Our body maintains homeostasis when we are exposed to external and internal stimuli. However, chronic stress changes the sympathetic and parasympathetic nervous systems as well as endocrine systems such as the hypothalamic-pituitary-adrenal (HPA) axis, which leads to a loss of homeostasis. This maladaptation triggers several kinds of disorders, like major depressive disorder (MDD), psychosomatic disease, and immune dysfunction. In addition, excess stress is known to affect the function of several brain areas, including the hypothalamic paraventricular nucleus (PVN), which plays a critical role in the adaptation to stress. In particular, corticotropin-releasing hormone (CRH) neurons are most strongly implicated in the stress response because the HPA axis is activated by facilitating CRH-containing neurons. Although the activation of CRH^PVN neurons may play a role in immune cell homeostasis, little is known about how the direct activation of CRH^PVN neurons may suppress immune cells. In this study, we investigated whether the specific activation of CRH^PVN neurons by DREADD systems could affect the peripheral immune system. As a result, the number of CD4⁺ T cells and NK cells was influenced by the activation of CRH^PVN neurons. Furthermore, there were correlations between the immune cell population and plasma cortisol levels. Taken together, these findings provide further evidence that the modulation of stress-associated PVN-CRH neurons may affect the peripheral immune system via the HPA axis.

【Objective】Ceramide has been emerging as an anti-inflammatory lipid, and nanoscale delivery system of ceramides is a potential therapeutic strategy for inflammatory diseases. In this study, we evaluated the therapeutic effect of ceramide formulation (nanoliposomal ceramides) on allergic asthma in a mouse model.

【Methods】BALB/c mice sensitized with ovalbumin (OVA) were treated with nanoliposomal ceramides or ceramide-free liposomes followed by multiple OVA challenges. The numbers of mononuclear cells, eosinophils and neutrophils infiltrated in bronchoalveolar lavage fluids were counted. Airway remodeling was evaluated by staining with PAS and Masson trichrome. Type 2 cytokines in homogenized right lungs were measured by ELISA.

【Results】Treatment with nanoliposomal ceramides suppressed OVA-induced infiltration of mononuclear cells, eosinophils and neutrophils into the lung and development of airway remodeling. However, OVA-induced production of type 2 cytokines was not changed by nanoliposomal ceramides.

【Conclusions】Nanoliposomal ceramides are suggested to have anti-asthmatic effects by suppressing the development of airway remodeling. Our study gives insight into the development of ceramide-based therapy for allergic asthma.

Since the scratching assessment is the only way to estimate itching sensation in non-verbal experimental animal, it is utilized in the various research fields. However, current methods depend on human observation, which is laborious, low-throughput, and includes observer-bias. We here aimed to establish an automated scratching detection method of mice using neural network, an artificial intelligence technology which excels in image recognition.

Scratching was elicited by intradermal injection of a pruritogen, lysophosphatidic acid to BALB/c mice and their behavior was recorded with a video camera. Frame images were obtained from video data and classified into two classes: scratching or not. We then trained convolutional recurrent neural network (CRNN) with labeled datasets. Trained CRNN predicted scratching of first-look data with high accuracy (sensitivity: 81.6%, positive predictive rate 87.9%). We confirmed that the number and duration of predicted scratching bouts were comparable to those of human observation. Trained CRNN could also successfully detect scratching evoked by hapten-induced atopic dermatitis (sensitivity: 94.8%, positive predictive rate: 82.1%).

We here established a novel automated scratching detection method using artificial intelligence, which is applicable to the assessment of pathological mouse model.

Mas-relate G protein-coupled receptor b2 (Mrgprb2) and its human ortholog MRGPRX2 are specifically expressed in mast cells and widely recognizes cationic ligands such as neuropeptides, bacterial components and some drugs including vancomycin and morphine. These ligands of Mrgprb2/MRGPRX2 involved in (pseudo) allergic reactions, pain and infectious diseases. Previously, we reported that extracellular ATP potentiates antigen-induced mast cell degranulation via P2X4 receptor activation. In this study, we investigated the effect of P2X4 receptor signaling on degranulation induced by Mrgprb2 activation. Stimulation of mouse peritoneal mast cell (PMC) with Mrgprb2 agonist compound 48/80 (C48/80) induced degranulation in a concentration-dependent manner. C48/80-induced degranulation was potentiated by ATP but not by ADP, UTP and UDP. Similar results were obtained with another Mrgprb2 agonists substance P and PAMP-12. The potentiation by ATP was absent in PMC prepared from P2X4 receptor deficient mice. These results suggest that Mrgprb2-mediated mast cell activation is potentiated by the P2X4 receptor signaling.

【Background & Aim】 Antigen specific IgE is a fundamental factor in allergic reaction. In allergic reaction, prostaglandin D₂ (PGD₂) is known to play crucial role. PGD₂ acts on (chemoattractant receptor-homologous molecule on Th2 cells) CRTH2 receptor and its signaling is known to exacerbate allergic reaction by promoting antigen specific IgE production. However, it still be unknown the roles of PGD₂/CTH2 signaling in the production of antigen specific IgE. We aimed to reveal the role of PGD₂/CRTH2 signaling in allergic reaction.

【Methods & Results】 We sensitized wild type (WT) and CRTH2 deficient mice (Crth2^-/-) with ovalbumin (OVA) intradermally. The titer of OVA specific IgE in serum was lower in Crth2^-/- than WT after the sensitization. In the draining lymph node (dLN), the percentage of T follicular helper (Tfh) cells, a critical regulator of IgE production, was lower in Crth2^-/- than WT. These results suggested that CRTH2 signaling promotes differentiation of Tfh cells and IgE production in dLN. Intradermal administration of OVA increased the concentration of PGD₂ in the LN of WT. Immunostaining showed that the synthase of PGD₂, hematopoietic prostaglandin D synthase (HPGDS), was expressed in dendritic cells (DCs) in LN. Bone marrow derived dendritic cells released PGD₂ in response to OVA stimulation. These results suggested that antigen stimulation increased PGD₂ production in the DCs in dLN.

【Conclusion】 We found that PGD₂ derived from DC promotes antigen specific IgE production through CRTH2 signaling mediated Tfh differentiation in LN.

We have recently reported that systemic administration of the KCNQ (Kv7) K⁺ channel opener retigabine produces analgesic and antipruritic effects. In this study, we further explored the analgesic and antipruritic effects of retigabine behaviorally and analyzed its analgesic mechanisms electrophysiologically. In the cheek model, intraperitoneal injection of retigabine inhibited wiping behavior elicited by capsaicin injection reflecting pain sensation and scratching behavior elicited by chloroquine or compound 48/80 reflecting itch sensation. In the calf model, intrathecal injection of retigabine attenuated licking behavior after capsaicin injection reflecting pain sensation and biting behavior after chloroquine or compound 48/80 injection reflecting itch sensation, indicating that the spinal cord is an important site of action of retigabine. In dorsal horn neurons of spinal cord slices prepared from mice developing mechanical allodynia after partial sciatic nerve ligation, retigabine suppressed A-fiber-evoked monosynaptic EPSCs but not C-fiber-evoked EPSCs, while mEPSCs were not affected, suggesting that reduction of monosynaptic excitatory transmission from A-fibers contributes to the analgesic effect of retigabine, and that its antipruritic effect most likely involve spinal intrinsic neurons.

High mobility group box 1 (HMGB1), a nuclear protein, is released from various cells including macrophages (Mφ), and aggravates inflammation and pain. Given the involvement of HMGB1 and Toll-like receptor (TLR) 4 in the pathogenesis of rheumatoid arthritis (RA), we examined the effect of sulfasalazine (SSZ), an anti-RA agent, on TLR4-mediated inflammatory pain in mice, and on HMGB1 release from Mφ. The mechanical allodynia following intraplantar injection of lipopolysaccharide (LPS), a TLR4 agonist, was prevented by SSZ, an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of inactivating HMGB1, ethyl pyruvate, an inhibitor of HMGB1 release from Mφ, or liposomal clodronate (Clo), a Mφ depletor, and by inhibitors of CaMK kinase, JAK or NF-κB. In Mφ-like RAW264.7 cells, the LPS-induced HMGB1 release was suppressed by SSZ, but not its metabolite, 5-aminosalicylic acid, and by inhibitors of JAK, CaMK kinase and NF-κB. SSZ suppressed the LPS-induced phosphorylation of STAT3 and CaMK4, but not NF-κB p65, in RAW264.7 cells. These results suggest that SSZ relieves inflammatory pain by reducing TLR4-dependent HMGB1 release from Mφ through inhibition of JAK/STAT and CaMK signals.

Central post-stroke pain (CPSP) is defined as unbearable neuropathic pain that can subacutely occur after hemorrhage or ischemic cerebrovascular diseases in some central nervous regions, such as the thalamus and putamen. Since the pathophysiological mechanism underlying CPSP remains unclear, effective therapeutic targets are currently very limited. Recently, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) and its specific receptor (PAC1R) played an important role in the onset and maintenance of peripheral neuropathic pain, and therefore we developed novel small-molecule antagonists of PAC1R (PA-8, PA-915, etc.) using docking-based in-silico screening. Here, we attempted to analyze the association of CPSP and PACAP/PAC1R signaling using a thalamic hemorrhage mouse model. Intraperitoneal injections of PA-8 or PA-915 attenuated the hemorrhage-induced mechanical allodynia and astroglial activations in a dose-dependent manner. Furthermore, PACAP^-/- mice also developed reduced mechanical allodynia after thalamic hemorrhage. These results suggest that PACAP/PAC1R-evoked astroglial activation contributes to the thalamic hemorrhage-induced nociceptive behavior, and PAC1R antagonist can be a potential analgesic for CPSP.

Chronic pain including neuropathic pain increases risk of cognitive dysfunction. High-mobility group box 1 (HMGB1), a damage-associated molecular pattern, is involved in the development and maintenance of neuropathic pain and cognitive dysfunction. However, the relationship between HMGB1 and cognitive dysfunction in chronic pain state is unclear. The current study examined the effects of neutralizing antibody (nAb) against HMGB1 on cognitive dysfunction and related changes of neuronal morphology in mice with neuropathic pain.

Neuropathic pain was induced by partial sciatic nerve ligation (PSNL) of male ddY mice. Cognitive function was evaluated by novel object recognition test. Neuronal morphology in the hippocampus was assessed using Golgi-Cox staining. Anti-HMGB1 nAb was intranasally administered to mice to deliver into brain.

Nerve injury significantly evoked mechanical hypersensitivity and cognitive dysfunction compared with sham group. Repeated administration of anti-HMGB1 nAb to mice with neuropathic pain improved maladaptive neuroplastic changes, such as decreases of dendritic length and spine density, in the hippocampus and cognitive dysfunction.

The current study suggests that HMGB1-mediated maladaptive neuroplastic changes in the hippocampus could be involved in cognitive dysfunction under neuropathic pain state.

Intracerebral hemorrhage (ICH) is recognized as a serious clinical problem lacking effective treatment. High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity once released into the extracellular space from the nuclei. We previously demonstrated that intravenous injection of rat anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain injury induced by hemorrhage in the striatum of rat. In the present study, we examined whether and how humanized anti-HMGB1 mAb (OKY001) effects on ICH injury in common marmoset. The results show that administration of OKY001 inhibited the release of HMGB1 from brain into plasma, which was associated with the decrease of 4-HNE adduct accumulation in the brain and plasma. Besides, brain injury volume was ameliorated by treatment of OKY001 at 12 d after ICH induction. In vitro experiment showed that haptoglobin increased the uptake of hemoglobin and HMGB1 by THP1 cell respectively. However, recombinant HMGB1 inhibited the uptake of hemoglobin by THP1 cell, which suggested that abundant HMGB1 released form the brain impeded the absorption of hematoma. Moreover, OKY001 reduced body weight loss and improved the behavioral performance. Taken together, these results suggest that intravenous injection of OKY001 has potential as a novel therapeutic strategy for ICH disease.

[Background] SIRT1, an NAD⁺-dependent deacetylase, positively regulates autophagy, which maintains muscle mass and declines with age. We previously reported that treatment with resveratrol (RSV), an activator of SIRT1, restores muscle autophagy and preserves muscle mass in a mouse model of Duchenne muscular dystrophy. We hypothesized that RSV attenuates age-related muscle atrophy by activating SIRT1 and autophagy.

[Methods and Results] DDY mice were treated with either a control diet or a diet containing RSV (0.4 g/kg diet) from 23 weeks of age, and tissues of tibialis anterior muscle were analyzed at 60 weeks old. Rotarod running time was shortened with aging; however, RSV treatment preserved running time. RSV blocked the aging-related increase in acetylated lysine levels in muscles, suggesting that RSV restored deacetylase activity of muscle SIRT1. Compared with 20 weeks old mice, the myofiber diameter was decreased in untreated 60 weeks old mice. In addition, LC3-II/LC3-I ratio, a marker of autophagic activity, was decreased and p62 and ubiquitinated protein levels, which are degraded by autophagy, were increased in muscles of 60 weeks old mice, suggesting impaired autophagy by aging. RSV treatment preserved the myofiber diameter, increased LC3-II/LC3-I ratio, and decreased p62 and ubiquitinated protein levels. RSV did not change activities of signals that regulate autophagy including AMPK and mTORC1.

[Conclusion] These results suggest that RSV attenuates age-related muscle atrophy and motor dysfunction by activating SIRT1 and promoting autophagy.

Hypothalamic neurons in the central nervous system are pivotal regulators of body homeostasis by receiving and integrating perturbation in the levels of key hormones and primary nutrients (amino acid, glucose, and lipids). Here, we demonstrated that L-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor is important for systemic energy and bone homeostasis. The LAT1-dependent amino acid uptake system was functional in the hypothalamus, whose activity was impaired in a mouse model of obesity and diabetes. Mice whose LepR-expressing neurons lacked solute carrier transporter 7a5 (Slc7a5), which encodes LAT1, showed obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing ventromedial hypothalamus (VMH) neurons prior to the onset of obesity. Importantly, adenoviral introduction of Slc7a5 selectively in LepR-expressing VMH neurons corrected abnormal energy and skeletal homeostasis. Mechanistic target of rapamycin complex-1 (mTORC1) was identified as a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest the LAT1-mTORC1 axis in LepR-expressing VMH neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow.

Brown adipose tissue (BAT) is a major organ responsible for diet-induced thermogenesis, a phenomenon which converts excess energy intake into heat. Previous studies suggested that hepatocytes regulate diet-induced thermogenesis in response to changes in nutritional status. However, it is not clear how hepatocytes are involved in diet-induced thermogenesis.

Here, we demonstrate that liver nardilysin (NRDC) expression changes by nutritional state; it increases by fasting and decreases by re-feeding. Moreover, liver NRDC decreases upon high-fat diet (HFD) feeding. We have previously demonstrated that mice systemically deficient in a metallopeptidase nardilysin (NRDC) show reduced fat mass, enhanced energy expenditure and BAT activity. To clarify the liver-specific role of NRDC in diet-induced thermogenesis and energy metabolism, we established hepatocyte-specific Nrdc deficient mice (LKO). In HFD-fed state, the body weight gain was significantly suppressed, while glucose tolerance and insulin sensitivity were significantly improved in LKO. Measurement in metabolic cage demonstrated unchanged food intake and physical activity, while increased oxygen consumption rate and heat generation in LKO. BAT in LKO showed less fat accumulation and increased thermogenic genes such as UCP1 and PGC1a. Together, these results suggest that hepatic NRDC regulates diet-induced thermogenesis via BAT activity.

Caenorhabditis elegans is an experimental organism widely used for longevity studies.

Here, we have developed an automated system called "C-HAS" to measure individual lifespan, healthspan, and frailspan. This technology provides an intrapopulation analysis to classify individual nematodes based on the differences of quality of their lifespan. Furthermore, based on the analysis results, a novel index, "Health score (HaS™️)" helps to measure the "integrated health impact on individuals" of the examined agent. Previously, this system demonstrated that it could be applied to the discovery of drugs that regulate lifespan and healthspan, but it is unclear whether it can be targeted to novel genes. So, in this study, among the possible regulators of lifespan and healthspan extracted by integrative expression analysis with existing data sets, we focused on W09D10.4/PPTC7, a gene that has no reported association with lifespan. We show that, PPTC7 knockdown extended lifespan and healthspan and increased the high-healthspan population in WT nematodes (HaS^TM = 54.85), but not in AMPK inactivation mutants (HaS^TM = 0.05), suggesting that PPTC7 is a novel AMPK-associated healthspan modulatory factor in C. elegans. In conclusion, this study establishes that this technology can be used to identify novel lifespan and healthspan regulator genes.

In both developed and developing countries, incidence of type 2 diabetes is rapidly increasing due to occurrences of obesity and unhealthy lifestyles. A line of previous studies has shown that unbalanced diet and physical inactivity are also risk factors of diabetes. The development of diabetes is preceded by symptoms such as resistance to insulin and disturbances of glucose metabolism. Therefore, long-term monitoring of glucose metabolism is crucial to investigate the physiological basis of the symptoms and further find a method to prevent the pathogenesis of the disease. However, there are no realistic methods to continuously monitor glucose metabolism for days in laboratory conditions. Because periodical (e.g., every 4 minutes) sampling of blood from laboratory animals is stressful to the animal and laborious for the experimenters, we developed a novel wireless system to chronically monitor glucose levels.

In our system, a monitoring device is implanted subcutaneously in inguinal area of adult rats. Using the implanted device, blood glucose levels were measured every 4 minutes for up to 1 week. With the monitoring system, we observed an increase in the blood glucose level with oral administration of 15% glucose solution and decrease with subcutaneous injection of insulin aspart (0.5 µl/kg).

Continuous monitoring of glucose metabolism is applicable to human subjects, but it does not allow us to experimentally reveal the molecular and physiological mechanism underlying the glucose metabolism. Our system is applicable to laboratory animals and thus allows for a large variety of experiments such as pharmacological interventions in glucose metabolism and insulin-independent manipulation of glucose levels.

Memories are thought to be stored as plastic changes in synapses between neurons activated during memory formation. Although molecular imaging of such memory-related synapses would contribute to deciphering the molecular basis of memory, there is no established method for visualizing them. In this study, we developed a method to selectively visualize synapses between neurons that are activated during memory formation using the c-fos promoter-driven Tet-On system. Tag-fused synaptophysin(tagSyp) and FingR.PSD95 were expressed in presynaptic auditory cortex neurons and postsynaptic lateral amygdala neurons, respectively, in an activity dependent manner. We found that the number of synapses positive for both tagSyp and FingR.PSD95 in the lateral amygdala was 15-fold higher in mice with cued fear conditioning than in control mice. Thus, we concluded that tagSyp/FingR.PSD95 double positive synapses correspond to memory-related synapses. We also demonstrate that our method is compatible with immunohistochemistry and nanometer-resolution molecular imaging, including STORM, enabling quantification of endogenous synaptic proteins specifically at memory-related synapses. It is expected that our method will open the way for comprehensive analysis of molecular changes in synapses induced by memory formation.

We have recently reported that nicotine enhances object recognition memory (ORM) via acting on the medial prefrontal cortex (mPFC) in mice. However, it remains unclear whether this effect of nicotine is mediated by the activation of mPFC neurons. Additionally, since the mPFC sends excitatory afferents to the perirhinal cortex (PRH), it is hypothesized that this pathway is involved in the nicotine-induced enhancement of ORM. To address these issues, we performed the novel object recognition test (NOR) using adeno-associated virus (AAV) vector-mediated chemogenetic techniques in male C57BL/6J mice (7 – 16 weeks old). Systemic nicotine (0.1 mg/kg; s.c.) administration before the training session of NOR significantly increased ORM. Silencing of mPFC neurons with inhibitory DREADD, which were selectively expressed in excitatory neurons, before nicotine injection significantly inhibited the nicotine-induced ORM enhancement. Conversely, activation of mPFC neurons with excitatory DREADD before the training session enhanced ORM. Moreover, selective silencing of PRH-projecting mPFC neurons with inhibitory DREADD before nicotine administration also attenuated the nicotine-induced ORM enhancement. These data suggest that nicotine enhances ORM via activating mPFC neurons, which may then activate the PRH.

High salt (HS) intake is known as a risk factor for hypertension and dementia. Prostaglandin E2 (PGE2) has various effects on vascular function and central nervous system via four types of PGE2 receptors (EP1-EP4). However, an involvement of PGE2/EP1 signaling in the HS intake-induced hypertension and emotional and cognitive dysfunctions is still unclear. In this study, we confirmed the effect of HS intake on the blood pressure and emotional and cognitive functions in mice. Mice showed hypertension and impairments of social behavior in social interaction test and object recognition memory in novel object recognition test 12 weeks after HS intake. HS intake increased phosphorylation of tau, but decreased phosphorylation of Ca2+ / calmodulin-dependent protein kinase II and expression of PSD95 in the prefrontal cortex. HS intake increased expressions of mRNA of EP1 receptor in the kidney and prefrontal cortex. The HS intake-induced hypertension, abnormal behaviors and increased phosphorylation of tau were not observed in the EP1 heterozygous knockout mice. These findings suggest that PGE2/EP1-tau phosphorylation signaling is involved in the HS intake-induced hypertension and emotional and cognitive dysfunctions.

Major depressive disorder is one of the largest medical and social problems worldwide. Accumulating evidence indicates serotonin neurons in the dorsal raphe nucleus (DRN) play a critical role in pathophysiology of depression as well as its treatment. However, it is unclear whether and how chronic antidepressant treatment and stress alter transcriptome profile of the serotonin neurons, which should determine its activity and ultimately behaviors. To this end, we applied the Translating Ribosome Affinity Purification (TRAP) to isolate the mRNA of serotonin neurons selectively. We performed TRAP of the DRN serotonin neurons in naïve, a selective serotonin reuptake inhibitor (SSRI)-treated mice, as well as resilient and susceptible mice after chronic social defeat stress (CSDS). Differential expression analysis identified 49 presumably prodepressive genes whose expression was decreased in chronic SSRI treated mice and increased in CSDS susceptible mice. Among these, we focused on S100a10 which, in the cortex and lateral habenula, play a key role in depression. To investigate a causal relationship between S100a10 decrease in the DRN and antidepressive phenotype, we infected the viral vector which knockdown S100a10 in serotonin neurons. As expected, S100a10 knockdown mice showed significantly shorter immobility time in the tail suspension test without affecting general locomotor activity. These findings shed light on the molecular mechanism of depression in serotonin neurons and suggest new approaches for the development of antidepressants.

Hippocampal neurogenesis mediated by activation of tropomyosin receptor kinases B (TrkB) is thought to play important roles in repair of brain function injury caused by neurodegenerative disorders. Therefore, stimulation of TrkB-mediated neurogenesis would be a possible therapeutic target. In the present study, we examined effect of overexpression of TrkB gene in the brain on hippocampal neurogenesis and any behavior change in vivo after systemic gene transduction with an aim to clarify possible treatment strategy. For such purpose blood-brain barrier permeable adeno-associated virus serotype PHP.eB (AAV-PHP.eB) was used to transduce TrkB gene in the brain. Flag-tagged mouse TrkB gene was first inserted to pAAV-CMV vector, followed by co-transfection with AAV-PHP.eB vector to construct the final AAV construct (AAV-PHP.eB-mTrkB-flag). Transfection with the transgene plasmid in neuroblastoma cell line Neuro2a increased expression of TrkB gene product, the phosphorylation of which was increased in the presence of a TrkB agonist 7,8-dihydroxyflavone, confirming activation of exogenously transfected TrkB. Intravenous administration of AAV-PHP.eB-mTrkB-flag not only increased expression of TrkB, but also tended to increase area of new-born neuron marker Dcx-positive cells in hippocampus compared to control AAV-treated mice, implying possible promotion of neurogenesis. Thus, AAV-mediated TrkB gene transduction would be the possible treatment of neurodegenerative disorders by performing further analyses of its pharmacological actions.

Chronic neuropathic pain is often caused by peripheral nerve injury. We previously demonstrated that spinal expression of serum and glucocorticoid-inducible kinase-1 (SGK-1) is associated with exacerbation of pain hypersensitivity in nerve-injured mice, but there are no available strategies to inhibit SGK-1 in the spinal cord. In this study, we attempted to search for clinically approved drug that has the ability to inhibit SGK-1 activity. As result of screening 1,271 clinically approved drugs, sulfasalazine (SSZ) was identified as potent inhibitor of SGK-1. SSZ is a prodrug composed of 5-aminosalicylic acid and sulfapyridine linked by an azo bond, but the azo bond in SSZ was necessary for its inhibitory action against SGK-1.  Although intrathecal injection of SSZ to nerve-injured mice alleviated mechanical pain hypersensitivity, no anti-neuropathic pain effects of SSZ were detected after oral administration due to its low bioavailability and limited spinal distribution, which were associated with efflux by breast cancer resistance protein (BCRP). Febuxostat (FBX) has been demonstrated to inhibit BCRP. Concomitant oral administration of SSZ with FBX increased the spinal concentrations of SSZ and allowed to exert its anti-neuropathic pain effects in nerve-injured mice, suggesting that FBX can improve the distribution of SSZ to the spinal cord after its oral administration by inhibiting the function of BCRP in intestinal epithelial cells and blood–cerebrospinal fluid barrier.

High mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, participates in pathological pain including chemotherapy-induced peripheral neuropathy (CIPN) and diabetic peripheral neuropathy (DPN). Endothelial thrombomodulin (TM) causes thrombin-dependent degradation of HMGB1, and TMα, a soluble form of TM, prevents CIPN in mice, an effect reversed by thrombin inhibitors. Intriguingly, repeated treatment with anticoagulants aggravates CIPN itself in mice. Thus, we examined the effect of TMα and/or thrombin on DPN in mice, and retrospectively analyzed the association between the use of anticoagulants and DPN in diabetic patients. In reptin receptor-deficient db/db mice, known as a model for type 2 diabetes, repeated treatment with TMα prevented the development of DPN, an effect reversed by argatroban, a thrombin inhibitor. Of 1036 diabetic patients hospitalized in Kansai Medical University Hospital in 2018, 615 who met inclusion criteria were enrolled in this study. Multivariate logistic regression analysis after propensity score matching showed that prescription of anticoagulants was significantly associated with the development of DPN. Thus, our data suggest that the TM/thrombin system functions to reduce DPN, and its inhibition by anticoagulants is a risk for DPN.

[Aim] Lenvatinib (LEN) is an oral tyrosine kinase inhibitor used for the treatment of thyroid cancer and hepatocellular carcinoma. Although fatigue is a common adverse reaction resulting in discontinuation of LEN, the mechanism underlying its development remains poorly understood. To address this issue, we investigated the effect of LEN on carnitine disposition and mitochondrial damage.

[Methods] Male Wistar rats were orally administered with vehicle or LEN (0.2 or 2 mg/kg) once daily for 14 days. In vitro experiments, differentiated C2C12 myotube cells were cultured with or without LEN (10 nM­­­–10 µM) for 24 hours.

[Results] Treatment with LEN did not affect serum concentration and urinary excretion of carnitine, but decreased mRNA and protein expression levels of CPT1, CPT2, CACT and OXPHOS in the skeletal muscles, but not the kidney, of rats. Consistently, LEN caused cell death and decreased mitochondrial protein expression in C2C12 cells in a dose-dependent manner.

[Conclusion] LEN may directly cause mitochondrial impairment in the skeletal muscle, suggesting one possible mechanism for the development of fatigue.

Background: Esophageal cancer (EsC) often recurs early after surgery. Recent studies shown that commensal bacteria affect the treatment for cancer through the mediation of host immune system and become a biomarker for recurrence of cancer after surgery in colorectal cancer. We, therefore, hypothesize that commensal bacteria are a potential biomarker of postoperative recurrence of EsC. This study leads to clarifying the immunological mechanism of the EsC microenvironment. Method: We collected the stool sample from patients that underwent surgery for esophageal cancer at the Department of General and Gastroenterological Surgery of Showa University, between January 2017 and September 2018. Patients were followed for twelve months to allow time to assess recurrences of EsC. We investigated the composition of the bacterial microbiome in the stool of patients with EsC by using 16s rRNA sequencing. Furthermore, we identified specific microbiotas as a risk factor in the recurrence of EsC by using AI/machine learning. Result: 16s analysis with Qiime2 showed a significant difference between recurrence of EsC group and non-recurrence of EsC group in the bacteria A in the Odoribacteraceae family. AI/machine learning showed a significant difference between the two groups in 8 species including bacteria A in the Odoribacteraceae family and bacteria B in the Actinomyces family. Bacteria A was significant higher in the recurrence group with the two analysis methods. Conclusion: Commensal bacteria become a potential biomarker of postoperative recurrence of EsC. Among commensal bacteria, bacteria A has been suggested to be a biomarker as a risk factor for EsC recurrence.

Measurement of plasma drug concentrations at a clinical site is essential for personalized medicine. For such purpose, the conventional liquid chromatography-mass spectrometry (LC-MS) method is unlikely to be suitable, owing to time and cost consumption. In this study, we describe an approach to rapid, easy, and low-cost drug monitoring with a boron-doped diamond (BDD) electrode, an advanced electrochemical material. As a test drug, we selected pazopanib, an inhibitor for multiple tyrosine kinase types. A small size sensor system with a simple BDD plate electrode of ~26 mm² without any chemical modifications determined the drug concentration in a measurement time of ~35 s from 100 µL rat plasma, which was exogenously mixed with pazopanib. We showed that this system was also applicable to blood samples collected from healthy rats orally administrated with pazopanib as well as drug-treated patients with different cancer types. Notably, all the procedures, including sample preparation and electrochemical measurement, were completed in a short time of ~10 min. The pharmacokinetics data obtained by the BDD electrode were similar to the data determined by LC-MS. Finally, we fabricated a prototype of a palm-sized system, which successfully analyzed ~60 µL of rat blood. This strategy may contribute to advances in on-site drug monitoring.

Based on them, we evaluated plasma samples from five rats orally administered pazopanib and eight clinical patients treated with the drug at our institution. The plasma concentrations and other pharmacokinetic parameters obtained by the BDD method were generally consistent with the LC-MS.

Because this strategy by the BDD sensor is more compact and inexpensive than the LC-MS setup, the electrochemical approach described here can be commonly used and contribute to tailor-made medicine.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of motor neurons. In the SOD1^G93A mouse model of ALS, abnormal proteins such as misfolded SOD1 and SOD1 oligomers are detected inside and outside of motor neurons. The concept named "glymphatic system" has recently been proposed as a waste clearance mechanism where aquaporin-4 (AQP4) is essential for generating a convective water flow in the brain. Recently we reported the overexpression and mislocalization of AQP4 in SOD1^G93A mice during the disease progression.

First, we investigated whether SOD1 oligomers were cleared by the glymphatic system using AQP4-deficient mice. The clearance of SOD1 oligomers in AQP4-deficient mice was reduced compared to that in WT mice. Next, to evaluate how aberrant AQP4 affect the clearance capacity of SOD1^G93A mice, fluorescently labeled ovalbumin (OVA) was injected into the CSF. The observed OVA clearance was evidently delayed in SOD1^G93A mice compared with WT mice.

These results suggest that the glymphatic system is abnormal and that waste clearance is delayed in SOD1^G93A mice. Since we have also observed OVA uptake by perivascular macrophages (PVM), the innate immune cells residing in the CNS, we will discuss the possible roles of PVM-mediated waste clearance in SOD1^G93A mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by Aβ deposition and neural networks disruption in the brain. We previously found that diosgenin, a constituent of Dioscorea Rhizoma, restored Aβ-induced axonal atrophy in neurons (in vitro) and recovered memory deficits in a mouse model of AD, 5XFAD. In the present study, we investigated whether diosgenin promoted long-distance axonal regeneration toward their intrinsic target area in 5XFAD brains, and clarified molecular mechanisms for accurate pathfinding of injured axons.

Retrograde tracing revealed that 14-day administration of diosgenin promoted axonal regeneration from the hippocampus to the prefrontal cortex, a neural circuit for memory formation, in 5XFAD mice. Subsequently, naïve neurons and axon-regenerated neurons in the brain slices were individually captured by laser microdissection to serve DNA microarray. Overexpression of the gene, whose expression was the most elevated in axon-regenerated neurons, to the hippocampal neurons promoted axonal regeneration in the brain and recovered memory deficits in 5XFAD mice.

Our study showed that axons in AD brains have capacities to regenerate toward long-distance original target by diosgenin administration. This finding proposes a novel therapeutic strategy to promote axonal regeneration for AD treatment.

In recent years, cannabis is known to affect the central nervous system and designated as an illegal drug and its possession or use is prohibited in many countries and regions. Δ 9-Tetrahydrocannabinol (THC), the main component of cannabis, acts on CB1 receptors, which are highly expressed in presynaptic terminals in the central nervous system. The prefrontal cortex (PFC) is known to be involved in a cognitive function in a number of studies. However, the role of CB1 receptors in the PFC on cognitive function remains unclear. In this study, we administered ACPA (arachidonylcyclopropylamide), a CB1 receptor agonist, topically to mice PFC and examined the involvement of CB1 receptors in the PFC in cognitive functions.

A guide cannula was placed in the PFC of C57BL/6J mice to locally administer ACPA. After a week's recovery period from surgery, various behavioral tests were performed.

In object location test evaluating spatial memory, spatial memory impairment was observed when ACPA was administered topically to the PFC at all three stages of memory; acquisition, consolidation, and retrieval. In addition, spatial memory impairment was observed when ACPA was administered locally to the PFC during memory acquisition. This study suggests that CB1 receptors in the PFC are important for the acquisition of spatial memory. Analysis of CB1 receptor in the PFC may reveal the effects of cannabis on the brain and neuronal circuits in PFC memory formation.

Glioblastoma, a primary brain tumor, is resistant to chemotherapy and can develop into a fatal space-occupying lesion. Glioma stem cells (GSCs) are thought to be responsible for tumor growth, chemo-resistance, and recurrence. Clonal glioma sphere (GS) culture, in which GSCs are enriched and self-renew as GS clone populations, provides us with quantitative details regarding GS clone survivability and changes in growth during GS/GSC population self-renewal. Previously, we proposed a novel chemotherapeutic paradigm, temozolomide (TMZ) and OTS964 in combination (T&O), and showed that T&O efficiently eliminated self-renewing GS clones and significantly suppressed the regrowth of TMZ-sensitive GS clones. However, it remained unclear whether T&O would be effective in treatment of TMZ-resistant GSC populations. T&O did not suppress T98-GS clone growth during population self-renewal, suggesting that TMZ-like growth suppression is necessary for the long-term control of GSC population size. In this study, we tested the PI3K inhibitor LY294002, which is thought to suppress GSC self-renewal, alone and in combination with OTS964 (L&O) against T98G-GS populations. LY294002 alone suppressed T98G-GS clone growth for 2–3 weeks, while allowing the clones to survive. By contrast, L&O efficiently eliminated two-thirds of the T98G-GS clones and continuously suppressed T98G-GS clone regrowth for 2–3 times longer than LY294002 alone, suggesting that L&O represents an alternative to T&O. Our findings indicate that this quantitatively validated combination paradigm could control growth of TMZ-resistant GSC populations through immediate and sustained shrinkage of heterogeneous GSC populations.

Human pluripotent stem cells (hPSCs) combine the features of robust proliferation with precise differentiation capacity. The molecular mechanisms orchestrating the differentiation process are poorly understood. In a previous shRNA screen, Aurora-B, the enzymatic component of the chromosomal passenger complex (CPC), has been identified as a candidate involved in pluripotency of mouse PSCs. Here we show that inhibition of Aurora-B kinase activity results in spontaneous differentiation of hPSCs. Sustained Aurora-B kinase activity results from the stabilization of the CPC components Aurora-B and Borealin due to low APC/C^Cdh1 activity in hPSCs. During hPSC differentiation, CPC activity is terminated by activated APC/C^Cdh1. RNA-seq analysis reveals that CPC activity maintained the epithelial characteristics of hPSCs via inhibition of an epithelial-to-mesenchymal transition (EMT) program. In conclusion, sustained CPC activity plays a critical role in the maintenance of pluripotency. We therefore propose an interphase role of the CPC in regulating embryonic pluripotency.

Parathyroid hormone receptor 1 (PTH1R) is a class B G-protein-coupled receptor (GPCR), consisting of extracellular domain (ECD) and transmembrane domain (TMD). PTH1R is activated by two endogenous peptide hormones called PTH and PTHrP. These hormones share similar sequences and activate the stimulatory G-protein (Gs) signaling pathway but show different physiological functions by the differences in the ligand dissociation kinetics. However, the structural basis for ligand recognition and ligand kinetics remains elusive.

We revealed the activated PTH1R structure binding the two

endogenous hormones, respectively. The structures and mutagenesis revealed distinct molecular recognition for each ligand and conserved active mechanism of PTH1R. Moreover, these structures elucidate molecular switch toggling signaling periods, responsible for the different pharmacological effects. Furthermore, we revealed five distinct structures PTH-PTH1R-Gs toward inactive transition. These sequential structures and molecular dynamics simulations revealed that an unwinding middle region of PTH induces PTH dissociating from PTH1R. This is the first GPCR structure that suggests the ECD allosterically modulates the activation of the receptor. Our structure provides structural insight for a different signal duration and another strategy for drag development for fine-tuning a duration of the receptor activation.

Metastasis is a major cause of cancer morbidity and mortality. Cancer cells can avoid detachment-induced apoptosis (anoikis) and survive under floating conditions. However, the mechanism by which cancer cells evade anoikis has not been fully established.

Here, we found that intracellular Na⁺,K⁺-ATPase α3-isoform (α3NaK) was abnormally expressed in intracellular vesicles of human cancer cells. Interestingly, the α3NaK-containing vesicles were translocated to the plasma membrane (PM) upon loss of anchorage. Pharmacological and biochemical studies showed that the translocation of α3NaK is mediated by FAK- and NAADP-dependent Ca²⁺ mobilization. The PM expression of α3NaK was also found in metastatic cancer cells obtained from peritoneal fluids of gastric and colon cancer patients. The α3NaK knockdown accelerated the detachment-induced apoptosis of cancer cells. In the mouse model, overexpression of α3NaK significantly inhibited cancer cell growth and lung metastasis. In downstream of the PM-translocated α3NaK, AMPK was found to be phosphorylated (pAMPK). These results suggest that intracellular α3NaK is translocated to PM via detachment-induced FAK/NAADP/Ca²⁺ pathway and that the PM-α3NaK and pAMPK are contributed to the survival of the detached cancer cells.

Ninjinyoeito (NYT) and Juzentaihoto (JTT) are immunomodulatory Kampo medicines, and clinically used to support cancer therapy. Our have previously shown that these medicines can suppress the differentiation of Myeloid-derived suppressor cells (MDSC), which is known to be accumulated in tumor and potently inhibits T-cell and NK-cell activity. In the tumor-bearing state, MDSC migrates to the tumor microenvironment (TME) and becomes an activated state that produces immunosuppressive factors, to promote tumor growth. In this study, we have investigated the effects of NYT and JTT on migration and activation in TME. To prepare MDSC, bone-marrow cells were isolated from C57BL/6J mice and differentiated into MDSC by the treatment with IL-6 and GM-CSF. The treatment of 4T1 breast cancer cells-conditioned media to MDSC markedly increased expression of immumosuppressive factors, arginase-1 and iNOS, and these increase in mRNA inhibited by NYT and JTT. In Transwell assay, migration of MDSC was stimulated by treatment of 4T1 cells or 4T1-conditioned media, NYT and JTT significantly inhibited this migration. These data indicated that, NYT and JTT suppressed not only differentiation, but also the activation and the migration of MDSC in TME. The multi-steps inhibition may be important in the immunomodulatory effects of these Kampo medicines.

Astrocytes, a non-neuronal cell in the central nervous system, participate in the purinergic signaling. As lowered ATP release from astrocytes is related to depressive-like behavior, basal ATP transmission is thought to play important roles in physiological brain function. Since a single astrocyte interacts with up to 140,000 synapses in rodents, spatial property of ATP release is essential to understand purinergic signaling within brain architecture, which is not well understood. Using two-photon microscopy, we investigated spatiotemporal properties of ATP release in astrocytes with a novel genetically encoded extracellular ATP sensor, GRAB_ATP. In neuron-astrocyte coculture, TTX insensitive ATP release events were detected. In acute slices in which astrocytes expressed GRAB_ATP sparsely by means of in utero electroporation, we also observed spontaneous ATP release events, which were suppressed by astrocyte specific toxin, fluorocitrate, and insensitive to TTX and vesicular release blocker, bafilomycin A1. Typical ATP release spread over 50­–200 µm² with concentration roughly ranged 0.5–5 µM. Simultaneous monitoring with intracellular calcium revealed that ATP release and Ca²⁺ event rarely co-occurred. In conclusion, our findings indicate that astrocytes spontaneously release ATP in acute slices via mainly non-vesicular Ca²⁺ independent pathway, which possiblly activates purinergic receptors in nearby hundreds of synapses.

Astrocytes become reactive upon injury and inflammation in the brain to alter their molecular profiles, morphologies and functions. Reactive astrocytes alter the expression of receptors which are responsible for their functions especially communications in neuron-glia and glia-glia. Among such receptors, the expression of P2Y1 receptors (P2Y1) is upregulated in many neurological diseases including epilepsy and Alzheimer's disease, in which neuronal hyperexcitability is commonly observed. We have previously shown that P2Y1 upregulation in astrocytes trigger neuronal hyperexcitability by enhancing neuron-astrocyte communications. However, the mechanism underlying the upregulation of P2Y1 in astrocytes remains unknown. We investigated the role of microglia in enhanced P2Y1R signaling in astrocytes during pathological conditions. To ask whether microglia play a role in P2Y1 upregulation in astrocytes, we depleted microglia by treatment with PLX5622 and found much larger Ca²⁺ elevation evoked by a P2Y1 agonist and more P2ry1 transcripts in astrocytes. Microglia depletion enhanced extracellular ATP level presumably through impairment of degradation of ATP. These findings suggest that microglia should has an important role to control P2Y1 receptor expression in astrocytes and negatively regulate neuron-astrocyte communication.

Alexander disease (AxD) is a rare neurological disorder caused by mutations of GFAP gene, an astrocyte selective intermediate filament, and AxD astrocytes show abnormal aggregates, i.e., Rosenthal fibers (RFs), a main pathological finding. AxD brain also shows neuroinflammation, where microglia are activated.　In this study, we show that manipulation of microglia would be a potential therapeutic strategy for the treatment of AxD using AxD model mice with human GFAP mutation (R239H) (60TM), To achieve this, we used an ON/OFF protocol of PLX5622 (PLX), a selective CSF-1 receptor antagonist. PLX-ON depleted almost all resident microglia, and subsequent PLX-OFF caused almost complete recovery of microglia, indicating that the old microglia should be replaced with newly repopulated microglia in the AxD brain. The amount of RF stained with Fluoro Jade B was significantly reduced by this replacement, suggesting that microglia should become more protective by their repopulation. Furthermore, microglial replacement significantly decreased the expression of Lipocalin2, the most upregulated molecule in 60TM astrocyte, and other inflammatory molecules in 60TM. Taken together, the ON/OFF protocol of PLX allows us to replace old microglia with newly repopulated ones in the AxD brain, which can dramatically ameliorate AxD pathogenesis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease. During demyelination, oligodendrocyte precursor cells (OPCs) can proliferate, migrate to the site of injury, differentiate into mature oligodendrocytes, and generate new myelin. Since increased numbers of OPCs are observed in MS lesions, insufficient differentiation of OPCs is considered to be one of the causes of demyelination and axonal degeneration. However, it has been reported that OPCs could promote the disruption of the BBB or release inflammatory cytokines under inflammatory conditions, and the involvement of OPCs in MS is not fully understood. In this study, we investigated the role of OPCs in the acute phase of MS by removing OPCs. We employed a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which was induced by immunization with myelin oligodendrocyte glycoprotein (35-55). We depleted OPCs by intraperitoneally injecting tamoxifen and diphtheria toxin (DT) in Pdgfra^CreER/+:Rosa26^DTR/+ mice. When DT was injected from the next day of disease onset, EAE severity was significantly reduced in OPC-depleted mice. Quantitative RT-PCR analysis revealed that the expression levels of pro-inflammatory cytokines and the marker of helper T cell subset Th17 were suppressed in the spinal cord of OPC-depleted group. These data suggest that OPCs are involved in CNS inflammation, T cell response and the development of EAE.

Glaucoma is a leading cause of blindness worldwide, which is caused by the degeneration of retinal ganglion cells (RGCs). An elevated intraocular pressure (IOP) is widely recognized as one of the major risk factors for glaucoma. We have reported that purinergic P2Y₁ receptor is essential for IOP reduction and P2Y₁ receptor knockout (P2Y₁KO) mice show hypertensive glaucoma-like phenotype. We already reported that P2Y₁ receptor is located in ciliary body(CB) and trabecular meshwork (TM), and controls production and outflow of aqueous humor, respectively. We also found that ocular hypertension itself did not solely cause RGC degeneration at young ages, so we hypothesized IOP-independent pathogenic mechanism. Immunohistochemical analysis revealed that P2Y₁ receptors were expressed in Müller cells in the retina in addition to CB and TM. To test the role of P2Y₁ receptors in Müller cells for RGC damages, we made Mlc1-tTS::P2ry1^tetO/tetO mice (Müller cell-specific P2Y₁ receptor conditional knockout mice; MC-cKO). We measured the IOP of control (P2ry1^tetO/tetO) and MC-cKO mice and found no difference between them. Next, RGC damage was compared by counting the number of Rbpms-positive cells. Howeve, MC-cKO mice at 12 months old showed significantly higher number of RGCs loss than that in age-matched control mice. Accompanied this, the number of apoptotic cells significantly increased in the MC-cKO mice. Taken together, our results demonstrated that the lack of P2Y₁ receptors in Müller cells promotes RGC loss without IOP elevation, suggesting an importance of Müller cells for pathogenesis of glaucoma. (1303/1350 words)

During the critical period of development of the central nervous system, synaptic connections are first excessively generated and then reduced gradually via selective synaptic pruning. However, the rule how select the target synapses of pruning remains incompletely known. The candidate mechanisms include the Hebb's rule, which suggests that synaptic connections between neurons that fire synchronously would survive from developmental pruning. To experimentally verify this well-known theory, we developed a new method that enables to induce synchronous firing in in vivo layer 2/3 neurons of the mouse somatosensory cortex that sparsely expressed channelrhodopsin2 (ChR2) through in utero electroporation. We transcranially stimulated ChR2-positive neurons on postnatal days 9-to-13 and measured the connection probability between these neurons using in vitro patch-clamp recordings on postnatal days 21-to-28. The neocortex that received chronic photostimulation exhibited higher probabilities of synaptic connections between ChR2-positive neurons, compared to non-stimulated neocortex. The results are consistent with the Hebb's rule.

The desmosome is an intercellular junctional structure that provides strong adhesive forces between cells. Desmoplakin, an integral core protein of the desmosome, is expressed not only in epithelium and cardiac muscle but also in hippocampal neurons, which do not have desmosomes. The desmoplakin and the plakoglobin in cultured hippocampal neurons is shown to be coimmunoprecipitated with N-cadherin. However, there is still little evidence for the localization or function of desmoplakin in hippocampal neurons. Here, we found that three variants of Desmoplakin are expressed in the hippocampus. One of them corresponded to Desmoplakin Ia, which has only been identified in humans. We also confirmed that Desmoplakin mRNA is expressed in mature granule cells in the dentate gyrus of the mouse hippocampus by in situ hybridization. Desmoplakin immunoreactivity was localized to ciliary structures and dendrites of the granule cells. Exploration of the specific function of Desmoplakin in the granule cells of dentate gyrus may provide insight into the architecture and plasticity of hippocampal neural network.

Orexin neurons in the hypothalamus regulate physiological functions, including energy homeostasis and wakefulness, and are also related to motivation. Here, we examined the roles of orexin neurons in motivated behaviors. We measured the activities of orexin neurons using fiber photometry under a free-moving condition, in which the rats were subjected to the fixed ratio (FR) or progressive ratio (PR) schedule of a touchscreen-based automated operant task. To measure the activities of orexin neurons, AAV-FLEX-GCaMP7s was injected into the hypothalamus of Orexin-Cre rats. We found that under FR5 conditions in which rats were able to obtain a food pellet by touching the screen consecutively five times, the activity in orexin neurons was increased after the fifth screen touch (after which one food pellet is delivered). The activity peaked before rats obtained reward, and then decreased after food intake. Next, we included non-reward trials in the FR5 test in which the rat was not able to earn reward even after touching the screen five times. The orexin activities in non-reward trials were also increased after the fifth screen touch, but the decrease after food intake was diminished compared to those in reward-trials. In the PR schedule test, the orexin activities were gradually increased. Together, these observations suggest that the orexin activities are associated with motivational behaviors, and that orexin neurons may be involved in craving and reward prediction, and satisfaction.

It is known that stress suppresses neurogenesis in the hippocampus. Neurotrophin-3 (NT-3), a neurotrophic factor, has been reported to be upregulated in the hippocampus of adult mice by stress and corticosterone administration. In order to investigate the effects of increased NT-3 on neurogenesis in the hippocampus and stress-induced behaviors, we generated NT-3 overexpressing mice in the hippocampus by administering adeno-associated virus carrying the NT-3 gene (AAV-NT-3). NT-3 mRNA was expressed more than 7 times higher in the hippocampus by AAV-NT-3 administration compared to control hippocampus. NT-3 expression was mainly localized in hippocampal hilus. After 4 weeks of AAV-NT-3 administration, the number of proliferating cells in the hippocampal dentate gyrus was decreased in the NT-3 overexpression group compared to the control group. This result suggests that high dose of NT-3 may suppress proliferation of neuronal stem cells/progenitors in the dentate gyrus. In the future, we plan to investigate changes in neural differentiation and maturation in the hippocampus of NT-3 overexpressing mice to further clarify the role of NT-3 in neurogenesis. It is also necessary to clarify the effects of NT-3 on stress by examining stress-induced anxiety-like and depression-like behaviors in NT-3 overexpressing mice.

Interactions between the thalamus and cerebral cortex are crucial for relaying sensory signals, and their impediment is associated with neuropsychiatric disorders. However, the pathogenesis of these disorders, including autism spectrum disorder, remains unsolved due to the lack of in vitro models that mimic pathophysiological events of the human brain. Brain organoids, three-dimensional cell aggregates differentiated from pluripotent stem cells, have been shown to partly mimic the structure, function, and development of some brain regions in vitro. Here we report in vitro thalamocortical and corticothalamic interactions by generating assembloids, a 3D assembly of organoids, from human induced pluripotent stem cells (hiPSCs). We differentiated hiPSCs to both thalamic organoids and cortical organoids, each of which expressed brain region-specific markers. We then generated assembloids by fusing the thalamic and cortical organoids. Labeling the organoids with fluorescent proteins visualized reciprocal projections in the assembloids. In addition, rabies viral tracing demonstrated transsynaptic labeling between two organoids, suggesting the formation of synaptic connections in the assembloids. The in vitro models of neural circuits between the thalamus and cortex will help us understand neuropsychiatric disorders.

[Background] Chronic stress does not trigger depression in all individuals, as some remain resilient. However, the underlying mechanisms that contribute to stress sensitivity have been poorly understood. We found that Shati/Nat8l, N-acetyltransferase, levels increased in the dorsal striatum of stress-susceptible mice exposed repeated social defeat stress (RSDS). In the present study, we revealed the mechanism of regulation in stress sensitivity by Shati/Nat8l.

[Methods] C57BL/6J male mice were exposed RSDS using ICR mice, and the susceptible or resilient group were classified by social interaction test. We generated dorsal striatal Shati/Nat8l overexpression (STR-Shati OE) or knockdown mice (STR-Shati KD). These mice were assessed depression-like behaviors after RSDS. We investigated the relationship between Shati/Nat8l and serotonin in the striatum by pharmacological regulation of serotonergic system and in vivo microdialysis.

[Results] Striatal serotonin decreased in stress susceptible, not resilient mice. STR-Shati OE showed the vulnerability to social stress. Conversely, STR-Shati KD showed the resilience to social stress. The reduction of striatal serotonin was observed in STR-Shati OE. The vulnerability to stress in Shati OE recovered by modulation of serotonergic system.

[Conclusions] Striatal Shati/Nat8l controls stress sensitivity via regulation of serotonin in the striatum. Our study suggested the novel mechanisms underlying stress sensitivity, and striatal Shati/Nat8l could be a new target for medical tools for depression.

We propose that L-DOPA by itself is a neurotransmitter. Recently, a G-protein coupled receptor GPR143, a gene product of ocular-albinism1, was identified as a receptor for L-DOPA. In this study, to identify the physiological role of GPR143, we examined the phenotypic analysis using Gpr143-gene deficient (GPR143-KO) mice. We found that time spent in open arms using zero-maze test was decreased in GPR143-KO mice when compared to wild-type (WT) mice. The time spent in open arms was also decreased in indirect pathway striatal neuron specific GPR143-KO mice. To investigate the involvement of endogenous L-DOPA in this phenotype, we perform zero-maze test after treatment with alpha-methyl-para-tyrosine (α-MPT), a synthetic inhibitor of L-DOPA. Intraperitoneal injection of α-MPT at the dose of 3 mg/kg, which decreased the striatal content of L-DOPA without affecting that of dopamine, suppressed the time spent in open arms in WT mice. This effect of α-MPT was not observed in GPR143-KO mice. These results suggest that L-DOPA regulates anxiety-like behavior through GPR143 expressed in the striatal indirect pathway neurons.

Chronic social stress induces neuronal dysfunctions in the medial prefrontal cortex (mPFC) for emotional and cognitive disturbances. However, the subcellular mechanism remains elusive. Here we examined ultrastructural and multi-omics changes in the mPFC in a mouse model of social defeat stress. Acute stress induced dendritic membrane deformation with mitochondrial swelling in mPFC neurons, leading to dendritic atrophy after chronic stress. Synaptic, but not bulk tissue, proteomes in the mPFC differentiated naïve and stressed mice and further uncovered two distinct states in stressed mice. Proteins involved in mitochondrial metabolic functions mostly decreased with chronic stress regardless of the synaptic proteomic state. By contrast, proteins responsible for mitochondrial homeostasis increased in stressed mice with a specific synaptic proteomic state associated with behavioral resilience to chronic stress. These findings suggest that the balance between mitochondrial metabolic dysfunction and its maintenance at mPFC synapses determines stress susceptibility in mice.

Recent evidence has suggested that pituitary adenylate cyclase-activating polypeptide (PACAP) has critical roles in central and peripheral pathways, such as spino-parabrachio-amygdaloid and hypothalamic-pituitary-adrenal pathways, mediating stress-related negative emotional behaviors. Although it is well established that there is a great degree of comorbidity of chronic pain and negative emotional behaviors, the cellular mechanism underlying chronic pain and anxiety/depression interaction still remains to be elucidated. Here, we evaluated possible involvement of PACAP signaling in the development of anxiety- and depression-like behaviors after peripheral nerve injury in mice. We observed that spinal nerve ligation (SNL) induced anxiety- and depression-like behaviors lasting for at least 3 weeks in wild-type (PACAP +/+) mice. However, the development of SNL-induced anxiety- and depression-like behaviors was almost completely abrogated in PACAP -/- mice. Furthermore, we found that selective overexpression of PACAP by the infection of adeno-associated virus in the hypothalamic paraventricular nucleus (PVN), but not neighboring ventromedial hypothalamus, region resulted in the induction of anxiety-like behavior. In contrast, siRNA-mediated knockdown of PVN PACAP attenuated the development of SNL-induced anxiety- but not depressive-like behavior. Our data support that PVN PACAP signaling is involved in an important mechanism underlying the anxiety-like behaviors in peripheral neuropathic pain condition.

Psychiatric disorders, such as depression and anxiety related disorders, posed a significant burden worldwide. Therefore, it is necessary to develop additional safe and effective antidepressants. Accumulating evidence indicates that PACAP (pituitary adenylate cyclase-activating polypeptide) and its preferring receptor PAC1 are involved in psychiatric disorders, especially stress-related disorders. Recently, we developed novel small-molecule, non-peptide, and high-affinity PAC1 antagonists and showed that the antagonists significantly attenuated mechanical allodynia in mice. In this study, we aimed to characterize the PAC1 antagonist as a new therapeutic reagent for stress-related disorders and conducted behavioral pharmacological experiments in mice. A single dose of the PAC1 antagonist significantly improved anxiety-like and depressive-like behaviors in chronic social defeated stress mice, and this effect lasted long period which was similar to that of ketamine. In addition, the PAC1 antagonist did not exhibit behavioral impairments, including pre-pulse inhibition deficits and cognitive deficits in naïve control mice. These results indicate that the novel PAC1 antagonist may have a robust antidepressant effect and highly safe profile.

【Background】Oxytocin, a neurohypophysial hormone from the posterior pituitary, is known as an important factor for childcare and breastfeeding. Also, vasopressin has been reportedly involved in maternal behaviors through the vasopressin receptors of V1A and V1B subtypes. Previous studies demonstrated that the V1A receptor antagonist given into the median preoptic area of rat resulted in significant reduction of caregiving behavior of the mother. Although the studies on the oxytocin hormone and vasopressin/V1A receptor have been extensively conducted, our knowledge on the V1B receptor in maternal behavior is still limited.【Purpose】We intended to clarify a role of the V1B receptor in mother-child interaction during lactating period.【Methods】We compared exploratory behavior between nonpregnant females of control mice and those of the V1B knockout mice in open field test. After giving a birth, the mothers were examined on their behavior in pup retrieval test. Moreover, massive amounts of data from behavioral recordings were visualized and mother-infant relationship was analyzed by deep learning strategy.【Results and discussion】After training about 3000 images, our deep learning model successfully classified mother and babies with 99% accuracy. The analysis results by deep learning model were in good agreement with the observational results by an investigator. Together, we propose that this new method can be applied further to other areas of behavioral study to overcome the limitations and increase the efficiency for analyzing complex behaviors.

【Objective】NPSLE is an intractable autoimmune disease with neuropsychiatric symptoms, such as depression. Recent, studies reported that LPA reduces neuroinflammation. MRL/lpr mouse has been used as an animal model of NPSLE because of behavioral abnormalities. In this study, we examined the effects of LPA on NPSLE model mice.

【Methods】15-week-old MRL/lpr mice were treated with or without LPA for 2 weeks. In another study, the mice were pretreated with or without ki16425 (an antagonist of LPA receptors 1 and 3) and they were treated with LPA for 2 weeks. After treatment, the behavioral tests were performed as indices of depression. Histological examinations were performed in the harvested brain tissues.

【Conclusions】The treatment with LPA significantly reduced the depressive behaviors in MRL/lpr mice. Pretreatment with ki16425 negated the effects of LPA. The expressions of Iba1 and CD68 (microglial markers) were increased in the hippocampus and prefrontal cortex of MRL/lpr mice compared to controls and LPA treatment suppressed the increases of Iba1 and CD68. Pretreatment with ki16425 negated the inhibitory effects of LPA. These findings suggest that LPA receptor stimulation by LPA may suppress microglial activation and depressive behaviors in NPSLE model mice.