The serotonergic neurons derived from rhombomere 2 are localized in the median raphe and project to the dorsal pallium in zebrafish.

The serotonergic neurons in the raphe nucleus are implicated in various cognitive functions such as learning and emotion. In vertebrates, the raphe nucleus is divided into the dorsal raphe and the median raphe. In contrast to the abundance of knowledge on the functions of the dorsal raphe, the roles of the serotonergic neurons in the median raphe are relatively unknown. The studies using zebrafish revealed that the median raphe serotonergic neurons receive input from the two distinct pathways from the habenula and the IPN. The use of zebrafish may reveal the function of the Hb-IPN-median raphe pathway. To clarify the functions of the median raphe serotonergic neurons, it is necessary to distinguish them from those in the dorsal raphe. Most median raphe serotonergic neurons originate from rhombomere 2 in mice, and we generated the transgenic zebrafish which can label the serotonergic neurons derived from rhombomere 2. In this study, we found the serotonergic neurons derived from rhombomere 2 are localized in the median raphe and project axons to the rostral dorsal pallium in zebrafish. This study suggests that this transgenic system has the potential to specifically reveal the function and information processing of the Hb-IPN-raphe-telencephalon circuit in learning.

Transgenic tools targeting the basal ganglia reveal both evolutionary conservation and specialization of neural circuits in zebrafish.

The cortico-basal ganglia circuit mediates decision making. Here, we generated transgenic tools for adult zebrafish targeting specific subpopulations of the components of this circuit and utilized them to identify evolutionary homologs of the mammalian direct- and indirect-pathway striatal neurons, which respectively project to the homologs of the internal and external segment of the globus pallidus (dorsal entopeduncular nucleus [dEN] and lateral nucleus of the ventral telencephalic area [Vl]) as in mammals. Unlike in mammals, the Vl mainly projects to the dEN directly, not by way of the subthalamic nucleus. Further single-cell RNA sequencing analysis reveals two pallidal output pathways: a major shortcut pathway directly connecting the dEN with the pallium and the evolutionarily conserved closed loop by way of the thalamus. Our resources and circuit map provide the common basis for the functional study of the basal ganglia in a small and optically tractable zebrafish brain for the comprehensive mechanistic understanding of the cortico-basal ganglia circuit.

Neural mechanism of experience-dependent sensory gain control in C. elegans.

Animals' sensory systems adjust their responsiveness to environmental stimuli that vary greatly in their intensity. Here we report the neural mechanism of experience-dependent sensory adjustment, especially gain control, in the ASH nociceptive neurons in Caenorhabditis elegans. Using calcium imaging under gradual changes in stimulus intensity, we find that the ASH neurons of naive animals respond to concentration increases in a repulsive odor 2-nonanone regardless of the magnitude of the concentration increase. However, after preexposure to the odor, the ASH neurons exhibit significantly weak responses to a small gradual increase in odor concentration while their responses to a large gradual increase remain strong. Thus, preexposure changes the slope of stimulus-response relationships (i.e., gain control). Behavioral analysis suggests that this gain control contributes to the preexposure-dependent enhancement of odor avoidance behavior. Mathematical analysis reveals that the ASH response consists of fast and slow components, and that the fast component is specifically suppressed by preexposure for the gain control. In addition, genetic analysis suggests that G protein signaling may be required for the regulation of fast component. We propose how prior experience dynamically and specifically modulates stimulus-response relationships in sensory neurons, eventually leading to adaptive modulation of behavior.

Clinical investigation of patients with jaw deformity with comorbidities.

BACKGROUND: With improvements in the safety and stability of surgeries, the number of orthognathic surgeries is increasing. Most patients who undergo orthognathic surgeries are younger, and the number of orthognathic surgeries for patients with comorbidities is also increasing. We report a survey and clinical investigation of patients with comorbidities who underwent orthognathic surgeries at our department to improve the safety of orthognathic surgery. RESULTS: The participants included 296 men and 712 women, with a mean age of 28 years (13-19 years, n=144; 20-29 years, n=483; 30-39 years, n=236; 40-49 years, n=102; 50-59 years, n=39; ≥60 years, n=4). In total, 347 patients underwent one-stage Le Fort type I osteotomy and sagittal split ramus osteotomy (SSRO), 243 underwent SSRO, 287 underwent plate removal, 126 underwent genioplasty and plate removal, and five underwent other surgeries. In total, 529 patients had comorbidities (52%), including allergic diseases (n=220, 33%), respiratory diseases (n=107, 16%), neurologic and psychiatric diseases (n=69, 10%), gynecologic diseases (n=28, 4%), hematologic diseases (n=27, 4%), cardiovascular diseases (n=24, 4%), digestive diseases (n=22, 3%), metabolic and endocrine diseases (n=18, 3%), spinal diseases (n=11, 2%), ophthalmologic diseases (n=11, 2%), renal and urological diseases (n=9, 1%), and other diseases (n=117, 18%). Among the patients with comorbidities, 11 with hemorrhagic diatheses (hemophilia and von Willebrand disease), arrhythmia (atrioventricular block), psychiatric disease (adjustment disorder), and metabolic disease (diabetes) required cautious perioperative management. The patient with hemophilia was managed with regular low-dose recombinant factor VIII replacement therapy, and the patient with type I diabetes mellitus was administered continuous insulin infusion and sliding-scale insulin therapy; both patients had an uneventful course. CONCLUSIONS: The study findings suggest that with the increase in orthognathic surgeries, oral and maxillofacial surgeons should adequately manage cases requiring cautious perioperative control and highlight the importance of preoperative screening. Despite the well-established safety and postoperative stability of orthognathic surgeries, oral surgeons should adopt appropriate additional preventive measures for patients with comorbidities.

Calcium Imaging of Neuronal Activity under Gradually Changing Odor Stimulation in Caenorhabditis elegans.

Olfactory behavior is among the most fundamental animal behaviors both in the wild and in the laboratory. To elucidate the neural mechanisms underlying olfactory behavior, it is critical to measure neural responses to odorant concentration changes resembling those that animals actually sense during olfactory behavior. However, reproducing the dynamically changing olfactory stimuli to an animal during such measurements of neural activity is technically challenging. Here, we describe technical details and protocols for odor stimulation during calcium imaging of the sensory neurons of the nematode Caenorhabditis elegans. In this system, the neuronal activity of C. elegans is measured using ratiometric calcium imaging during exposure to quantitatively controlled olfactory stimuli over time. Temporal changes in odor concentrations around the animal are precisely controlled according to a predesigned temporal odor gradient to reproduce a realistic odor concentration change during olfactory behavior in a behavioral arena. By monitoring neural activity in response to the realistic olfactory stimulus, it is possible to elucidate the mechanisms by which olfactory input is processed by neural activities and reflected in behavioral output.

STEFTR: A Hybrid Versatile Method for State Estimation and Feature Extraction From the Trajectory of Animal Behavior.

Animal behavior is the final and integrated output of brain activity. Thus, recording and analyzing behavior is critical to understand the underlying brain function. While recording animal behavior has become easier than ever with the development of compact and inexpensive devices, detailed behavioral data analysis requires sufficient prior knowledge and/or high content data such as video images of animal postures, which makes it difficult for most of the animal behavioral data to be efficiently analyzed. Here, we report a versatile method using a hybrid supervised/unsupervised machine learning approach for behavioral state estimation and feature extraction (STEFTR) only from low-content animal trajectory data. To demonstrate the effectiveness of the proposed method, we analyzed trajectory data of worms, fruit flies, rats, and bats in the laboratories, and penguins and flying seabirds in the wild, which were recorded with various methods and span a wide range of spatiotemporal scales-from mm to 1,000 km in space and from sub-seconds to days in time. We successfully estimated several states during behavior and comprehensively extracted characteristic features from a behavioral state and/or a specific experimental condition. Physiological and genetic experiments in worms revealed that the extracted behavioral features reflected specific neural or gene activities. Thus, our method provides a versatile and unbiased way to extract behavioral features from simple trajectory data to understand brain function.

Neuronal, mathematical, and molecular bases of perceptual decision-making in C. elegans.

Animals process sensory information from the environment to make behavioral decisions. Although environmental information may be ambiguous or gradually changing, animals can still choose one behavioral option among several through perceptual decision-making. Perceptual decision-making has been intensively studied in primates and rodents, and neural activity that accumulates sensory information has been shown to be crucial. However, it remains unclear how the accumulating neural activity is generated, and whether such activity is a conserved decision-making strategy across the animal kingdom. Here, we review the previous perceptual decision-making studies in vertebrates and invertebrates and our recent achievement in an invertebrate model animal, the nematode Caenorhabditis elegans. In the study, we analyzed temporal dynamics of neuronal activity during perceptual decision-making in navigational behavior of C. elegans. We identified neural activity that accumulates sensory information and elucidated the molecular mechanism for the accumulating activity, which may be relevant to decision-making across the animal kingdom.

Calcium dynamics regulating the timing of decision-making in C. elegans.

Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca2+]i), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+]i rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.

In actio optophysiological analyses reveal functional diversification of dopaminergic neurons in the nematode C. elegans.

Many neuronal groups such as dopamine-releasing (dopaminergic) neurons are functionally divergent, although the details of such divergence are not well understood. Dopamine in the nematode Caenorhabditis elegans modulates various neural functions and is released from four left-right pairs of neurons. The terminal identities of these dopaminergic neurons are regulated by the same genetic program, and previous studies have suggested that they are functionally redundant. In this study, however, we show functional divergence within the dopaminergic neurons of C. elegans. Because dopaminergic neurons of the animals were supposedly activated by mechanical stimulus upon entry into a lawn of their food bacteria, we developed a novel integrated microscope system that can auto-track a freely-moving (in actio) C. elegans to individually monitor and stimulate the neuronal activities of multiple neurons. We found that only head-dorsal pair of dopaminergic neurons (CEPD), but not head-ventral or posterior pairs, were preferentially activated upon food entry. In addition, the optogenetic activation of CEPD neurons alone exhibited effects similar to those observed upon food entry. Thus, our results demonstrated functional divergence in the genetically similar dopaminergic neurons, which may provide a new entry point toward understanding functional diversity of neurons beyond genetic terminal identification.

Neonatal Estrogen Receptor ß Is Important in the Permanent Inhibition of Epithelial Cell Proliferation in the Mouse Uterus.

Estrogen receptor α (ERα) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ERß inhibits cell proliferation. ERß mRNA is expressed in neonatal uteri and vaginae; however, its functions in neonatal tissues have not been ascertained. In this study, we investigated the ontogenic mRNA expression and localization of ERß, and its roles in cell proliferation in neonatal uteri and vaginae of ERß knockout (ßERKO) mice. ERß mRNA and protein were abundant in the uterine and vaginal epithelia of 2-day-old mice and decreased with age. In uterine and vaginal epithelia of 2-day-old ßERKO mice, cell proliferation was greater than that in wild-type animals and in uterine epithelia of 90- and 365-day-old ßERKO mice. In addition, p27 protein, known as a cyclin-dependent kinase inhibitor, was decreased in the uteri of 90- and 365-day-old ßERKO mice. Inhibition of neonatal ERs by ICI 182780 (an ER antagonist) treatment stimulated cell proliferation and decreased p27 protein in the uterine luminal epithelium of 90-day-old mice but not in the vaginal epithelium. These results suggest that neonatal ERß is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ERß function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.