultaneously with the formation of lipid bilayers. Using this method, ionic currents were detected in less than 1 minute; moreover, some channel properties could be measured at the single channel level. In addition, we developed an automated system based on this novel method. In this system, a driving device automatically moves the gold electrode, depending on the current detected. This automation could be the basis of a system that makes multiple measurements.

Actin filaments in cells are essential for many cell functions such as cell migration, proliferation, and contraction. We wondered if we could regulate actin polymerization non-invasively, we could manipulate the mechanical force required for cell growth, motility, and cytoskeletal rearrangements. To this end, we noted the Rap1GAP protein. The Rap1GAP is known as GTPase-activating protein specific for Raps that are small monomeric GTP-binding proteins. The purpose of this study was to develop the genetically-encoded photoswitchable protein, which inhibited actin polymerization.

We used the near-infrared-responsive BphP1-QPAS1 optogenetic pair (1). The BphP1 was combined with plasma membrane translocating peptide, and the enzymatic domain of Rap1GAP was fused to QPAS1 and fluorescent protein (Rap1GAP-QPAS1). Then they were co-transfected to the HeLa cells. Firstly, the Rap1GAP-QPAS1 was localized at cytoplasm, whereas irradiation of 740 nm allowed Rap1GAP-QPAS1 to translocate to the plasma membrane. Moreover, the area of cells was becoming smaller than that of before irradiation. These results indicated that the enzymatic domain of Rap1GAP is translocated to the plasma membrane by irradiation of infrared, and inhibited the actin polymerization by suppressing the Rap1.

(1) Redchuk TA et al., Nat. Chem. Biol. 13, 633-639 (2017)

Evaluations of locomotor activity, body temperature and gastrointestinal motility in monkeys or dogs are useful to understand effects of candidate drugs on the central nervous and gastrointestinal systems. Here we describe less-invasive evaluation methods using the small device, nano tag^® (15×14×7 mm).

Nano tag was subcutaneously implanted in cynomolgus monkeys. Gelatin capsule containing nano tag was orally administered in beagle dogs. Then body temperature and the amount of locomotor activity were simultaneously and continuously measured by nano tag and a telemetry system (PONEMAH system). The measured profiles obtained by nano tag approximately corresponded with those by the telemetry system, suggesting data obtained by nano tag are comparable to telemetry data. Moreover, nano tag could detect drug-induced changes of locomotor activity and body temperature in animals treated with caffeine, ketamine or thiopental. As to gastrointestinal motility, gastrointestinal residence time of nano tag was evaluated in dogs. The gastrointestinal residence time became shortened and extended by treatment with pilocarpine and loperamide, respectively. The proposed less-invasive methods using nano tag could help to evaluate effects of drugs on the central nervous and gastrointestinal systems in monkeys and dogs.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to take place of animal models for the assessment of drug-induced cardiotoxicity because of no possible prediction errors arising from species differences. However, currently, the qualities of hiPSC-CMs are inconsistent among product lots and must be controlled by well-trained experimenters. This labor-intensive process prevents high throughput screening. To tackle this problem, we developed an automated method for controlling the qualities of the contraction of cultured hiPSC-CMs using machine learning.

After 5–7 days of culture of hiPSC-CMs, a total of 556 bright-field videos of the hiPSC-CMs were obtained. The contractile qualities of the hiPSC-CMs were inspected by four well-trained experimenters and were labelled as either ‘normal' (n = 366 videos) or ‘abnormal' (n = 190 videos). The contractile properties of hiPSC-CMs were measured using the absolute changes in the pixel intensity between video frames, and these dimensions were reduced to 2 using uniform manifold approximation and projection (UMAP). We then trained the support vector machine (SVM) algorithm to classify normal or abnormal hiPSC-CMs. We found that fast Fourier transformation and data augmentation improved the classification scores of SVM. In summary, we demonstrated that the machine learning approach is applicable to the control of contractile qualities of hiPSC-CMs.

Vancomycin is a glycopeptide antibiotic that kills bacteria by blocking the construction of the cell wall and used to treat different bacterial diseases including meningitis and methicillin‐resistant Staphylococcus aureus infections. Because this antibiotic can sometimes induce renal failure and hearing loss, the plasma concentration is monitored to adjust the dose applied to individual patients. In this study, we show a rapid and simple procedure with an electrochemical approach. The sensor we used consisted of a boron-doped diamond electrode, which elicits more stable reaction than classical materials such as carbon and gold. With this sensor we examined guinea-pig plasma containing vancomycin at different concentrations. The procedure we developed allowed us to complete a series of measurement in 35 sec. Time necessary for all the processes including a sample's pretreatment did not exceed 10 min. The sensor detected the drug concentration of 1 to 50 µM, which falls into the range of the therapeutic window. Moreover, we found that the sensor was repeatedly usable for the measurement with minimal impairment of the sensitivity. The methodology described here may contribute to not only advances in personalized medicine but also reduction of the cost for therapeutic drug monitoring.

Ca²⁺ plays important roles as a second messenger in a wide range of biological phenomena including neurotransmission. Near infrared (NIR) fluorescence is suitable for in vivo Ca²⁺ imaging because of its high tissue penetration, low light scattering, and minimal autofluorescence. Many types of NIR chemical probes have been developed but they lack selectivity for labeling of particular cell types upon multi-cell bolus loading. We developed DeQODE chemical tag system as a new chemical biology tool, in which a small-molecular QODE probe can visualize Ca²⁺ signals with NIR fluorescence selectively inside the target cells expressing DeQODE tag. In an application of our DeQODE tag system to primary cultures of rat hippocampal neurons, neurons expressing DeQODE tag were selectively labeled by QODE probe. We successfully visualized Ca²⁺ signals of the target neurons in response to electrical stimulation at 10 Hz. We will perform ex vivo and in vivo application of our chemical tag system.

Recently, decoding that predicts behavior from an electroencephalogram(EEG) has been many reported, and decoding an electromyogram(ECG) from an EEG is one of them. However, there are many waveforms in the brain such as noise from experiment environment, and the EEG signal is not only directly related to the EMG but also includes the waveform reflecting another role. Filtering of Wavelength has been mainly used as a method for classifying such as EEG, but in recent years, a denoised method using deep learning has also been used for waveform analysis. The purpose of this study is to evaluate decoding accuracy of denoised EMG using deep learning and to classify the ratio of EEG reflected in muscle activity and EEG reflected in different activity.

We recorded primary motor cortex(M1) EEG and 4 or 5 regions of EMG in the rat brain. Next, the denoising EMG using Latent Factor Analysis via Dynamical Systems(LFADS), a deep learning method reported using EMG analysis. After denoising, EMG and EEG were coded each other to evaluate their accuracy.

Denoised EMG using deep learning showed higher decoding accuracy than just filtering. Moreover, the reflection ratio of EEG to electromyogram was evaluated by mutual decoding of EMG and EEG. This study suggested　that the usefulness of noise removal by machine learning and the EEG classification can be made more accurate.

Alzheimer's disease (AD) is one of neurodegenerative diseases and the most common cause of dementia. Among pathology of AD, synaptic dysfunction has most correlation with cognitive dysfunction. Drebrin is an actin binding protein and stabilizes actin filaments. Drebrin-decorated stable actin filaments accumulate in dendritic spines and are thought to be crucial for synaptic plasticity but drebrin has been decreased at onset of dementia in AD. We therefore hypothesized that loss of drebrin, that is, loss of stable actin filaments from dendritic spines elicits synaptic dysfunction and causes dementia in AD. Here we used the App knock-in mouse model of AD (App^NL-G-F mouse), to analyze the details of abnormal synapse in AD. First we performed immunohistochemical analysis using App^NL-G-F mice brains. We focused on the cortex and found no drebrin immunoreactivity around amyloid plaques in the App^NL-G-F mice brains. We further used primary hippocampal cultured neurons derived from the App^NL-G-F mice (App^NL-G-F neurons) and evaluated synaptic status based on drebrin cluster number using high-content imaging analysis. Our data showed App^NL-G-F neurons had less drebrin clusters indicating low functionality of synapse. These data suggest that the loss of drebrin from the dendritic spine in AD brains causes synaptic dysfunction.

The dopamine (DA) D1 receptors in the nucleus accumbens (NAc) is implicated in cocaine-induced conditioned place preference (CPP), which is driven by the cocaine-associated cue. However, the effect of the cocaine-associated cue on DA release in the prefrontal cortex (PFC) has little been studied. The present study examined the effects of cocaine-associated cues on the extracellular DA levels in the NAc and PFC using in vivo microdialysis. Furthermore, the role of D1, D2, and ionotropic glutamate receptors in cocaine (7.5 mg/kg i.p.)-induced CPP, locomotor and DA release were investigated. Cocaine-associated cues increased DA levels in the PFC, but unexpectedly had no effects on DA levels in the NAc. Pharmacological inhibition of D1 and D2 receptors and chemogenetic inhibition of D1 receptor-expressing cells by Gi-DREADD in the PFC during the cocaine conditioning procedure suppressed the cocaine-induced locomotor response, but did not affect the CPP. Pharmacological modulation of ionotropic glutamate receptors by antagonists and agonists in the PFC suppressed the cocaine-induced CPP, but did not affect the locomotor response. The DA response to the cocaine-associated cue in the PFC was abolished by pharmacological inhibition of ionotropic glutamate receptors, but not of D1 or D2 receptors.

The present results demonstrate that the cocaine-induced CPP and locomotor response are induced by the distinct neural pathways in the PFC mediated through ionotropic glutamate receptor and DA receptor signaling, respectively.

The nucleus accumbens (NAc), a major terminal area of mesolimbic dopaminergic system, expresses α₂-receptors (-Rs). We have previously shown that local administration of α₂-R ligands into the NAc failed to alter accumbal extracellular dopamine and noradrenaline levels of freely moving rats (Saigusa T et al., 2012). The NAc contains cholinergic interneurons that might receive inputs from noradrenergic projections (Gonzales KK & Smith Y, 2015) and α-R ligands modulate accumbal cholinergic activity-dependent locomotion of rats (Ikeda H et al., 2007). In the present study, to investigate the involvement of α₂-Rs in the regulation of accumbal cholinergic neural activity we analysed the effects of the α₂-R agonist UK 14304 and the α₂-R antagonist RX 821002 on acetylcholine (ACh) efflux in the NAc of freely moving rats using in vivo microdialysis. Drugs were infused directly into NAc through the dialysis membrane. Doses of compounds indicate total amount infused (mol) during 60 min infusions. UK 14304 (300 pmol) reduced accumbal ACh efflux by around 70%. RX 821002 (600 and 6000 nmol) induced a dose-related increase in accumbal ACh efflux by around 175%. These results suggest that accumbal α₂-Rs may exert inhibitory roles in the control of cholinergic neural activity in the NAc.

The nucleus accumbens (NAc), a major terminal area of mesolimbic dopaminergic projections, receives inputs from orexin neurons. Behavioural studies suggest that accumbal orexin receptors (-Rs) play modulatory roles in the regulation of accumbal dopaminergic activity-dependent locomotion in rats (Kotani et al., 2008). We studied the effects of intra-accumbal application of orexin-R ligands on accumbal dopamine (DA) efflux in freely moving rats, using in vivo microdialysis, to analyse the roles of orexin-Rs in regulating accumbal dopaminergic neural activity. Orexin-R ligands were applied into NAc though a microinjection needle attached with a dialysis probe. The OX₁- and OX₂-R agonist orexin-A failed to alter DA levels in NAc. However, the OX₁- and OX₂-R antagonist MK-4305 induced a dose-related increase in DA levels. This MK-4305-induced increase in accumbal DA levels was suppressed by infusion of TTX through the probe and was inhibited by local co-administration of orexin-A. These results suggest that intra-accumbal application of MK-4305 could enhance accumbal DA efflux by antagonism of OX₁- and/or OX₂-Rs. The present study also indicates that accumbal OX₁- and/or OX₂-Rs could play inhibitory roles in the regulation of accumbal dopaminergic neural activity.

In the brain, neurons communicate with each other at excitatory and inhibitory synapses. In each excitatory synapse, a dendritic spine receives the excitatory neurotransmitter released by an axon terminal. An extracellular high K⁺ induces robust depolarization and mimics massive synaptic transmission. A dendritic spine transiently shrinks in high K⁺ solution. In this study, we investigated the mechanisms of the spine shrinkage using cultured hippocampal neurons. The spine shrinkage observed in response to high K⁺ was inhibited in the presence of extracellular Co²⁺ or Cd²⁺. Then we investigated Ca²⁺-signaling pathway because these metal ions are supposed to be wide spectrum Ca²⁺-channel blockers. We preincubated neurons with ROCK inhibitor Y27632 or CaMKII inhibitor KN-93. However, neither of them inhibited the spine shrinkage. Finally, we tested whether myosin is involved in the spine shrinkage. A myosin ATPase inhibitor, blebbistatin, did not inhibit the spine shrinkage in high K⁺. Taken together, extracellular high K+-triggered Ca²⁺-influx seem to induce the spine shrinkage; however, the downstream signaling pathway or the motor machinery has not yet been determined.

The second-order neurons in the nucleus tractus solitarius (NTS) receive excitatory inputs from the broncho-pulmonary afferents and serve as a gate in the cough reflex pathway. Although central antitussives are thought to inhibit the cough center including the NTS, their sites of action and mechanisms are not fully understood. In our previous study, dextromethorphan (DEX) inhibited tractus solitarius (TS)-evoked synchronous release of glutamate in the second-order NTS neurons independently of its agonistic effect on the sigma receptor. However, there is still limited knowledge of its cellular mechanisms. To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and tractus solitarius-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin (adenylyl cyclase activator) counteracted the inhibitory effect of DEX on mEPSCs. 8-Bromo-cAMP (cAMP analog) and N-ethylmaleimide (Gi/o protein inhibitor) also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. The present study demonstrates for the first time that the cAMP-dependent pathway regulates the excitatory synaptic transmission in the NTS neurons and the distinct mechanisms underlie the DEX-induced inhibition of spontaneous and synchronous release of glutamate in the second-order NTS neurons.

Whole-cell patch-clamp recordings were made from striatal cholinergic interneurons (ChINs) in P10-17 mice brain slices with restricted expression of channelrhodopsin-2 (ChR2) in the striatal medium spiny neurons (MSNs). Neurons were voltage clamped at -60 mV. Light stimulation evoked postsynaptic currents in the presence of glutamate and glycine receptor antagonists. These postsynaptic currents were blocked by GABA_A receptor antagonist, bicuculline, suggesting they were GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs). A muscarine receptor agonist, carbachol (1 μM), suppressed IPSCs by 49.5 ± 7.8% (n = 5). To examine the changes in GABA release probability, we calculated coefficient of variation (CV). The CV was not increased after application of bicuculline, suggesting the action site of bicuculline was postsynaptic GABA_A receptors. On the other hand, CV after application of carbachol was significantly increased (p = 0.004). In addition, carbachol (10 μM) did not affect inward currents evoked by puff-applied GABA (100 μM). These results suggest that activation of M1 muscarine receptors presynaptically inhibits GABA release from MSNs onto ChINs.

Astrocytes regulate neuronal excitability by maintaining ion homeostasis and secreting neuroactive substances. We previously showed that expressional knockdown of inwardly rectifying potassium (Kir) 4.1 channels elevates brain-derived neurotrophic factor (BDNF) expression in astrocytes (Int. J. Mol. Sci., 19, 3313, 2018). In order to explore the neural factors influencing of the Kir4.1 expression, we investigated the effects of serotonergic agents on Kir4.1 mRNA levels in primary cultured astrocytes. Treatment of astrocytes with serotonin (5-HT; 100 μM) inhibited Kir4.1 expression. The 5-HT_1A agonist (±)-8-OH-DPAT, 5-HT₂ agonist (±)-DOI, 5-HT₃ agonist SR-57227 or 5-HT₆ agonist WAY-208466 showed no effect on the astrocytic Kir4.1 expression. However, the 5-HT₇ agonist LP-211 significantly inhibited the expression of Kir4.1 in a dose-depended manner. In addition, the 5-HT₇ antagonist SB-258719 did not affect the Kir4.1 expression by itself, but antagonized the inhibition of Kir4.1 expression induced by 5-HT₇ agonist LP-211. The present results strongly suggest that 5-HT inhibits Kir4.1 expression via the activation of 5-HT₇ receptor in astrocytes.

Astrocytes maintain extracellular milieu, provide nutrients and release signaling molecules to regulate neuronal excitability. Dysfunction or augmentation of astrocytic roles is implicated in neuronal hyperexcitablity found in many neurological disease, such as epilepsy and Alzheimer's disease. In disease, astrocytes generally become reactive and reactive astrocytes show more Ca²⁺ signals with increase in Gq-protein coupled receptors (GqPCRs). To investigate the role of augmented GqPCR-mediated Ca²⁺ signals in neuronal excitability, we engineered mice whose astrocytes specifically overexpress P2Y1 receptor, one of major GqPCRs in astrocytes in adult brain, (astrocyte P2Y1OE). Using dual color Ca²⁺ imaging of neurons and astrocytes by two different colors of genetically encoded Ca²⁺ indicators, we found that astrocytes showed robust Ca²⁺ signals in response to neuronal stimuli and neurons showed enhanced dendritic responses in P2Y1OE, both of which depend on P2Y1 receptor signaling. Extracellular glutamate increased by neuronal stimuli was also enhanced in P2Y1OE. Astrocyte P2Y1OE mice show higher susceptibility to pilocarpine-induced status epilepticus. Overall data suggest that astrocytic Ca²⁺ signals mediated by GqPCR lead to neuronal hyperexicitability through enhancement of glutamatergic signaling.

Chronic neuroinflammation in the peripheral/central nervous system is important for the molecular basis of neuropathic pain. In particular, roles of macrophage and microglia have been well demonstrated. In this study, we evaluated the region, time, and sex-dependent effects of macrophages/microglia on neuropathic pain using mice that can induce Gi-DREADD driven by macrophages/microglia-specific cx3cr1 promoter (CX3CR1-hM4Di). Neuropathic pain model mice were generated by partial sciatic nerve ligation (PSL) or paclitaxel treatment, and mechanical allodynia was evaluated using von Frey test.

In CX3CR1-hM4Di mice after PSL, expression of hM4Di was localized in both F4/80 positive macrophages and Iba1 positive microglia. Intraperitoneal or intrathecal administration of clozapine-N-oxide (CNO), a ligand for hM4Di, improved mechanical allodynia in male CX3CR1-hM4Di mice after PSL or paclitaxel treatment. These anti-allodynic effects was observed in male, but not in female mice. These results support the notion that sex-dependent roles of macrophage/microglia in neuropathic pain and the pharmacological inhibition of these cells might be effective therapeutics for neuropathic pain.

Morphine, a µ Opioid receptor (MOPr) agonist, is one of the powerful analgesics, but the development of analgesic tolerance by the chronic use or under some inflammatory diseases such as neuropathy would be a clinical problem. Recently, we have demonstrated that the increase of the receptor transporter protein 4 (RTP4), one of the GPCR chaperone molecules, will facilitate the MOPr-DOPr heteromer formation and thus lead to the analgesic tolerance in neuronal cells. Interestingly, RTP4 is highly expressed in macrophages, so here we focus on the role of RTP4 in the microglial cells, the brain-resident macrophages. In this study, we determined the changes in RTP4 mRNA levels after the treatment of morphine or DAMGO (10 µ M, 24 hrs) in SIM-A9 microglial cell line. In addition, we determined the effects of inflammatory stress by use of lipopolysaccharide (LPS) (1 ng/mL to 1 µg/mL, 24 hrs). As a result, RTP4 mRNA levels in SIM-A9 cells were decreased by morphine or DAMGO, while they were significantly increased by LPS treatment. These results suggest that the regulation of RTP4 expression in microglial cells is differed between under the chronic MOPr stimulation and the inflammatory stress. The role of RTP4 in microglial cells in the development of analgesic tolerance to morphine in these states would be determined in the future.

Brain ischemia leads to irreversible tissue necrosis and central nervous system (CNS) dysfunction. On the other hand, many studies have demonstrated that CNS shows reparative potential after brain injury. We previously reported that brain pericytes in the ischemic core area acquired multipotent stem cell activity, and termed the cells "ischemia-induced multipotent stem cells (iSCs)." iSCs can differentiate into various cells, including neurons, astrocytes, oligodendrocytes, and microglia in vitro. In this study, we analyzed the behavior and functions of iSCs in the ischemic cortex, using ischemic model mice. In the ischemic core area, there were some Iba1+ cells, which expressed Nestin (iSCs marker). Parabiotic analysis revealed that Iba1+ cells in the ischemic core area were not derived from peripheral blood cells. Iba1+/Nestin+ cells in the ischemic core area abundantly expressed genes that are involved in vascular development. These results suggest that iSCs differentiate into microglia in the ischemic core area, which contribute to maintenance of iSCs niches.

The blood-brain barrier (BBB) restricts the transport of substances between vasculature and brain. Recent studies have clarified that various kinds of cells in neurovascular unit are related to the BBB functions. In this study, we investigated the roles of microglia in BBB functional maturation using in vitro BBB model comprised of endotherial cells, pericytes, and astrocytes (Ⓒpharmacocell).  When we added primary microglia to the brain side of the model during the maturation period, trans-endothelial electrical resistance (TEER) and the expression level of claudin-5, were significantly increased. On the other hand, when we added LPS-activated microglia, the TEER and the expression levels of tight junction proteins (TJs) were significantly decreased.  We next investigated involvement of cytokines/chemokines in the effects of microglia. We clarified that microglia-induced increase in TEER was mediated by VEGF, while the increases in TJs were mediated by the inhibition of fractalkine signaling. In the developing brain, a lot of microglia surroud the capillaries. Our data suggest that microglia contribute to the developmental maturation of the BBB. We are currently investigating the accurate time course of BBB maturation and the roles of microglia in the BBB maturation at the developmental stage by in vivo experiments.

Amyloid-β (Aβ) accumulation in the brain is the first trigger for the onset of Alzheimer's disease (AD), and its prevention and elimination are promising strategies for AD therapy. Previously, we demonstrated that injection of mouse bone marrow (BM)–derived microglia-like (BMDML) cells into the brain decreases Aβ and ameliorates cognitive impairment in a mouse model of AD. In this study, considering majority of AD patients are elderly and less invasive ways for preparing autologous microglia-like cells are needed, we focused on hematopoietic stem cells (HSCs) in peripheral blood (PB). Mouse HSCs were mobilized from BM to PB by administration of granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist and were collected from PB. Collected HSCs were subsequently differentiated into microglia-like cells upon stimulation with colony- stimulating factor 1 (CSF-1) and interleukin-34. The PB-derived microglia-like (PBDML) cells expressed macrophage/microglia markers and effectively phagocytosed Aβ. We further found that PBDML cells injected into the hippocampi of AD model mice diffused in the brain with phagocytosing Aβ, and contributed to the reduction of brain Aβ and improvement of cognitive impairment. These results suggest that PBDML cells could be promising candidate source for the development of cell therapy against AD.

Glaucoma is second leading cause of blindness worldwide which is characterized by progressive degeneration of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is one of the highest risk factors and IOP-lowering agents are used to prevent glaucoma. New molecular target is required because of the side effects, drug resistance, and insufficiency for IOP reduction by a part of pre-existing agents. Here, we report that P2Y₁ receptor (P2Y₁R) activation induces IOP reduction and knock out of P2Y₁R (P2Y₁KO) causes sustained IOP elevation associated with age-dependent RGC degeneration. Topical application of MRS2365, selective agonist for P2Y₁R, caused significant reduction in IOP in wild-type (WT) mice but not in P2Y₁KO mice. We also found that P2Y₁KO mice showed significantly higher IOP level than that in WT mice. Because sustained IOP elevation is one feature of hypertensive glaucoma, we checked RCG damages and found that the number of RGCs in P2Y₁KO mice was comparable at 3 months old but significantly smaller at 12 months old. Furthermore, optical coherence tomography (OCT) revealed that 12-month-old P2Y₁KO mice showed thinner ganglion cell and inner plexiform layers, general diagnostic feature of glaucoma patients. Taken together, our results demonstrated that (1) P2Y₁R activation reduces IOP; (2) loss-of-function of P2Y₁R causes sustained elevation in IOP and (3) hypertensive glaucoma-like phenotypes in middle-aged mice.

Parkinson's disease (PD) is the second most common neurodegenerative disorders, which mainly affects the motor system. The cause of PD is suggested to be the gradual degeneration of the dopaminergic neurons in substantia nigra pars compact and microglia, a key player in the innate immune system, have been shown to be involved in the pathophysiology of PD. Voltage-dependent Ca²⁺ channels (VDCCs) are generally known to be active only in excitable cells like neurons and muscle cells, however recently they have been reported to be also functional in non-excitable cells such as microglia. Cav1.2 channels are an L-type VDCC, which is sensitive to the calcium antagonists. In vitro experiments revealed that Cav1.2 channels are expressed in MG6, a microglial cell line derived from mouse, and that an increased neuroinflammatory M1 transition and a decreased neuroprotective M2 transition were induced by treating the MG6 cells with calcium antagonists. Besides, by intoxicating mice with MPTP, a neurotoxin that induces Parkinsonism, we have found an increased degeneration of dopaminergic neurons and the accompanying behavioral deficits in microglia-specific Cav1.2 knockdown mice. These results suggest that microglial Cav1.2 channel may have neuroprotective roles under physiological conditions.

hiPSC-derived neural networks (hiPSC-networks) on dish are expected to provide the improvement of human predictability in the drug development. To date, we have established the stable protocol to reproduce hiPSC- networks. However, little data are available whether astrocytes are also functional or not in these hiPSC-networks. In this study, we examined the role of astrocytes in hiPSC-networks. qRT-PCR study indicated that the mRNA levels of GFAP, AQP4 and L-glutamate (L-Glu) transporter were increased along with culture days and their expression changes were obtained later than those of neuronal genes. GFAP(+)Nestin(-) astrocytes appeared at DIV 49 and L-Glu transporters were detected in these cells immunohistochemically at DIV 63. We examined whether L-Glu transporters were functional or not at DIV 63. When we applied 100 microM of L-Glu to the sample, the concentration was decreased to less than 15 microM in 1hr. In microelectrode array experiments at DIV 63, the treatment with non-specific EAAT blocker TFB-TBOA decreased firing activities. These results suggest that astrocytes are functionally differentiated and indispensable for homeostatic control of extra-synaptic L-Glu concentrations of hiPSC-networks. Currently we are identifying EAATs subtypes in the hiPSC-networks used in this study.

Background; Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood.

Methods; Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection.

Results; In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group.　

Conclusions; Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Diabetes mellitus is one of the major causes of peripheral neuropathy, which can reveal the spontaneous allodynia. Our study was designed to determine a potential involvement of spinal cholecystokinin-8 (CCK-8) and histamine in mediating streptozotocin (STZ)-induced diabetic allodynia in mice. STZ (200 mg/kg)-induced mechanical allodynia was evoked significantly 7 day after intravenous (i.v.) injection. The mechanical allodynia elicited by STZ was concentration-dependently inhibited by intrathecal (i.t.) administration of antisera against CCK-8 (1:100 – 1:25) and histamine (1:200 – 1:50). No significant of mechanical allodynia induced by i.v. administration of STZ was shown in histidine decarboxylase deficient mice. The mechanical allodynia elicited by STZ were suppressed by i.t. administration of agmatine (40 – 160 pmol), antagonists for the NMDA receptor polyamine-binding site. The inhibitor of the activation and proliferation of microglia, minocycline (0.25 – 2 nmol, i.t.), inhibited the mechanical allodynia elicited by STZ in a dose-dependent manner. The present results suggest that the STZ-induced mechanical allodynia are mediated through the spinal CCK-8 and histamine and are elicited via activation of NMDA receptors in glial cell.

The histamine H3 receptor (H3R), an inhibitory G-protein coupled receptor, is exclusively expressed at axon terminals in the brain. H3R negatively regulates histamine release as an autoreceptor and also inhibits the release of other neurotransmitters including GABA and acetylcholine as an heteroreceptor. Recent evidence indicates that H3R inverse agonists, which can induce the release of histamine and other neurotransmitter, alleviate mechanical allodynia. However, the mechanism(s) by which H3R inverse agonists attenuate allodynia remain unclear.

Here, we aimed to uncover the mechanism of action for JNJ-10181457 (JNJ1), an H3R inverse agonist, in mechanical allodynia. The intraperitoneal administration of JNJ1 to Chung-model mice significantly increased paw withdrawal threshold, confirming the therapeutic effect of H3R antagonists on allodynia. However, JNJ1 could not alleviate pain symptom in histamine-deficient mice. Pharmacological assays showed that the therapeutic action of JNJ1 were diminished by H1R antagonist and H2R antagonist. When we deleted H3R around the periaqueductal grey matter (PAG) by AAV-based gene recombination, the therapeutic action of JNJ1 was diminished.

These results indicated that histamine release around PAG by JNJ1 activated H1R and H2R and subsequently exerts an antinociceptive effect in allodynia.

Gabapentinoids such as gabapentin and pregabalin, which act on the α₂δ subunit of voltage-gated Ca²⁺ channels, are widely used to treat neuropathic pain. They are known to activate the descending noradrenergic pain inhibitory system. Here, we investigated the analgesic effect of mirogabalin, a novel gabapentinoid recently launched for treatment of peripheral neuropathic pain including diabetic peripheral neuropathy and postherpetic neuralgia. Mirogabalin besilate produced analgesic effects on mechanical and thermal hypersensitivity developing after partial sciatic nerve ligation in mice, when it was administered systemically (10 and 30 mg/kg, i.p.) and locally (10 and 30 μg, i.c.v or i.t.). In particular, its analgesic effects (30 mg/kg, i.p. and 30 μg, i.c.v.) were largely reduced by pretreatment with yohimbine HCl (3 μg, i.t.). Moreover, in mutant mice with the substitution of arginine at position 217 with alanine (R217A) on the α₂δ-1 subunit, the analgesic effects of pregabalin and mirogabalin besilate (30 μg, i.c.v., respectively) on mechanical hypersensitivity were almost completely suppressed. These results demonstrate clearly that mirogabalin also employs the descending noradrenergic system, and the binding to the α₂δ-1 subunit is crucially important in the supraspinally mediated analgesic effects of gabapentinoids.

Morphine with its potent analgesic property has been widely used for the treatment of various kinds of acute pain and for long-term treatment of severe chronic pain. However, the chronic use of morphine is complicated by unwanted side-effect, including a paradoxical increase in pain sensitivity (i.e. hyperalgesia and allodynia). Indeed, continuous morphine infusion by osmotic pump caused allodynia in mice. The morphine-induced allodynia was inhibited by an antisera against dynorphin. However, the selective k-opioid receptor antagonist, nor-BNI did not prevent the morphine-induced allodynia. Dynorphin is rapidly degrated by a dynorphin-converting enzyme (cystein protease), to leucine-enkephalin (Leu-ENK). The morphine-induced allodynia was inhibited by an antisera against Leu-ENK. We also showed that the morphine co-administrated with Leu-ENK-converting enzyme inhibitors, phosphoramidon and bestatin produced much stronger behavioral responses than morphine alone. Furthermore, the morphine-induced allodynia was inhibited by intrathecal injection of the non-selective delta-opioid receptor antagonist, naltrindole or selective delta-2 opioid receptor antagonist, naltriben, while the selective delta-1 opioid receptor antagonist, BNTX had no effect. Taken together, these results suggest that continuous morphine infusion-induced allodynia may be triggered through the delta-2 opioid receptors activated by Leu-ENK which is formed from dynorphin in the spinal cord.

Nerve injury induces neuropathic pain such as allodynia and hyperalgesia. Effects of current treatments for the neuropathic pain are limited. Previously, we reported that partial ligation of the infraorbital nerve (pl-ION) induced cortical hyperexcitation in responses to mandibular molar pulp stimulation. In the present study, we performed optical recording with a voltage-sensitive dye to investigate the effects of oxytocin and low-level laser therapy (LLLT) on pl-ION-induced cortical hyperexcitation. Oxytocin (500 nmol) and/or LLLT (diode laser, 810 nm, 0.1 W, 500 sec, continuous mode) were locally applied to the injured nerve. Cortical responses to electrical stimulation of the mandibular molar pulp under urethane-anesthesia were recorded 3 days after pl-ION. Both the amplitude and area of excitation in the somatosensory and insular cortices were increased by the pl-ION. The amplitude of cortical excitation enhanced by pl-ION was suppressed by applications of oxytocin or LLLT. The area of cortical excitation enhanced by pl-ION was suppressed by the combined application of oxytocin and LLLT but not by OXT or LLLT alone. These results suggest that oxytocin and LLLT have positive effects in relief of abnormal pain induced by the nerve injury.　COI: Osada Lightsurge Square (OSL-S) was provided by Osada (Tokyo, Japan).

Glycosphingolipids (GSLs) are abundant in the nervous system and play important roles in cellular interaction and intracellular signal transduction. We found that gene expressions of several glycosyltransferases in GSLs biosynthesis pathway were increased in the spinal cord one day after inflammation caused by complete Freund's adjuvant (CFA). In naïve mice, intrathecal injection of GSLs, sulfatide and b-series gangliosides, synthesized from enzymes encoded by upregulated genes caused allodynia. Inhibitors of glial activation blocked or TNFα blocked sulfatide-induced allodynia. On the other hand, gene expressions of glycosyltransferases including sulfatide synthase were decreased 15 days (chronic inflammation) after CFA injection. Thus, we examined the effects of intrathecal sulfatide injection on chronic inflammatory pain. 21 days after CFA treatment, mice that were received daily injection of sulfatide showed reduced inflammatory pain. It appears that sulfatides play the different roles in early or chronic inflammatory pain. However, the molecular mechanisms underlying the change of function of sulfatide remain unclear. Future studies are needed to reveal how sulfatide induced both algesic effects in early inflammatory pain and analgesic effects in chronic inflammatory pain.