ADRA is an alternative method developed based on the principle of covalent bonding between sensitizer and proteins in the early stage of the mechanism of skin sensitization. The Direct Peptide Reactivity Assay (DPRA) with same principle previously listed in the OECD test guidelines and uses a seven-amino acid peptide containing cysteine or lysine as a nucleophilic reagent. Therefore, DPRA’s detection sensitivity in measurement by HPLC-UV is not sufficient, so it is necessary to prepare a high-concentration test chemical. Therefore, there are problems such as precipitation of the test chemical in the reaction solution and co-elution of the peptide and the test chemical. While, instead of DPRA, the ADRA was developed using two chemically synthesized nucleophilic reagents-namely, N‐(2‐(1‐naphthyl)acetyl)‐L‐cysteine (NAC) and α‐N‐(2‐(1‐naphthyl)acetyl)‐L‐lysine (NAL)-in which naphthalene rings with a high molar absorbance coefficient in the ultraviolet range have been introduced to N‐termini of the cysteine and lysine amino acids that react with the test chemical. Therefore, in March 2016, we set up a validation team consisting of JaCVAM, universities, chemical companies, and domestic and foreign experts with the aim for listing in the OECD guidelines. First, experimental technology was transferred to participating facilities, troubles that occurred at each facility were shared, and the protocol was improved so that highly reproducible results could be obtained at any facility, and then validation tests were conducted. After preparing a validation report, a third-party evaluation meeting and two commenting rounds, ADRA was able to be included in the OECD Test Guidelines in June 2019. In addition, since the introduction of naphthalene which has a high obsorption has made it possible to reduce the concentration, enabling the following items. (1) Decrease in the frequency of precipitation of the test chemicals in the reaction solution (2) Decrease in the frequency of co-eluting of the nucleating reagent and the chemical (3) It is possible to evaluate chemicals with unknown molecular weight using the gravimetric approach (4) High-sensitivity detection of nucleophilic reagents by the fluorescence method (5) Evaluation the mixture by a combination of the gravimetric approach and fluorescence detection (6) Evaluation for photo-allergy chemicals, (7) Evaluation of respiratory sensitizing chemicals. Since ADRA is in chemico method, there is no need to prepare animals or cultured cells, and not only can it be evaluated easily, but it can also be implemented with 96-well microplates, so high throughput system is possible. Therefore, it is considered that it can be applied to drug toxicity screening, and it is expected that it will be used in the pharmaceutical field in the future.

Alternative test, which comes from mind of animal welfare, is replacement of an animal test with one that uses non-animal systems for purpose of research and education (Replacement), and includes reduction of animal use (Reduction) and to lessen or eliminate pain or distress to animals (Refinement). Alternative test is effective the abolition of useless animal experiment, economic evaluation of a large number of new compound, risk evasion with searching a toxic unknown compound in animal experiment, study of the mechanism of action for extrapolation to human. In recent years, from the viewpoints of animal welfare and promotion of efficiency of research and development, the reconsideration of animal experiment, reduction of the used animal number, and international development of the alternative experimental animal which can replace it from mammals, are studied positively. In this symposium, I will discuss "New approach of alternative to animal experiments in Japan".

Next generation sequencing of human cancers have revealed the presence of characteristic mutational signature that reflect their etiology. Furthermore, when a strong strand bias is observed in a mutational signature, it is suggested that DNA adducts might contribute to the mutation induction at the relevant lesions of contextual mutations. These DNA adducts could be considered as "driver adducts" for human cancer development. Therefore, screening and identification of driver adducts that can be linked to the mutational signatures is a promising method to unveil the environmental factors-human cancer relationship. Recently, we have reported unique mutational signatures with C:G to T:A transitions observed in cholangiocarcinoma among printing industry workers in Osaka, Japan. Moreover, we analyzed mutational signatures and driver adducts of 1,2-dichloropropane (DCP) that have been suggested to be responsible for occupational cholangiocarcinomas, by using a multidisciplinary approach, such as whole genome and a comprehensive DNA adducts analyses. Based on the results obtained from the present study, 1,2-DCP might contribute to the occupational carcinogenesis of cholangiocarcinoma.

Asbestos is naturally occurring mineral fiber and one of representative harmful and tumorigenic materials. Occupational or environmental exposure to asbestos is still a global issue to cause asbestos-related diseases (ARD), in particular malignant mesothelioma is a serious disease with poor prognosis. It takes a long period of about 40 years to develop mesothelioma after inhalation exposure to asbestos, which is known to accumulate in regional lymph nodes. Those clues motivated us to examine immunological effects of asbestos exposure by cull culture experiments, omics data and broad analyses for peripheral blood of patients with mesothelioma. Our studies clarified several key events of asbestos-caused functional impairment in NK, helper T (Th) and cytotoxic T cells (CTL) as well as augmentation in regulatory T cells, associated with decrease in NKp46, IFN-gamma, CXCR3 and perforin and increase in CTLA-4, which were also observed in patients with malignant mesothelioma. In addition, it was also found that diffuse pleural thickening, a benign type of ARD, showed better characteristics of anti-tumor immunity compared with malignant mesothelioma, including high expression of perforin in CTL and low expression of GITR on cell surface of Treg. Those findings indicate that asbestos exposure has a potential to cause suppressed immune functions, which might contribute to the pathogenesis of malignant mesothelioma together with directly tumorigenic effect of asbestos on mesothelial cells. On the basis of our obtained knowledge, immunological scoring devices with mathematical formulae were also developed, in which M-score successfully distinguished patients with mesothelioma from other people. Moreover, our recent study about transcriptome of human cytotoxic T cell line demonstrated that continuous exposure to asbestos causes gradual decline in IFN-gamma expression and shared alteration in expression of transcripts among the types of asbestos fiber. All of our obtained findings together with more those in future will contribute to early diagnosis of and therapeutical strategy for mesothelioma in people exposed to asbestos.

Airborne particulate matter is produced artificially or naturally from boilers, incineration plants, depository sites, automobiles, cigarettes, soil and volcanic eruptions, and causes air pollution. Inhalation of particulate matter is known to exacerbate respiratory allergic disorders such as bronchial asthma. On the other hand, recent studies suggest that inhaled particulate matter reaches the brain through the nasal epithelial layer, and in addition epidemiological studies show that exposure to particulate matter induces extension of hospitalization period and high case fatality rates after onset of ischemic stroke, suggesting that the brain is an important target of inhaled particulate matter.

Cerebral edema is one of significant factors to determine the prognosis of cerebral ischemic stroke. We reported that polycyclic aromatic hydrocarbons, which are components of airborne particulate matter, activate immune cells and cause inflammation. Furthermore, inflammation in the central nervous system (CNS) reportedly induce rupture of blood-brain barrier, leading to vasogenic edema. Based on these findings, we have investigated the relationship between exposure to airborne particulate matter and the prognosis of ischemic stroke focusing on neuroinflammation and cerebral edema. In this symposium, we will report the effects of particulate matter collected in Beijing on pathophysiology of photothrombosis mouse models and would like to discuss the action of airborne particulate matter in the CNS.

There are many chemicals around us, and some of them affect our health, but most of them are still unknown. Some of these chemicals may have serious effects on the developing brain, and it is hoped that they can be clarified. To clarify the effects of chemicals on the developing brain, it is essential to develop evaluation indices to determine whether or not they have serious effects on the developing brain. In the process of investigating the neurotoxic mechanism of low concentrations of organotin using cultured cortical neurons isolated from rat fetuses, we found that GluA2 was reduced from an early stage before the neurons were damaged and that this was due to a decrease in the transcription factor, nuclear respiratory factor-1. On the other hand, to explore other evaluation indices, we used methylmercury as a known developmental neurotoxin and examined changes in mRNA expression encoding 12 molecules that have already been reported to play important roles in neurodevelopment until day 21 of culture. The results showed that low concentrations of methylmercury repressed the expression of three genes, Dlg4, Syp, and Bdnf, at specific time points. By investigating whether these gene expression changes are also observed in other developmental neurotoxicants and whether these phenomena are reproduced in human iPS neurons, it is expected that the developmental neurotoxicity assessment index will be useful.

Epidermal growth factor receptor (EGFR) is most intensively studied receptor tyrosine kinase. Several EGFR mutations and modifications have been known to induce abnormal self-activation, which plays a central role in carcinogenesis. Environmental air pollutants, which are associated with cancer and respiratory diseases, can also activate EGFR. For example, the environmental electrophile 1,2-naphthoquinone (1,2-NQ) has previously been shown to impact EGFR signaling. Humans are exposed to NQs through combustion of fossil and diesel fuel and from tobacco smoke. In addition, 1,2-NQ was recently detected in atmospheric PM2.5. 

In this symposium, we will present that 1,2-NQ is a novel activator of EGFR but not other types of EGFR family proteins. We found that 1,2-NQ forms a covalent bond with Lys80 located in extracellular domain I of EGFR. This modification leads to the activation of EGFR-Akt signaling in a human lung adenocarcinoma cell line. Our study reveals a novel mode of EGFR pathway activation and suggests a link between abnormal EGFR activation and environmental pollutant-associated diseases such as cancer.

G protein-coupled receptors (GPCRs) play pivotal roles in converting physicochemical stimuli due to environmental changes to intracellular responses. After ligand stimulation, many GPCRs are desensitized and then recycled or degraded through b-arrestin-dependent internalization, an important process to maintain protein quality control of GPCRs. However, it is unknown how GPCRs with low β-arrestin sensitivity are controlled. Here we unmasked a b-arrestin-independent GPCR internalization, named Redox-dependent alternative internalization (REDAI), using b-arrestin-resistant purinergic P2Y₆ receptor (P2Y₆R). Natural isothiocyanates (ITCs) covalently bind with Cys²²⁰ in the intracellular 3^rd loop of P2Y₆R, and promote internalization and degradation of P2Y₆R through ubiquitination of Lys¹³⁷ in the 2^nd loop. P2Y₆R is highly expressed in macrophage and pathologically contributes to the development of colitis in mice. Endogenous electrophiles, such as S-nitrosoglutathione, also induce P2Y₆R degradation leading to anti-inflammation in macrophages. Prevention of Cys²²⁰ modification on P2Y₆R resulted in aggravation of the colitis. Accordingly, targeting REDAI on GPCRs will be a breakthrough strategy for the prevention and treatment of inflammatory diseases.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. In this presentation, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI. We also present our recent work for development of other class of reactive warheads for selective covalent inhibition of cellular proteins.

Most GPCR form protein families composed of functionally and/or structurally related subtypes. The subtype-selective ligands are highly desired for both pharmacological tools and drug discovery. However, high protein sequence homologies within a receptor family hamper developing subtype-selective ligand. Our purpose is to develop new methods for selective activation of target receptor subtype using chemogenetic approach in which the target receptor is genetically engineered to interact with a designed chemical partner selectively. In this direction, we have recently developed chemogenetic methods for activating mGlu1, a metabotropic glutamate receptor subtype. This approach allowed chemogenetic regulation of mGlu1 endogenously expressed in mice. Although powerful, it may be hard to apply this method in vivo due to the unclear pharmacokinetics of the designed ligands. In this context, we are now developing new chemogenetic methods for in vivo application. In this symposium, I will present the recent progress of our GPCR chemogenetics and discuss perspectives toward covalent drugs using our chemogenetics.

The serotonin hypothesis of depression has existed since over 50 years ago. However, it remains unclear whether decreased serotonin levels in the brain could cause depression mainly because of ethical or technical limitations. For example, we and others have demonstrated using optogenetic or chemogenetic methods that two origins of serotonergic projections to the forebrain, the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN), play distinct roles in cognitive and emotional function (Ohmura et al., 2014, Int J Neuropsychopharmacol; Teissier et al. 2015, Cell Rep; Ohmura et al., 2020, Neuropharmacology; Ohmura et al., 2021, Curr Biol), indicating that selective manipulation of each nucleus is required. Moreover, recent studies showed that serotonin neurons release glutamate as well as serotonin, suggesting that previous studies have failed to manipulate serotonin levels selectively. To overcome the above limitations, we separately knocked out the tryptophan hydroxylase 2 (TPH2) gene in each serotonergic origin (DRN and MRN) by taking advantage of in vivo gene editing with CRISPR/Cas9 and examined whether the reduction of serotonin in each nucleus could induce depression-like behavior in adult mice.

Repeated exposure to emotional stress can lead to a variety of mental disorders. The social defeat stress model is an animal model that is frequently used to investigate the mechanisms of chronic stress-related mental disorders. In this model, however, it is difficult to study the effects of emotional stress itself since the subject animals are exposed to both physical and emotional stress at the same time. Recently, a novel psychosocial stress model has been established that exposes mice to emotional stress without physical distress by witnessing socially defeated conspecifics. It has been reported that animals witnessed social defeat scenes display behavioral and physiological changes similar to those observed in actually defeated animals.

On the other hand, we found that emotional stress activated different brain regions compared to physical stress. In this symposium, we will focus on the insular cortex, the region activated only during emotional stress, and report our findings on the roles of the insular cortex in emotional stress-induced behavioral changes.

There are many studies on the effects of antidepressants in normal animals, but a more relevant study is to evaluate the effects of the drugs in paradigms designed to mimic symptoms of human depression. Animal models of repeated stress exposure simulate the presumed etiology of depression, and this stress may be produced by exogenous corticosterone administration. Additionally, rodents reared in isolation from early life have marked behavioral differences from socially reared animals. Despair is a common symptom of depression, and the forced swim and tail suspension tests are widely used to measure this behavior in rodents. Anhedonia, or markedly diminished interest or pleasure, is also a characteristic feature of depression, and tests that assess this behavior are important for studying the etiology of depression. Thus, we have developed a novel and simple method, which we have termed the female encounter test, for assessing motivation in adult mice. We have recently investigated the pharmacological action of (R)-ketamine, (S)-ketamine, and their metabolites in mouse models of depression. However, our and other studies on antidepressant-like effects of some ketamine metabolites show inconsistent results. In this symposium, we would like to discuss about what we are looking for in animal models of depression.

Among the fields of medical treatment, pharmacological therapy is most the therapy that nurses is most concerned in. In the basic education of nursing care, we learn pharmacological efficacy and adverse effects, methods of administrations, and management methods of pricinpal medical drugs in the clinical pharmacology. As pharmacology for nursing care, we lean comprehensive understanding of diseases, life style, and psychological situations. In addition, actual nursing education under clinical scenes, we have to learn knowledge and skill of performing safety pharmacological treatment of patients. In the clinical scenes, nurses are often the final performer of drug administration to patients.

This means that the nurses, performing drug administration according to the indication of doctors, hold responsibility as performer in common: and that they are required to have high level of expert knowledge and judgement ability, Improving those ability leads to secure quality of pharmacological treatment,

Espescially in the clinical scenes of a highly acute phase, nurses are concerned in pharmacological treatment of patients in connection with performing the administration of anti-cancer drugs having serious side effects and drugs of a high level of clinical research. The quarantee of the nursing quality in the pharmacological treatment is important.

In our hospital, we perform an interchange of personnel from clinical fields to a faculty, as link in the chain of activity of nursing career, which nursing association, is managing in co-operation with health faculty.

In this symposium, I am going to introduce the efficacy of an interchange of personnel in our hospital and clinical scenes regarding to pharmacological treatment, as issuers to be required for the education of pharmacology for nursing care form the clinical scenes, from the standpoint of the thema:"Issusses required to the clinical nursing scenes promoting human and practical pharmacological education."

In 2015, personal accreditation system for Japanese midwives was started by Japan Institute of Midwifery Evaluation. In the present practice, midwives are not able to acquire advanced competencies. It is required to enhance midwifery competencies in a planned manner. Because of Japan's declining birthrate and uneven distribution of work places for midwives, opportunities for midwives to experience midwifery practice are decreasing and proficiency cannot be enhanced easily. The Japanese Nursing Association realized the need for strategic enhancement of midwifery practice, and consequently developed the Clinical Ladder of Competencies for Midwifery Practice (CLoCMiP) to be made available wherever it was needed. Five midwifery-related associations (Japanese Nursing Association, Japanese Midwives Association, Japan Academy of Midwifery, Japan Society Midwifery Education, and Japan Institute of Midwifery Evaluation) each shared a sense of crisis that the current situation hindered the ability of midwives to improve their ability and performance, so they held multiple meetings and decided to establish a certification system based on CLoCMiP. CLoCMiP evaluates 'ethical thinking', 'maternity care' and 'professional autonomous skills' as midwifery core competencies and comprises 5 levels from Novice to Level IV. The CLoCMiP Certification System started in August 2015 by certifying midwives at Level III, who are considered capable of providing midwifery care autonomously.

To be linked to this, designation rules of midwifery education was changed by Ministry of Health, Labor and Welfare. It increases from 28 credits to 31 credits, and applied from 2022.It should be strengthened in perinatal mental health, clinical judgment skills, Comprehensive support for the child-rearing generation in the community and so on. Then, we reviewed our midwifery program and created core curriculum in preferred midwifery education by Japan Society Midwifery Education.

I will show the requirements of Advanced-midwife and let's think about Interprofessional Continuing Education.

It is well recognized among medical professions that a patient-centered multidisciplinary collaboration in pharmacotherapy is important to maximize the efficacy of drug therapy and minimize the adverse drug reactions. It is true especially for cancer chemotherapy because anticancer drugs have severe side effects and its management may affect the outcome of treatment. In this symposium, I will introduce our collaborative approach in cancer chemotherapy for outpatients currently implemented at Nagoya University Hospital, and discuss the future direction.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. In ~97 % of ALS cases, cytoplasmic aggregation of TDP-43 characterizes degenerating motor neurons. Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.

Aggregated α-synuclein (α-Syn) triggers development of synucleinopathies and it has also known that pathological α-Syn can propagate in the brain like as a prion protein. However, the molecular mechanisms remain unclear. We have recently reported that RNA G-quadruplex (G4RNA) initiates phase transition of fragile X tremor ataxia syndrome (FXTAS)-related prion-like proteins, resulting in neuronal dysfunction (Sci Adv. 2021). Here, we introduce α-Syn phase transition induced by G4RNA. α-Syn can bind to guanin-enriched RNA sequences in randomized RNA and specifically form a complex with G4RNAs, but not other RNAs. Purified α-Syn underwent liquid-liquid phase separation (LLPS), and liquid-to-solid transition of α-Syn was significantly facilitated by adding G4RNAs under the condition of molecular crowding. In addition, cellular stress markedly increased G4RNA levels and G4RNA assembly caused α-Syn aggregation in α-Syn-expressed cells. Moreover, we demonstrated that G4RNA is co-aggregated with phosphorylated α-Syn in dopaminergic neurons of the substantia nigra, and that G4 ligand attenuates α-Syn aggregation and propagation in α-Syn preformed fibril (PFF)-injected mice. These results suggest that G4RNA is a key factor for the onset of synucleinopathies, and propose a novel therapeutic approach.

One of the characteristic pathologies of Alzheimer disease (AD) is senile plaque, which is mainly composed of amyloid-β peptide (Aβ). Since the deposition of the aggregated Aβ is known as an initiator of the cascade of neurodegenerative events, the aggregation inhibition and the clearance of already deposited Aβ have been focused as therapeutic strategy for AD.

For the therapeutic strategy, we have developed the artificial photo-oxygenation system using photo-catalysts that selectively bind to cross-β sheet structure, which is the characteristic structure of the amyloid fibrils. These catalysts are activated by the light irradiation only when the compounds bind to the cross-β sheet structure. The activated catalysts generate the singlet oxygen, resulting in the oxygenation of the amyloid aggregates nearby the compounds. We showed that the photo-oxygenation of synthetic aggregated Aβ using these catalysts attenuated the further aggregation potency, leading to lower the neurotoxicity of Aβ in vitro. To verify the effects of the photo-oxygenation on deposited Aβ, we carried out the in vivo photo-oxygenation using AD model mice. We found that the photo-oxygenation decreased the amount of Aβ in the brain. The photo-oxygenated Aβ aggregates were cleared faster than that of non-modified Aβ aggregates in a microglia-dependent manner. These data suggest that the photo-oxygenation has a potential as novel useful strategy against AD. We would like to elucidate the molecular mechanism of the photo-oxygenation to further improve this strategy for AD therapy.

Alzheimer's disease is neuropathologically characterized by neuronal loss, gliosis, and the deposition of misfolded proteins such as amyloid plaques and tau tangles. Reactive astrocytes and activated microglia are observed surrounding amyloid plaques and tau tangles in postmortem AD brains. These activated glial cells secrete pro-inflammatory cytokines and reactive oxygen species, which may contribute to neurodegeneration. Therefore, in vivo imaging of glial response by positron emission tomography (PET) combined would provide new insights to monitoring glial response disease-specific therapeutics, as well as aid in the differential diagnosis, and better understand the disease process. Recently, we developed a novel PET tracer, [¹⁸F]SMBT-1 for imaging monoamine oxidase-B (MAO-B), which is predominantly expressed in the mitochondrial membranes of astrocytes and upregulated in not only AD but also various neurodegenerative conditions. Here, we introduce the development of [¹⁸F]SMBT-1 and its applications.

The number of patients with chronic kidney disease is estimated to be 13 million, and the number of dialysis patients exceeds 340,000. The annual medical cost of dialysis alone is close to 1.7 trillion yen, and the quality of life of patients is not good. The number of patients is increasing year by year, and if the situation is left unchecked, it is expected to become catastrophic. Prevention against developing severe stage is an important strategy, but drug therapy for renal disease is limited. Although potential breakthrough drugs such as SGLT2 inhibitors and bardoxolone methyl are currently being tested for pharmacological effects and efficacy, efforts to increase the number of options are needed. In this talk, I would like to introduce the current status and future of these renal drug therapies.

Vasopressin/cAMP/protein kinase A (PKA)/aquaporin-2 (AQP2) signaling in the kidneys is a canonical pathway that determines AQP2 activity in response to body fluid balance. AQP2 phosphorylation by PKA increases water reabsorption from urine for the prevention of further water loss. However, critical mediators in this phosphorylation process remain unknown.

We used several PKA activators as screening tools to generate various phosphorylation patterns of PKA substrates in renal collecting ducts. We extracted the most important PKA substrate (Protein X) whose phosphorylation levels were almost perfectly correlated with those of AQP2. We confirmed the renal physiological role of Protein X by generating Protein X^-/- mice. AQP2 was not phosphorylated even by the administration of exogenous vasopressin in Protein X^-/- mice, resulting in polyuric phenotype.

Protein X unexpectedly had a characteristic of an anchor protein that interacted with PKA (AKAP). We had previously reported that an AKAP–PKA interactions inhibitor, FMP-API-1/27, can directly activate PKA/AQP2 in renal collecting ducts (PMID: 34224008). Notably, not only FMP-API-1/27 but also vasopressin preferentially dissociated the Protein X–PKA interaction rather than other AKAPs–PKA interactions. Therefore, Protein X is a potential novel therapeutic target in the development of anti-aquaretic drugs.

Inflammation contributes to the pathogenesis of a wide variety of disorders including kidney diseases. Recent advances have shown that neural pathways are able to regulate immunity and inflammation. The cholinergic anti-inflammatory pathway is a well-studied neural circuit involving the vagus nerve that is thought to contribute to the response to inflammatory disorders. Indeed, we have so far elucidated the renal protective effect of vagus nerve stimulation and the importance of α7 nicotinic acetylcholine receptor in macrophages, and we identified novel factors existing downstream of α7 nicotinic acetylcholine receptor. Furthermore, we found that sympathetic nerves are important for the renal protective and anti-inflammatory effects of C1 neurons in the medulla oblongata, and that macrophages exert anti-inflammatory and renal protective effects through β2 adrenergic receptors. In this session, I will briefly show anti-inflammatory and organ protection mechanisms mediated by the autonomic-immune system. In particular, I will present the latest findings including the data obtained by single cell RNA-seq and I would like to discuss them with you.

All chronic inflammation of the kidney leads to end stage renal failure via renal interstitial fibrosis. In diabetic nephropathy, changes in renal glucose metabolism have been noted to affect interstitial fibrosis. However, the effects of other metabolic pathways on renal interstitial fibrosis are not clear.

To find the metabolic pathways that are altered in renal interstitial fibrosis, we performed metabolomic analysis using a mouse model of unilateral ureteral obstruction (UUO). The pathway of arginine and proline metabolism, and arginine biosynthesis were significantly enhanced in UUO mice. Therefore, we focused on arginase 2 (ARG2), a key enzyme in arginine metabolism.

In UUO mice, ARG2 and spermidine, a degradation product of L-arginine by ARG2, was increased in the renal tubules. When the UUO model was created using Arg2 knockout (KO) mice, renal interstitial fibrosis was significantly exacerbated in Arg2 KO mice. Arg2 KO kidneys were expected to have high inflammation, but the antioxidant transcription factor Nrf2 was reduced, and the expression of the target gene HO-1 was also decreased. To investigate arginine metabolism and Nrf2 activation, spermidine was added to human renal tubule HK-2 cells, and nuclear translocation of Nrf2 was strongly induced. ARG2 is increased in tubules during renal interstitial fibrosis, was predicted to exert its nephroprotective effect by activating Nrf2 through its metabolite spermidine.

SARS-CoV-2-causing COVID-19, of which outbreak was initially reported from China at the end of 2019, became a large-scale global epidemic in 2020. To cope with COVID-19, a novel type of vaccines has been developed with unprecedented speed and has been very useful in many countries, including Japan, for controlling the severity of this infectious disease. Two items of the coronavirus-modified uridine RNA vaccine, in which mRNA encoding the SARS-CoV-2 spike protein is embedded in lipid nanoparticles (LNP), are currently being marketed in Japan. Each of them has very high efficacy and almost no serious side effects, although fever and fatigue that subside in about 2-3 days are observed. In this symposium, I would like to talk about the characteristics of the mRNA/LNP vaccine, which was generated by combining the best of various sciences and technologies, based on publicly available information. Unlike conventional virus vaccines, the above mRNA/LNP vaccine does not contain any components directly derived from living organisms and is more like a chemical medicine. However, the vaccine is still subject to official verification at the time of lot release. The global standard that must be implemented at the time of lot release of a vaccine is the review of the Summary Lot Protocol (SLP),which summarizes the manufacturing and quality testing records for each lot, to determine whether it conforms to the approved content of the product. The SLP review is also conducted for the mRNA/LNP vaccines by the National Institute of Infectious Diseases.

Novel coronavirus infections have spread as a pandemic around the world since the first case was reported at the end of 2019. While several new coronavirus vaccines that are effective in controlling pandemics have been developed overseas and vaccination is progressing, domestic vaccines have yet to be commercialized. Having the ability to develop and produce vaccines and therapeutic drugs domestically is extremely important not only for contributing to the health of the people, but also from the perspective of diplomacy and security.

Shionogi has been trying to provide solutions to a broad range of viruses, including HIV, influenza viruses, norovirus, and respiratory syncytial virus (RSV). The infectious disease treatments that Shionogi has given to society so far, such as Tivicay and Xofluza, are the growth drivers that are currently supporting us. We are currently putting top priority on employing our knowhow to come up with solutions for COVID-19.

Shionogi has participated in the vaccine business since 2017 and succeeded in developing a  rhabdovirus-free BEVS under a capital and business alliance with UMN Pharma Inc. Immediately after that, a pandemic of COVID-19 occurred, we are currently making every effort to research and develop the COVID-19 vaccine.

Even if a superior vaccine becomes available, it will not completely control the infection, more effective antivirus therapeutic drugs will be needed. As a result of research using various drug discovery approaches targeting specific proteins of COVID-19, over the course of a few months we generated several interesting possibilities in the areas of medium-sized molecules, as well as some promising small-molecule compounds. Among these, in July 2021, we began clinical trials on a small-molecule compound that could become an orally administered antiviral drug.

In this session, I will report on the development of these vaccines and therapeutic agents, including some stories of hardship.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. Effective treatments for COVID-19 have not yet been established. The main pathway of infection is that the Spike protein on the surface of SARS-CoV-2 binds to its recognition receptor, angiotensin converting enzyme (ACE) 2, on the host cell. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike protein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. These results provide the potentiality of clomipramine for improving COVID-19 aggravation and sequelae.

New approaches methodologies (NAMs) have been developed to predict adverse events in humans more accurately. In vitro methods, such as human iPS cell technology, are expected to accelerate the efficiency of drug development and ensure the patients' safety and speed-up of the review process. We have evaluated the iPSC-based tools to assess safety and effectiveness of chemicals from the viewpoint of regulatory science. Based on the current COVID-19 pandemic situation and animal alternative methods, we have made various iPSC models, such as cardiomyocytes, blood-brain barrier, and intestine, to evaluate the effects of COVID-19 drugs. Here we would like to summarize the current status and future perspectives of iPSC-based system for COVID-19 in drug development.

To elucidate neural circuit dysfunction behind behavioral abnormalities in brain disorders, it is essential to understand how external and internal information is represented in healthy and diseased brains by measuring the large-scale neural circuit activity of behaving animals with high resolution. Over years, we developed a procedure for in vivo deep brain imaging, transgenic mice expressing fluorescent calcium indicator proteins in the brain, and a virtual reality system for head-fixed mice to visualize the hippocampal neural circuit activity during virtual navigation by single-cell resolution two-photon calcium imaging. In basic neuroscience, virtual reality has long been used for behavioral tasks in human and primate brain research, and its importance has increased in recent years as an experimental paradigm for rodents as well. In my introductory talk, I will briefly outline the background of this methodology and present our recent study that investigated the formation and plasticity of hippocampal representation for reward and landmark in a mouse model of autism spectrum disorders.

Functional breakdown of neural microcircuits due to deposition of pathogenic proteins leads to cognitive dysfunctions in Alzheimer's disease (AD). AD patients show spatial and temporal cognitive dysfunctions as early symptoms, in which the hippocampus plays an essential role. However, the precise process leading to the breakdown of the hippocampal neural circuits of AD remains unclear. To address this issue, we performed chronic two-photon calcium imaging under a virtual reality environment to monitor spatiotemporal representations using AD model mice (App^NL-G-F) expressing a fluorescent calcium protein G-CaMP7. The activity of ~700 hippocampal CA1 pyramidal neurons was detected over months from the same neuronal populations. In AD mice, the rate of stable place cells started declining at 4 months before the number of place cells declined, and hyperactive cells increased from 7 months. Place cells were especially impaired near deposits of Aβ, suggesting toxicity of Aβ aggregates. In contrast, cells with temporal representation showed no impairment. These results are due to compensatory hyperactivity of the place cells near the Aβ aggregates, leading to the overall reduction of active place cells. Our chronic imaging using G-CaMP7-expressed AD mice could provide a novel view to evaluate the effects of a drug for AD progression.

Functional connectivity (FC) can provide insight into cortical circuit dysfunction in neuropsychiatric disorders. Since FC is usually measured during rest (e.g. resting state fMRI), locomotion-based tasks on FC thus remains unexplored. To investigate FC in behaving states, we developed an integrated calcium imaging and virtual reality environment. We found evidence of profound and rapid reorganization of functional cortical networks in response to changing behavior states. Locomotion induced a rapid decoupling of sensory regions and greater coupling between motor regions, resulting in more pronounced modular network organization, compared to rest. Remarkably, 15q dup mice, a model of autism spectrum disorder (ASD), displayed a globally hyper-connected functional cortical network with their correlation changed poorly in association with locomotion. Furthermore, highly accurate machine learning classification identified FC patterns involving the secondary motor area as the most distinguishing features of the ASD mice from wild-type mice. Our findings highlight the importance of motor areas in cortical FC dysfunction during spontaneous behavioral switching in autism.

Cognitive and behavioural activities are underpinned by dynamic neural activities on large-scale brain network architectures. Thus, atypical cognitions and behaviours seen in neuropsychiatric conditions should be accounted for by characteristic brain network dynamics and controlled by modifying such brain state dynamics. Here, I will present empirical, numerical and causal evidence for such a notion by taking autism spectrum disorder as an example. First, I will talk about energy landscape analysis that enables us to elucidate global neural dynamics behind seemingly unstable and fluctuating cognitive and behavioural tendencies. Next, I will show that the data-driven analysis method identified overly stable brain state dynamics as a key mechanism of both the symptoms and unique intelligence of high-functioning autistic individuals. Finally, I will introduce a brain-state-driven non-invasive neural stimulation method and recent results based on the device. The observations indicate the causal roles of the brain state dynamics on human behaviours and the possibility that modifying the large-scale neural dynamics can mitigate atypical cognition and behaviours in some neuropsychiatric disorders in humans.

Sequential firing is a prominent feature of population activity in exploratory behavior. During the subsequent immobility and sleep period, the sequential firings spontaneously are replayed accompanying high-frequency field oscillations called sharp-wave (SW) ripples. The replays of sequential firing and SWs are considered a key mechanism to achieve normal memory function. However, how a downstream neuron decodes this spatiotemporally organized activity remains unexplored in subcellular resolution. To monitor the calcium activity of dendritic spines from multiple dendritic trees of a neuron in an ex vivo network, we established a new optical recording technique for functional multiple-spine calcium imaging (fMsCI). Using fMsCI, we discovered that neurons that fired spikes frequently in SWs received increasing synaptic inputs during SWs. The synaptic barrages during SWs were composed of a rich repertoire of sequential structures on nearby synapses. Our data suggest that sequential spikes of hippocampal memory engrams may excite neighboring spines of a postsynaptic neuron in a different order, which may partially explain the specific activation of SW participants during SWs.

In the long history of science, the unique research originated from each researcher's revolutionary ideas has often brought about innovative developments in scientific progress. As such research matures and becomes more influential in the surrounding research fields, it will be established as a new scientific field. Such areas come to the subject of project research and become promoted according to policy demands, social needs, or the intentions of the majority of the scientific community. Large-scale projects built on such a foundation will advance that area further by investing large amounts of budgets. However, behind that, new seeds of science that would revolutionize the next era are secretly born. Large-scale project research and small individual research by individual researchers, like the two wheels of a car, are of equal value to scientific progress, although there are significant differences in size and investment. Based on this understanding, it is important to take a bird's-eye view of the master plan and the large-scale academic research plan and consider the flow of contemporary science, the ideal form of Japanese science, and an individual's research itself. The Science Council of Japan has formulated the "Master Plan" to systematize large-scale research plans with high academic significance and serve as guidelines for Japan's large-scale research plans and academic policies. The ones that have high priority and should be promoted immediately are selected as priority large-scale research plans. This introduction provides an overview of these topics discussed at this symposium.

Visualization (or "Understanding" or "Researchinto") of physiological and pathological phenomena has taken a leapforward by recent advanced optical bio imaging technique. Traditionally,physiological phenomena including cell division, cell function, moleculartransport and cell death were detected by biochemical methods. However, thisinformation lacked high resolution spatial and temporal information. Usingrecent optical techniques, both spatial and temporal information can beintegrated across the molecular, cellular and systems levels that allow us tofurther investigate the hierarchical interaction of brain systems thatultimately reveal the pathological mechanism. In this session, we will discussthe current and potential future state of the construction of bio-imagingcenter, and will further discuss by understanding hierarchical physiologicalsystem that leads to the creation of healthy society.

In this project, various imaging technologies will be developed to visualize the complicated life systems by integrating multiple disciplines such as physics, chemistry, biology, information science, and computational science. The trans-scale-scope, one of the core facilities, is equipped with the ability to capture single-shot images of more than millions of cells at microscopic spatial resolution, allowing to investigate the emergence mechanism of each cell or individuals characteristics, that cannot be achieved by conventional technologies. This technology leads to a deeper understanding of the principle of life. Across the world, there are large-scale projects such as the Francis Crick Institute (UK) that was established with a capital of 100 billion yen and an annual budget of 17 billion yen. It is of vital importance to develop and share an unrivaled measurement equipment that can contribute to a broad range of research across the globe. This project is expected to contribute to all aspects of medical applications, such as personalized medicine, precision predictive medicine, and to solving problems directly related to human health and welfare, and the safety and security of the people. In this symposium, the latest research results related to this idea are introduced, and future problems and prospects are discussed.

Immune responses are supposed different between individuals and at different times. Personal health histories and unique environmental conditions should collectively determine the present state of immune cells. However, the cellular and molecular system mechanisms underlying such heterogeneity remain largely elusive. Here, we conducted a systematic time-lapse single-cell analysis, using 171 single-cell libraries and 30 mass cytometry 41 datasets intensively for 7 healthy individuals. We found substantial diversity in immune cell populations and their gene expression patterns between different individuals. These patterns showed daily fluctuations even within the same individual spending a usual life. Similar diversities were also observed for the T/B cell receptor repertoires. Detailed immune cell profiles at healthy statuses should give an essential background information to understand their immune responses, when the individual is exposed to various environmental conditions. To demonstrate this idea, we conducted the similar analysis for the same individuals on the vaccination of Influenza and SARS-CoV-2. Single cell immune cell profile data should make fundamental data resource to understand variable immune responses, which are unique to each individual. Discussion will also cover the importance of integrating analytical platforms, exemplifying the case of Medical Genomics Initiative of University of Tokyo, where researchers from a total of 15 departments and two hospitals join.

T-cell receptor (TCR) signaling abnormalities and gut dysbiosis are thought to be involved with systemic autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). However, it is not known whether these mechanisms are interrelated. We have explored the impact of defective TCR signaling on microbiota-driven immune responses and the consequent triggering of systemic autoimmunity. SKG mice, which harbor a point mutation in the zeta-chain-associated protein kinase 70, develop RA on the BALB/c background and SLE on C57BL/6 (B6) background. Interestingly, development of RA and SLE in these mice were significantly attenuated in microbially clean condition. Gut microbiota in these mice were altered due to the alteration of thymic selection by defective TCR signaling, which attenuated the positive selection of microbiota-reactive T cells while facilitating the positive selection of otherwise negatively deleted selected self-reactive T cells. Thus, defective TCR signaling leads to impaired immunity against gut microbiota and self-reactivity, both of which promote the development of systemic autoimmune diseases. In this seminar, gene-microbiota interactions for the development of systemic autoimmunity will be discussed.

Diabetic kidney disease is a major cause of renal failure with urgent need for a breakthrough of management. Diabetes causes significant changes in an array of plasma metabolites, and in the humans, renal SLCO4C1 in the proximal tubules is the only transporter contributes to transport them into urine. We found that phenyl sulfate (PS) was increased with the progression of diabetes and was decreased in Tg rats with limited proteinuria. In diabetes models, PS administration induced albuminuria with podocyte damage due to the mitochondrial dysfunction, suggesting the cause of renal damage in DKD.

In the gut, phenol is synthesized from tyrosine by gut bacterial-specific tyrosine phenol-lyase (TPL) and phenol is metabolized to PS in the liver. We then administered TPL inhibitor and found the reduction not only serum PS level but also albuminuria in diabetic mice. TPL inhibitor also ameliorated renal dysfunction in adenine-induced renal failure model without changing gut microbial community.

In a diabetic patient cohort (U-CARE, n=362), the serum PS level was related with the basal albuminuria level. Logistic regression analysis showed that among known ACR predictive factors, PS was the only factor which related 2-year progression of albuminuria in patients with microalbuminuria. These data suggested that PS is not only a modifiable cause but an early diagnosis marker, therefore a target for the treatment of DKD. Reduction of phenol production should represent another aspect for developing drugs preventing for DKD.

The emergence of antimicrobial-resistant (AMR) bacteria poses an imminent threat to our society and requires urgent actions. We here propose that a bacteriophage-derived protein called endolysin can be exploited as a new antimicrobial agent to tackle this global public heath issue. Endolysins are hydrolytic enzymes that cleave bacterial cell-walls (peptidoglycan layers), allowing for rapid cell lysis in a matter of minutes. Not only this swift response makes a stark contrast with conventional antibiotics, but the protein nature of endolysins makes them amenable to modifications. For example, modular domains of endolysin can be recombined to create new artificial endolysins. Using in-house high-throughput screening system, we show that we can quickly discover multiple endolysins against methicillin-resistant Staphylococcus aureus (MRSA) with improved activity compared to previously reported ones. We also discuss the prospects for the development of antimicrobial endolysins against any potential pathogens using our proprietary microbial single-cell genome sequencing platform, bit-MAP^(R).

The human microbiome is an emerging research area of interest with a series of reports on associations with various diseases and health, mainly due to advances in comprehensive analysis with the advent of next-generation sequencers. Fecal microbiota transplantation, a powerful method for modulating gut microbiota, has been reported to be surprisingly effective for certain disease populations that cannot be treated with conventional drugs. These results provide the possibility of microbiome modulation as a new approach for drug discovery. In the United States, the Human Microbiome Project was initiated in 2010 and many human microbiome data have been acquired, archived, and published. This has led to the emergence of a number of biopharmaceutical companies with clinical stage pipelines. Some of the most advanced pipelines are in the late clinical stage. However, issues such as the variability of data generated by different assays/protocols and the lack of data from healthy individuals as a reliable reference dataset should be addressed to further facilitate microbiome research and drug discovery based on human microbiome research. In Japan, in particular, established pharmaceutical companies tend to be reluctant to use these new approaches, as seen in human genome-based drug discovery. This may result in increased barriers to entry into emerging markets and a potential loss of opportunity. Since human microbiome research, especially gut microbiome research, has long been addressed by various research groups in Japan, microbe expertise and technology is high mainly in academia. It is important for Japanese industry to utilize the existing expertise of academic research groups. To accelerate the industrial use, Japan Microbiome Consortium (JMBC) was established in 2017, and its member companies include 33 pharmaceutical, chemical, food, and testing companies in Japan, including major companies and bio-venture companies. In this lecture, I will introduce the background of the establishment of JMBC and share the activities of the project for establishment of the measurement infrastructure and the project for construction of the database of healthy persons, which have been common goals. I would like to summarize the recent global trends and activities on drug discovery based on the human microbiome, as well as issues for obtaining approval and launching the drug. We would also like to discuss how we can activate drug discovery activities as one of JMBC's activities and lead to the creation of innovative new drugs.

The primary cilium is a hair-like microtubule-based structure that protrudes from the surface of nearly every cell and performs a wide range of sensory functions across species. A variety of signaling receptors localize to primary cilia and receive extracellular stimuli including Hedgehog morphogens, odorants, and light. Ciliary dysfunction is known to cause human diseases termed ciliopathies, which are characterized by a broad spectrum of symptoms, including polydactyly, cranio-facial abnormalities, brain malformation, obesity, polycystic kidney, anosmia, hearing loss, and retinal degeneration; however, treatment for ciliopathies remains a clinical challenge due to a poor understanding of the underlying mechanism. The assembly, disassembly, maintenance, and function of cilia rely on ciliary protein transport systems. Recently, we identified several post-translational modification enzymes as regulators of ciliary protein transport. In this presentation, we introduce the current understanding of the roles of these post-translational modification enzymes in the regulation of ciliary protein trafficking as well as their regulatory mechanisms, physiological significance, and involvement in human diseases.

In the interaural direction, linear acceleration is loaded during lateral translational motion, and gravity acceleration is loaded during lateral tilting movement. The otolith is a sensory organ that responds to linear acceleration, i.e., the two acceleration types. Otolith induces otolith-ocular reflex. Otolith-ocular reflex is a reflex that produces compensatory eye movements in response to head movement. The eye movements are equal in amplitude and speed to the head movement, but in the opposite direction. According to Einstein's equivalence principle, the two acceleration types cannot be discriminated. However, the frontal-eyed animals such as humans and monkeys are typically able to discriminate the two acceleration types perceptually, and act in space accordingly. My study revealed that humans use two types of otolith-ocular reflexes that control the gaze to ensure stable perception of the environment during movement. One type of otolith-ocular reflex induces horizontal eye movement during lateral translational motion and the other type induces ocular counter rolling (OCR) during lateral tilting movement.

My study also revealed that lateral-eyed animals such as mice only exhibited one type of otolith-ocular reflex, OCR, both during translational linear motion and during tilting movement.

The ancestor of modern primates, frontal-eyed shoshonius, evolved from oldest primates, lateral-eyed purgatorius. The progressive frontalization of the eyes resulted in overlap of the left and right visual fields, which led to stereopsis that enabled the perception of depth in the overlapped narrow visual field. Because clear vision during translational motion in humans and monkeys requires stabilization of gaze in space via eye movement to compensate for translational motion, the primitive otolith-ocular reflex, i.e., OCR evolved into an additional type of otolith-ocular reflex that compensates for translational motion, i.e., horizontal eye movement by combining signals from multiple sensory pathways, such as semi-circular canals and the visual system.

Sodium taste, a distinct taste modality induced by sodium ions (Na⁺), elicits attraction to sodium salts, and thereby regulates the amount of salt consumption. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na⁺ sensor located in the apical membranes of taste cells dedicated to sodium taste, which we can refer to as sodium cells. However, the identity of sodium cells, their intracellular signaling cascade, and neurotransmission mechanism remain long-standing enigmas. In this study, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na⁺ influx without changing the intracellular Ca²⁺ concentration, and form an atypical chemical synapse with afferent neurons involving the voltage-gated ion channel CALHM1/3 as the conduit for neurotransmitter release, which we have termed the "channel synapse". Genetic elimination of ENaC in Calhm1-expressing cells (ENaC cKO) as well as global Calhm3 knockout (KO) abolished the amiloride-sensitive component of the gustatory nerve responses and attenuated behavioral attraction to NaCl. Together, cells expressing ENaC and CALHM1/3 constitute sodium cells, where the entry of oral Na⁺ elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, from the sensor to neurotransmission process, sodium taste signaling bypasses Ca²⁺ signals by involving only Ca²⁺-independent ion channels: ENaC, voltage-gated Na⁺ channels, and CALHM1/3.

The anti-nociceptive effects induced by aromatic compounds derived from plant explants have attracted pharmacologists as the seeds of drug discovery. For example, salicylic acid was originated in the extract of White Willow (Salix Alba). However, most of the effects were purely brought by the direct interaction of the compounds with functional molecules in our body, thus the effects are independent of their odor. Then, are there any functional benefits of "odor" for anti-nociception?

To address the question, we performed pain-related behavior tests under odor exposure in mice. We found that the odor of linalool, one of the terpene-alcohol found in lavender extract, induced significant analgesic effects. The effects disappeared after olfactory deprivation, indicating that the olfactory input evoked by linalool odor triggers the effects. Interestingly, the effects were not observed in mice lacking Orexin peptides, which are produced by hypothalamic neurons, indicating the pivotal role of the orexinergic transmission for the effects. Furthermore, the intrathecal injection of orexin 1 receptor selective antagonist canceled out the effects, indicating the contribution of the orexinergic descending inhibitory pathway in the analgesic effects. These findings set up the neuronal circuit mechanisms underlying the odor-induced analgesia.

Prevalence rate of some psychiatric disorders, including post-traumatic stress disorder, is higher in women than men. However, the biological mechanism of the sex differences remains unclear. To clear the biological mechanism of the sex differences, we have studied sex differences in neural circuits and molecular mechanism of fear memory and extinction. We found that female mice showed a resistance to fear extinction and a low stability of extinction memory when compared to male although acquisition and retrieval of fear memory did not differ between sexes. After fear extinction training, females showed lower neural activation in the CA3 region of the dorsal hippocampus (dHIP) and posterior basolateral amygdala than males. Females also showed low activation levels of extracellular signal-regulated kinase (ERK) 2, which is related to fear extinction, and high expression levels of immune-related genes in the dHIP when compared to males. Injection of immunosuppressant dexamethasone into the dHIP enhanced fear extinction in females. Thus, the resistance to fear extinction in females may be due to high activity of immune system in the dHIP. Additionally, we found some sex-specific drug or stress effects on fear memory or extinction. In this presentation, I will discuss sex differences in biological mechanism of fear regulation.

Breaking into individuals' personal space by others sometimes evoke negative emotional responses like anxiety or discomfort, while contact with familiar ones might elicit emotion with positive valence. Knowing the mechanisms regulating emotion and behavior in response to personal space intrusion is important to understand social behavior. We identified a discrete population of neurons expressing neuropeptide B/W receptor 1 (Npbwr1) in the central nucleus of the amygdala (CeA) (Npbwr1^CeA neurons) as an essential component for circuit regulating social behavior in response to change in distance between individuals. We found that in vivo calcium activity of Npbwr1^CeA neurons was increased when social distance is shortened and vice versa. Npbwr1^CeA neurons receive direct input from ventrolateral entorhinal cortex (vlEC), ventral CA1 (vCA1) and bed nucleus of the stria terminalis (BNST) and send projections to the parabrachial nucleus (PBN), the nucleus of the solitary tract (NTS) and microcellular tegmentum (MiTg) as potential postsynaptic targets. Pathway specific manipulation of Npbwr1^CeA neurons projecting to the MiTg modulated the social distance and the time in social contact with a conspecific mouse. To understand the physiological role of Npbwr1 in the CeA, we utilized focal expression of NPBWR1 with or without single nucleotide polymorphism (A404Y SNP) in Npbwr1^CeA neurons of Npbwr1-deficient mice and found that the SNP affects social distance and behavior. These observations suggest that Npbwr1^CeA neurons comprise a discrete neural circuit that plays an important role in regulating social distance and behavior and show a possibility to develop a new drug to treat the abnormal sociability sometimes seen in social phobia or adjustment disorder.

The dopaminergic system in the central nervous system is known to play significant roles in motor coordination and affective behaviors. The extended amygdala, which includes the oval nucleus of the bed nucleus of the stria terminalis (BNST_OV) and the lateral part of the central nucleus of the amygdala (CeL), receives dense innervations of DAT⁺ fibers from the dorsal raphe (DR) and periaquductal grey (PAG) regions. We found that DAT^DR-PAG neurons consisted of two subpopulations: DAT⁺/TH⁺/VIP^- and DAT⁺/TH^-/VIP⁺. Next, we examined the properties of synaptic transmission of DAT^DR-PAG and VIP^DR-PAG innervations in BNST_OV and CeL by whole cell patch clamp recording from acute brain slices obtained from DAT-cre::Ai32 or VIP-cre::Ai32 mice that express ChR2 in DAT⁺ or VIP⁺ fibers. Both in BNST_OV and CeL, the photo stimulation evoked excitatory synaptic currents. Finally, we measured the activity of DAT^DR-PAG and VIP^DR-PAG neurons during the head-fixed classical fear conditioning by using the GCaMP photometry technique. The unconditioned stimulus (US) was air puff, and the conditioned stimulus (CS) was tone. DAT⁺ and VIP⁺ neurons were transiently activated during US presentation, while CS presentation did not alter the activities during the habituation, training session, and test session. These results suggest that DAT⁺ neurons including VIP⁺ neurons in PAG-DR region respond to aversive stimuli and mediate aversive information to their downstream targets; BNST_OV and CeL.

After the retrieval, consolidated fear memory undergoes reconsolidation to maintain the memory when it still necessary to remember, otherwise extinction to reduce fear response. In pathophysiology of fear-related disorders, such as PTSD and phobia, heightened/persistent fear memory has been well established. Therefore, the development of a method by which facilitates extinction or suppresses reconsolidation seems useful for the treatment of the aforementioned diseases. In this context, we show here that the activation of δ-opioid receptor (DOP) facilitates extinction and disrupts reconsolidation of contextual fear memory in mice. In addition, DOPs in the amygdala and infralimbic subregion of the medial prefrontal cortex (IL) are participate in facilitation of fear extinction. Along with the behavioral changes, excitatory synaptic transmission in the IL, a brain region positively stimulates the extinction, is enhanced by DOP activation. These results suggest that DOPs can be a target molecule for a desirable therapeutic agent for fear-related disorders.

In drug development, human predictability of non-clinical pharmacological evaluations should be improved for high success probability of clinical studies. In vitro in vivo extrapolation (IVIVE) is key methodology for this purpose. IVIVE has been eagerly studied to predict pharmacokinetics (PK) and pharmacodynamics (PD), thereby determining the therapeutic window of a drug. In the central nervous system (CNS), to examine whether or not candidate compounds could penetrate across the human blood-brain barrier (BBB), a variety of in vitro BBB models have been reported. On the other hand, in the CNS, specific regions are responsible for specific brain functions including higher-order functions. That means where and what candidate compounds are doing in the CNS is important to predict their efficacies and adverse effects. This information will also contribute to more accurate prediction of the therapeutic window. Currently we are focusing on in vivo imaging systems such as MRI, etc.. Now human neurons, such as human induced pluripotent stem cell-derived neurons (hiPSC-neurons), can be used in in vitro pharmacological evaluations. The human CNS predictability of non-clinical pharmacological evaluations is expected to be improved by unifying the multiphasic data described above.