視床下部GnRH ニューロンを中心とした真骨魚類生殖中枢制御機構に関する神経内分泌学的研究

学位の種別:課程博士University of Tokyo(東京大学

Morphological analysis of the axonal projections of EGFP-labeled Esr1-expressing neurons in transgenic female medaka

Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide Kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in non-mammalian species casts doubt on the regulation of GnRH1 neurons by Kisspeptin neurons. As a first step towards understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic medaka that expresses EGFP specifically in Esr1-expressing neurons (Esr1 neurons) and analyzed their axonal projections. We found that Esr1 neurons in the POA project to the GnRH1 neurons. We also demonstrated, by transcriptome and histological analyses, that these Esr1 neurons are glutamatergic and/or GABAergic, but not Kisspeptinergic. We therefore suggest that glutamatergic/GABAergic Esr1 neurons in the POA regulate GnRH1 neurons. This hypothesis is consistent with previous studies in mice that glutamatergic/GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic Esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-Esr1 neurons co-express progesterone receptors (PRs). These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone

Morphological analysis of the axonal projections of EGFP-labeled Esr1-expressing neurons in transgenic female medaka

Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide Kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in non-mammalian species casts doubt on the regulation of GnRH1 neurons by Kisspeptin neurons. As a first step towards understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic medaka that expresses EGFP specifically in Esr1-expressing neurons (Esr1 neurons) and analyzed their axonal projections. We found that Esr1 neurons in the POA project to the GnRH1 neurons. We also demonstrated, by transcriptome and histological analyses, that these Esr1 neurons are glutamatergic and/or GABAergic, but not Kisspeptinergic. We therefore suggest that glutamatergic/GABAergic Esr1 neurons in the POA regulate GnRH1 neurons. This hypothesis is consistent with previous studies in mice that glutamatergic/GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic Esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-Esr1 neurons co-express progesterone receptors (PRs). These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone

Distinct hypothalamic control of same- and opposite-sex mounting behaviour in mice

Animal behaviours that are superficially similar can express different intents in different contexts, but how this flexibility is achieved at the level of neural circuits is not understood. For example, males of many species can exhibit mounting behaviour towards same- or opposite-sex conspecifics, but it is unclear whether the intent and neural encoding of these behaviours are similar or different. Here we show that female- and male-directed mounting in male laboratory mice are distinguishable by the presence or absence of ultrasonic vocalizations (USVs), respectively. These and additional behavioural data suggest that most male-directed mounting is aggressive, although in rare cases it can be sexual. We investigated whether USV⁺ and USV⁻ mounting use the same or distinct hypothalamic neural substrates. Micro-endoscopic imaging of neurons positive for oestrogen receptor 1 (ESR1) in either the medial preoptic area (MPOA) or the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) revealed distinct patterns of neuronal activity during USV⁺ and USV⁻ mounting, and the type of mounting could be decoded from population activity in either region. Intersectional optogenetic stimulation of MPOA neurons that express ESR1 and vesicular GABA transporter (VGAT) (MPOA^(ESR1∩VGAT) neurons) robustly promoted USV⁺ mounting, and converted male-directed attack to mounting with USVs. By contrast, stimulation of VMHvl neurons that express ESR1 (VMHvl^(ESR1) neurons) promoted USV⁻ mounting, and inhibited the USVs evoked by female urine. Terminal stimulation experiments suggest that these complementary inhibitory effects are mediated by reciprocal projections between the MPOA and VMHvl. Together, these data identify a hypothalamic subpopulation that is genetically enriched for neurons that causally induce a male reproductive behavioural state, and indicate that reproductive and aggressive states are represented by distinct population codes distributed between MPOA^(ESR1) and VMHvl^(ESR1) neurons, respectively. Thus, similar behaviours that express different internal states are encoded by distinct hypothalamic neuronal populations

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic–pituitary–gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1–expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP–expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons—that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates

The Mouse Action Recognition System (MARS): a software pipeline for automated analysis of social behaviors in mice

The study of social behavior requires scoring the animals' interactions. This is generally done by hand-- a time consuming, subjective, and expensive process. Recent advances in computer vision enable tracking the pose (posture) of freely-behaving laboratory animals automatically. However, classifying complex social behaviors such as mounting and attack remains technically challenging. Furthermore, the extent to which expert annotators, possibly from different labs, agree on the definitions of these behaviors varies. There is a shortage in the neuroscience community of benchmark datasets that can be used to evaluate the performance and reliability of both pose estimation tools and manual and automated behavior scoring. We introduce the Mouse Action Recognition System (MARS), an automated pipeline for pose estimation and behavior quantification in pairs of freely behaving mice. We compare MARS's annotations to human annotations and find that MARS's pose estimation and behavior classification achieve human-level performance. As a by-product we characterize the inter-expert variability in behavior scoring. The two novel datasets used to train MARS were collected from ongoing experiments in social behavior, and identify the main sources of disagreement between annotators. They comprise 30,000 frames of manual annotated mouse poses and over 14 hours of manually annotated behavioral recordings in a variety of experimental preparations. We are releasing this dataset alongside MARS to serve as community benchmarks for pose and behavior systems. Finally, we introduce the Behavior Ensemble and Neural Trajectory Observatory (Bento), a graphical interface that allows users to quickly browse, annotate, and analyze datasets including behavior videos, pose estimates, behavior annotations, audio, and neural recording data. We demonstrate the utility of MARS and Bento in two use cases: a high-throughput behavioral phenotyping study, and exploration of a novel imaging dataset. Together, MARS and Bento provide an end-to-end pipeline for behavior data extraction and analysis, in a package that is user-friendly and easily modifiable

The Mouse Action Recognition System (MARS): a software pipeline for automated analysis of social behaviors in mice

The study of social behavior requires scoring the animals' interactions. This is generally done by hand-- a time consuming, subjective, and expensive process. Recent advances in computer vision enable tracking the pose (posture) of freely-behaving laboratory animals automatically. However, classifying complex social behaviors such as mounting and attack remains technically challenging. Furthermore, the extent to which expert annotators, possibly from different labs, agree on the definitions of these behaviors varies. There is a shortage in the neuroscience community of benchmark datasets that can be used to evaluate the performance and reliability of both pose estimation tools and manual and automated behavior scoring. We introduce the Mouse Action Recognition System (MARS), an automated pipeline for pose estimation and behavior quantification in pairs of freely behaving mice. We compare MARS's annotations to human annotations and find that MARS's pose estimation and behavior classification achieve human-level performance. As a by-product we characterize the inter-expert variability in behavior scoring. The two novel datasets used to train MARS were collected from ongoing experiments in social behavior, and identify the main sources of disagreement between annotators. They comprise 30,000 frames of manual annotated mouse poses and over 14 hours of manually annotated behavioral recordings in a variety of experimental preparations. We are releasing this dataset alongside MARS to serve as community benchmarks for pose and behavior systems. Finally, we introduce the Behavior Ensemble and Neural Trajectory Observatory (Bento), a graphical interface that allows users to quickly browse, annotate, and analyze datasets including behavior videos, pose estimates, behavior annotations, audio, and neural recording data. We demonstrate the utility of MARS and Bento in two use cases: a high-throughput behavioral phenotyping study, and exploration of a novel imaging dataset. Together, MARS and Bento provide an end-to-end pipeline for behavior data extraction and analysis, in a package that is user-friendly and easily modifiable

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic–pituitary–gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1–expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP–expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons—that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates