東 森生

Vasopressin (VP), which is one of the neurohypophyseal hormone, can play diverse roles via specific receptors belonging to a family of GPCRs. There are three subtypes of VP receptors–V1aR, V1bR, and V2R. Previous our studies reported that V1aR-KO female mice showed various reproductive abnormalities. In this analysis, we focused on the mechanism of the decrease of litter size and tried to identify the period when the fetuses drop out. First, we performed the staining of the implantation site (IS) at the day 5 of pregnancy (E5). However, the number of IS were not different. This observation suggests that the decrease of litter size observed in the V1aR-KO mice occurred after implantation. We next measured the litter size and the weights of fetuses and placentas at E14, E18, and E19. As a result, the weights of fetuses and placentas were smaller in the V1aR-KO mice compared with the wild-type. Intriguingly, aberrant fetuses are frequently observed at E14, E18, and E19 in the V1aR-KO mice and the litter sizes of the normal fetuses were smaller. Especially, omphalocele was observed in 13.5 % of E19 V1aR-KO fetuses. In general, fetuses with omphalocele die after birth. Therefore, the significant decrease in the number of litters observed in V1aR-KO mice may be due to the abnormal growth of the fetus in utero, such as omphalocele.

【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.

【Background】The basic principle of G-protein-coupled receptor signal involves G-protein-dependent and -independent, beta-arrestin-dependent pathways. Recent progress in structural study of chimeric beta-adrenaline receptor and beta-arrestin 2 complex proposed that the interaction went through two sequential steps. However, such steps in living cells and the practical application have not been provided, because these steps are difficult to analyze separately. Here, we report V1b vasopressin receptor as a valuable model to tackle this question.【Methods】Bioluminescent resonance energy transfer (BRET) between receptor-nanoluciferase and Venus-beta-arrestin 2 was employed to monitor interaction. Receptor dimers were further monitored by connecting two parts of split nanoluciferase to the receptor carboxyl terminus.【Results and Discussion】We detected two-step increases of BRET signal in the V1b receptor-beta-arrestin 2 interaction. Non-stimulated V1b receptor associated with beta-arrestin 2 and vasopressin further increased BRET. Heteromer formation between beta-arrestin 2-V1b receptor and mu-opioid receptor promoted morphine-induced analgesic tolerance in mice and in reconstituted cellular model. Our data indicates two-step interactions between V1b receptor and beta-arrestin 2 are the molecular target for alleviating morphine tolerance.

Melanin concentrating hormone (MCH) is a peptide conserved from fish to mammal, and well known as an orexigenic neuropeptide in mammals. Recently, we found MCH-immunopositive fibers in the median eminence of a basal actinopterygian fish, Polypterus senegalus, that is located in the phylogenetic branch of fishes and tetrapod. However, the functions of MCH on the endocrine cells in the adenohypophysis are unknown. In this study, we examined the expression of MCH receptors in the gland of this fish, and their intracellular signal transductions. Phylogenetic analysis showed that P. senegalus has two types of MCH receptor 1 (MCHR1) and two types of MCH receptor 2 (MCHR2), that are close to the MCH receptors of mammal or teleost. In situ hybridization showed that mammal-like MCHR2 (m-MCHR2) and teleost-like MCHR2 (t-MCHR2) are expressed in the endocrine cells of the pars distalis of the gland, that is homologous to the anterior pituitary gland of mammal. HEK293 cells, which were transfected with these receptors separately, showed that both MCHR2s increase intracellular Ca²⁺, and that t-MCHR2, but not m-MCHR2, inhibits the activity of the adenylate cyclase in a concentration-dependent manner. These findings suggest that two types of the MCH receptor 2 regulate release and/or production of adenohypophyseal hormone in this fish.

[Background] Drinking behavior after diuretics administration involves complex neuronal processes, in which participating hormonal signal is not well delineated. [purpose] To gain insight into frequency and timing of drinking behavior after diuretics, we employed the advantage and power of deep learning. [Method] Male mice with or without intraperitoneal injection of tolvaptan (20 mg/kg) were placed in metabolic cages and their behavior was monitored by video recording for six hours. Individual video frames were obtained using ffmpeg program. Mice were detected as an object and labelled as “drinking” or “away from water”. Labeled images were used to develop learning models of deep neural network (DNN) in Tensorflow and pyhton program. The trained model was evaluated its accuracy and further used to detect drinking behaviors of new mouse group in conda virtual environment. [Result] Our DNN model successfully detected a series of mouse behavior. We found a marked increase in the drinking behavior after diuretics treatment. [Discussion] Water-taking behaviors, which were composed of tens of thousands of images per mouse, can be automatically analyzed using trained DNN with high accuracy. Our results demonstrate the usefulness of deep learning in a pharmacological study of animal behavior.

【目的】バソプレッシン（VP）はオキシトシン（OT）に類縁の下垂体後葉ホルモンで，3種類のVP受容体サブタイプ（V1a，V1b，V2）を介して，その機能を発揮する。我々のこれまでの解析からV1a受容体欠損（KO）マウスでは有意な分娩遅延と産仔数の減少，そして分娩時の子宮収縮の減弱が示され，生殖生理にバソプレッシン受容体が重要な役割を持つことが示唆されている。本研究ではV1a KOマウスで観察された産仔数の減少がいつどのようにして生じるのかについて，さらなる解析を行った。【方法】産仔数減少の時期を特定するため，妊娠5日目に着床部位の染色，妊娠14日目，18日目，19日目に胎仔数と胎仔重量，胎盤重量の計測を行った。また，胎仔の組織異常を明らかにするために，妊娠19日目の胎仔の組織切片を作成し，観察を行った。【結果および考察】妊娠5日目の子宮の着床部位の染色からV1a KOと野生型マウスでは着床数に有意な差は見られなかった。妊娠14日目，18日目，19日目の胎仔数の比較からはV1a KOマウスで正常胎仔の減少が観察された。胎仔重量と胎盤重量は，妊娠14日目ではV1a KOマウスの胎仔のみで有意に小さかったが，18日目と19日目ではV1a KOマウスの胎仔と胎盤の両方で有意に小さかった。しかしながら，その差は18日目をピークとして縮まる傾向にあり，出産後には体重に有意差は見られない。さらに興味深いことに，V1a KOマウスでは子宮内で観察された異常胎仔の数が多いことがわかった。特に19日目の胎仔ではV1a KOマウスの総胎仔数の13.5％で臍帯ヘルニアが観察された。これまでの報告から臍帯ヘルニアを呈する遺伝子欠損マウスの多くが口蓋裂を伴うことが報告されているが，V1a KOマウスでは口蓋裂は観察されなかった。一般的に臍帯ヘルニアを患う胎仔は出産時に死んでしまう。このことから，V1a KOマウスで観察された有意な産仔数減少は臍帯ヘルニアなどの子宮内での胎仔の成長異常に起因すると考えられる。

Deposition of age-related fluorescent pigment in tissues was initially discovered by A. Hanover in 1842 and the pigments were subsequently called lipofuscin by W. Hueck in 1912 and M. Borst in 1922. It has been known for decades that the lipofuscin is found in several aged organs such as the eyes, brain and adrenal cortex of humans and rodents. However, how these normal aging processes proceed is not known. In a preliminary study, we found that in the adrenal gland of V1a vasopressin receptor-deficient mice (V1aKO), more lipofuscin deposits were accumulated with age compared with wild-type (WT) mice. To quantitatively examine lipofuscin deposits, we have collected 60 adrenal glands from V1aKO and WT mice of both sexes at 2, 6 and 12 months of age. The sections were stained to visualize nucleus and plasma membrane and examined by using confocal microscope. We found lipofuscin fluorescent signals in the adrenal gland of both V1aKO and WT mice. However, the amount of lipofuscin in V1a-deficient adrenal gland was significantly increased compared with the adrenal gland of WT mice in both sexes at all ages. In particular, at 6 month of age, lipofuscin deposition in WT and V1aKO males occupied 3.9 and 5.6 % of entire adrenal cortex areas. Also, we found that lipofuscin accumulation started in the cytoplasm. The mechanism of an increase in this deposit formation, especially its relationship to the V1a receptor, needs to be further examined.

Previous our work suggested that V1b receptor for neuropituitary hormone vasopressin enhances cellular signal leading to morphine tolerance by functionally interacting with mu-opioid receptor (MOR) and beta-arrestin 2. However, it has not been clarified how these three signaling molecules are arranged. Here, we explored a possibility of formation of three-molecule complex using bioluminescence resonance energy transfer (BRET) among split nanoluciferase and Venus fluorescent protein. To this end, two pieces of a split nanoliciferase were connected to carboxyl termini of V1b or MOR, and Venus to beta-arrestin2. We successfully detected BRET between V1b-MOR heteromer receptor and beta-arrestin 2-Venus. Importantly, this interaction was enough high at basal, indicating constitutive interaction without agonist. Furthermore, unlike the receptor tagged with a whole nanoluciferase, BRET signal from interaction among V1b-MOR heteromer with split nanolucifarese and beta-arrestin 2-Venus increased further after agonist treatment. These results indicated that we successfully developed screening method to search for a chemical that could suppress morphine tolerance by acting at V1b receptor in the V1b-MOR heteromer.

Parental behavior is widely preserved in living animals. A caregiving of mother is needed for behavioral and emotional development of child. Various responsive hormones have been identified to play the important roles on maternal behavior. One of neurohypophyseal hormone, arginine vasopressin, has been known to regulate maternal behavior mainly through activation of vasopressin V1a subtype receptors in the central nervous system. Moreover, administration of antagonist of another vasopressin receptor subtype, V1b receptor, into lateral ventricle changed maternal care. However, the knowledge of V1b receptors on maternal behavior is limited. Recently, machine learning algorithms have been applied in several scientific studies, in which the computer vision and deep learning are a brunch of this intelligence algorithm. Taking the advantage of computer science into account, we had employed this method to investigate a possible role of vasopressin in maternal behavior in mother and pup interaction during lactation period. In each image from video recording, one or more objects can be analyzed and classified by deep learning models. We found that intimal relationship between dam and pups can be analyzed in detail. Furthermore, our method can be applied to a large scale of dataset. In summary, we have successfully developed deep learning models to study the relationships between dam and pups of wildtype and gene knockout mice in free moving conditions. Furthermore, our developed method effectively analyzes the complex behavior of laboratory animals with increasing efficiency and consistency.

症例は患者が飼育中のアマゾン産淡水エイPotamotrygon leopoldiの水替え中に手背を刺されて来院したもので,刺傷後から疼痛が激しく,創部の洗浄と消毒に加え,海産エイ毒の解毒方法に準じて40℃の湯に創部を浸漬したところ,疼痛の軽減を図ることができた。その後,疼痛が数日の間は継続したが,広範な壊死を来すことはなく細菌感染などの併発もなく治癒した。エイ毒は複数の蛋白毒で構成され,疼痛・腫脹・壊死を来すほか,ショック症状・呼吸困難を来し,ときに死に至ることもある。一部の海産タンパク毒と同様に高温に弱く,患部を温水に浸漬することで症状を軽減できるといわれるが,種差・性差・成熟度による毒性・毒量を含めて不明な点が多い。本邦では淡水エイによる刺傷の報告が少なく,今回の症例のように後遺症を認めずに治癒する症例がある一方で,後遺症や手術を要した症例も報告されている。淡水エイの毒性については不確定なことが多く,症例および治療経験の蓄積が重要であると考えられた。(著者抄録)