Long-lasting redundant gnrh1/3 expression in GnRH neurons enabled apparent switching of paralog usage during evolution.

Expressed subtype of paralogous genes in functionally homologous cells sometimes show differences across species, the reasons for which have not been explained. The present study examined hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons in vertebrates to investigate this mechanism. These neurons express either gnrh1 or gnrh3 paralogs, depending on the species, and apparent switching of the expressed paralogs in them occurred at least four times in vertebrate evolution. First, we found redundant expression of gnrh1 and gnrh3 in a single neuron in piranha and hypothesized that it may represent an ancestral GnRH system. Moreover, the gnrh1/gnrh3 enhancer of piranha induced reporter RFP/GFP co-expression in a single hypophysiotropic GnRH neuron in both zebrafish and medaka, whose GnRH neurons only express either gnrh3 or gnrh1. Thus, we propose that redundant expression of gnrh1/3 of relatively recent common ancestors may be the key to apparent switching of the paralog usage among present-day species.

Involvement of the kisspeptin system in regulation of sexual behaviors in medaka.

In mammals, kisspeptin (Kiss1) neurons are generally considered as a sex steroid-dependent key regulator of hypothalamic-pituitary-gonadal (HPG) axis. In contrast, previous studies in non-mammalian species, especially in teleosts, propose that Kiss1 is not directly involved in the HPG axis regulation, which suggests some sex-steroid-dependent functions of kisspeptin(s) other than the HPG axis regulation in non-mammals. Here, we used knockout (KO) medaka of kisspeptin receptor-coding genes (gpr54-1 and gpr54-2) and examined possible roles of kisspeptin in the regulation of sexual behaviors. We found that the KO pairs of gpr54-1, but not gpr54-2, spawned fewer eggs and exhibited delayed spawning than wild type pairs. Detailed behavior analysis suggested that the KO females are responsible for the delayed spawning and that the KO males showed hyper-motivation for courtship. Taken together, the present finding suggests that one of the reproductive-state-dependent functions of the Kiss1 may be the control of successful sexual behaviors.

Zoology of Fishes.

The Zoological Society of Japan is one of the longest-standing scientific societies in Japan, and it has been publishing a unique prestigious international journal in zoology, Zoological Science, for a long period of time since its foundation in 1984 as the continuation of Zoological Magazine (1888-1983) and Annotationes Zoologicae Japonenses (1897-1983). One of the most salient features of the Society and the Journal may be the variety of species of animals used in the studies by the members of the society and the authors of the journal. Among various animal species, fish may have contributed to almost all disciplines of presentations and publications, including behavioral biology, biochemistry, cell biology, developmental biology, diversity and evolution, ecology, endocrinology, genetics, immunology, morphology, neurobiology, phylogeny, reproductive biology, and taxonomy. Owing to the recent advancement of modern molecular genetic methods in biology, not a few fish species have contributed to various research disciplines in zoological science as model animals. The present Special Issue includes various kinds of such studies in zoological science by taking advantage of a variety of fish species, which are contributed by authors of various generations ranging from junior to senior zoologists.

Neural Control of Sexual Behavior in Fish.

Many vertebrate species show breeding periods and exhibit series of characteristic species-specific sexual behaviors only during the breeding period. Here, secretion of gonadal sex hormones from the mature gonads has been considered to facilitate sexual behaviors. Thus, the sexual behavior has long been considered to be regulated by neural and hormonal mechanisms. In this review, we discuss recent progress in the study of neural control mechanisms of sexual behavior with a focus on studies using fish, which have often been the favorite animals used by many researchers who study instinctive animal behaviors. We first discuss control mechanisms of sexual behaviors by sex steroids in relation to the anatomical studies of sex steroid-concentrating neurons in various vertebrate brains, which are abundantly distributed in evolutionarily conserved areas such as preoptic area (POA) and anterior hypothalamus. We then focus on another brain area called the ventral telencephalic area, which has also been suggested to contain sex steroid-concentrating neurons and has been implicated in the control of sexual behaviors, especially in teleosts. We also discuss control of sex-specific behaviors and sexual preference influenced by estrogenic signals or by olfactory/pheromonal signals. Finally, we briefly summarize research on the modulatory control of motivation for sexual behaviors by a group of peptidergic neurons called terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which are known to be especially developed in fishes among various vertebrate species.

Acetylcholine Inhibits Spontaneous Firing Activity of Terminal Nerve GnRH Neurons in Medaka.

Vertebrates generally possess hypophysiotropic and non-hypophysiotropic gonadotropin releasing hormone (GnRH) neurons. The terminal nerve (TN) GnRH neurons are known to belong to the non-hypophysiotropic neurons and have been suggested to modulate sexual behaviors. These neurons show spontaneous pacemaker firing activity and release neuropeptides GnRH and neuropeptide FF. Since the spontaneous firing activities of peptidergic neurons, including GnRH neurons, are believed to play important roles in the release of neuropeptides, understanding the regulatory mechanisms of these spontaneous firing activities is important. Here, we analyzed firing activities of the TN-GnRH neurons in medaka during application of acetylcholine (ACh), which is one of the essential neuromodulators in the brain. Whole cell patch clamp recording of TN-GnRH neurons demonstrated that ACh induces hyperpolarization and inhibits their pacemaker firing. Electrophysiological analysis using an antagonist for acetylcholine receptors and in situ hybridization analysis showed that firing of TN-GnRH neurons is inhibited via M2-type muscarinic acetylcholine receptor. These findings, taken together with literature from several other fish species (including teleosts and elasmobranchs), indicate that ACh may generally play an inhibitory role in modulating spontaneous activities of TN-GnRH neurons and thereby sexual behaviors in fish.

Neuroendocrine regulation of reproduction by GnRH neurons: multidisciplinary studies using a small fish brain model.

After the discovery of GnRH, GnRH neurons have been considered to represent the final common pathway for the neural control of reproduction. There is now compelling data in mammals that two populations of kisspeptin neurons constitute two different systems to control the episodic and surge release of GnRH/LH for the control of different aspects of reproduction, follicular development and ovulation. However, accumulating evidence indicates that kisspeptin neurons in non-mammalian species do not serve as a regulator of reproduction, and the non-mammalian species are believed to show only surge release of GnRH to trigger ovulation. Therefore, the GnRH neurons in non-mammalian species may offer simpler models for the study of their functions in neuroendocrine regulation of reproduction, especially ovulation. Our research group has taken advantage of many unique technical advantages of small fish brain for the study of anatomy and physiology of GnRH neurons, which underlie regular ovulatory cycles during the breeding season. Here, recent advances in multidisciplinary study of GnRH neurons are reviewed, with a focus on studies using small teleost fish models.

Multi-Institutional Study of End-to-End Dose Delivery Quality Assurance Testing for Image-Guided Brachytherapy Using a Gel Dosimeter.

PURPOSE: To quantify dose delivery errors for high-dose-rate image-guided brachytherapy (HDR-IGBT) using an independent end-to-end dose delivery quality assurance test at multiple institutions. The novelty of our study is that this is the first multi-institutional end-to-end dose delivery study in the world. MATERIALS AND METHODS: The postal audit used a polymer gel dosimeter in a cylindrical acrylic container for the afterloading system. Image acquisition using computed tomography, treatment planning, and irradiation were performed at each institution. Dose distribution comparison between the plan and gel measurement was performed. The percentage of pixels satisfying the absolute-dose gamma criterion was reviewed. RESULTS: Thirty-five institutions participated in this study. The dose uncertainty was 3.6% ± 2.3% (mean ± 1.96σ). The geometric uncertainty with a coverage factor of k = 2 was 3.5 mm. The tolerance level was set to the gamma passing rate of 95% with the agreement criterion of 5% (global)/3 mm, which was determined from the uncertainty estimation. The percentage of pixels satisfying the gamma criterion was 90.4% ± 32.2% (mean ± 1.96σ). Sixty-six percent (23/35) of the institutions passed the verification. Of the institutions that failed the verification, 75% (9/12) had incorrect inputs of the offset between the catheter tip and indexer length in treatment planning and 17% (2/12) had incorrect catheter reconstruction in treatment planning. CONCLUSIONS: The methodology should be useful for comprehensively checking the accuracy of HDR-IGBT dose delivery and credentialing clinical studies. The results of our study highlight the high risk of large source positional errors while delivering dose for HDR-IGBT in clinical practices.

Estrogen upregulates the firing activity of hypothalamic gonadotropin-releasing hormone (GnRH1) neurons in the evening in female medaka.

The reproductive function of vertebrates is regulated by the hypothalamic-pituitary-gonadal axis. In sexually mature females, gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) are assumed to be responsible for a cyclic large increase in GnRH release, the GnRH surge, triggering a luteinizing hormone (LH) surge, which leads to ovulation. Precise temporal regulation of the preovulatory GnRH/LH surge is important for successful reproduction because ovulation should occur after follicular development. The time course of the circulating level of estrogen is correlated with the ovulatory cycle throughout vertebrates. However, the neural mechanisms underlying estrogen-induced preovulatory GnRH surge after folliculogenesis still remain unclear, especially in non-mammals. Here, we used a versatile non-mammalian model medaka for the analysis of the involvement of estrogen in the regulation of POA-GnRH (GnRH1) neurons. Electrophysiological analysis using a whole brain-pituitary in vitro preparation, which maintains the hypophysiotropic function of GnRH1 neurons intact, revealed that 17ß-estradiol (E2 ) administration recovers the ovariectomy-induced lowered GnRH1 neuronal activity in the evening, indicating the importance of E2 for upregulation of GnRH1 neuronal activity. The importance of E2 was also confirmed by the fact that GnRH1 neuronal activity was low in short-day photoperiod-conditioned females (low E2 model). However, E2 failed to upregulate the firing activity of GnRH1 neurons in the morning, suggesting the involvement of additional time-of-day signal(s) for triggering GnRH/LH surges at an appropriate timing. We also provide morphological evidence for the localization of estrogen receptor subtypes in GnRH1 neurons. In conclusion, we propose a working hypothesis in which both estrogenic and time-of-day signals act in concert to timely upregulate the firing activity of GnRH1 neurons that trigger the GnRH surge at an appropriate timing in a female-specific manner. This neuroendocrinological mechanism is suggested to be responsible for the generation of ovulatory cycles in female teleosts in general.

Multiple gonadotropin-releasing hormone systems in non-mammalian vertebrates: Ontogeny, anatomy, and physiology.

Three paralogous genes for gonadotropin-releasing hormone (GnRH; gnrh1, gnrh2, and gnrh3) and GnRH receptors exist in non-mammalian vertebrates. However, there are some vertebrate species in which one or two of these paralogous genes have become non-functional during evolution. The developmental migration of GnRH neurons in the brain is evolutionarily conserved in mammals, reptiles, birds, amphibians, and jawed teleost fish. The three GnRH paralogs have specific expression patterns in the brain and originate from multiple sites. In acanthopterygian teleosts (medaka, cichlid, etc.), the preoptic area (POA)-GnRH1 and terminal nerve (TN)-GnRH3 neuronal types originate from the olfactory regions. In other fish species (zebrafish, goldfish and salmon) with only two GnRH paralogs (GnRH2 and GnRH3), the TN- and POA-GnRH3 neuronal types share the same olfactory origin. However, the developmental origin of midbrain (MB)-GnRH2 neurons is debatable between mesencephalic or neural crest site. Each GnRH system has distinctive anatomical and physiological characteristics, and functions differently. The POA-GnRH1 neurons are hypophysiotropic in nature and function in the neuroendocrine control of reproduction. The non-hypophysiotropic GnRH2/GnRH3 neurons probably play neuromodulatory roles in metabolism (MB-GnRH2) and the control of motivational state for sexual behavior (TN-GnRH3).

Co-existing Neuropeptide FF and Gonadotropin-Releasing Hormone 3 Coordinately Modulate Male Sexual Behavior.

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertoires were delayed, not disrupted, in gnrh3 and npff single knockout males, while the double knockout appeared to alleviate the significant defects that were observed in single knockouts. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which co-existing peptides of the TN-GnRH neurons, NPFF, and GnRH3 coordinately modulate certain neuronal circuit for the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to sensory information from the external environments.

Comparison of Femtosecond Laser-Assisted Cataract Surgery and Conventional Phacoemulsification on Endothelial Cell Density When Using Torsional Modality.

PURPOSE: To compare the effects of femtosecond laser assisted cataract surgery (FLACS) and manual phacoemulsification on cumulative dissipated energy (CDE), torsional amplitude, and endothelial cell density (ECD). PATIENTS AND METHODS: This prospective, randomized study was conducted at Oka Eye Clinic (Fukuoka, Japan). Surgeries were performed using FLACS (with LenSx) or conventional technique in adults ≥20 years with grade 2-4 cataracts. Visits included preoperative, surgery day, and 5 postoperative visits (days 1, 4-10, 20-40, 60-120, and 150-210). Primary endpoint was CDE. Secondary endpoints included ECD percent change at day 150-210 versus preoperative visit and average torsional amplitude on surgery day. Exploratory endpoints included central corneal thickness and corrected distance visual acuity (CDVA). Superiority of FLACS to conventional technique was evaluated using t-tests based on a mixed model for repeated measures. RESULTS: Full analysis set included 53 eyes per group. Mean cataract grade was 2.92±0.58 in FLACS and 2.94±0.57 in conventional group. FLACS versus conventional method had significantly lower mean CDE (0.213±0.334 versus 1.718±0.898%-seconds, respectively; P<0.0001), demonstrating superiority of FLACS. Low endothelial cell loss (ECL) was achieved with both FLACS and conventional methods (1.5±5.6% and 2.7±5.2%; P=0.260). Torsional amplitude was significantly lower for FLACS versus conventional method (19.6±16.0% versus 31.1±6.6%; P<0.0001). Central corneal thickness was comparable for both methods at all visits except day 1; CDVA was comparable for both methods at all postoperative visits. CONCLUSION: FLACS achieved significantly lower CDE compared with the conventional surgical method (P<0.0001). Low ECL was achieved with both FLACS (1.5%) and conventional (2.7%) methods.

TMC4 is a novel chloride channel involved in high-concentration salt taste sensation.

"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

Establishment of open-source semi-automated behavioral analysis system and quantification of the difference of sexual motivation between laboratory and wild strains.

Behavioral analysis plays an important role in wide variety of biological studies, but behavioral recordings often tend to be laborious and are associated with inevitable human-errors. It also takes much time to perform manual behavioral analyses while replaying the videos. On the other hand, presently available automated recording/analysis systems are often specialized for certain types of behavior of specific animals. Here, we established an open-source behavioral recording system using Raspberry Pi, which automatically performs video-recording and systematic file-sorting, and the behavioral recording can be performed more efficiently, without unintentional human operational errors. We also developed an Excel macro that enables us to easily perform behavioral annotation with simple manipulation. Thus, we succeeded in developing an analysis suite that mitigates human tasks and thus reduces human errors. By using this suite, we analyzed the sexual behavior of a laboratory and a wild medaka strain and found a difference in sexual motivation presumably resulting from domestication.

Evaluation of differences and dosimetric influences of beam models using golden and multi-institutional measured beam datasets in radiation treatment planning systems.

PURPOSE: The beam model in radiation treatment planning systems (RTPSs) plays a crucial role in determining the accuracy of calculated dose distributions. The purpose of this study was to ascertain differences in beam models and their dosimetric influences when a golden beam dataset (GBD) and multi-institution measured beam datasets (MBDs) are used for beam modeling in RTPSs. METHODS: The MBDs collected from 15 institutions, and the MBDs' beam models, were compared with a GBD, and the GBD's beam model, for Varian TrueBeam linear accelerator. The calculated dose distributions of the MBDs' beam models were compared with those of the GBD's beam model for simple geometries in a water phantom. Calculated dose distributions were similarly evaluated in volumetric modulated arc therapy (VMAT) plans for TG-119 C-shape and TG-244 head and neck, at several dose constraints of the planning target volumes (PTVs), and organs at risk. RESULTS: The agreements of the MBDs with the GBD were almost all within ±1%. The calculated dose distributions for simple geometries in a water phantom also closely corresponded between the beam models of GBD and MBDs. Nevertheless, there were considerable differences between the beam models. The maximum differences between the mean energy of the energy spectra of GBD and MBDs were -0.12 MeV (-10.5%) in AcurosXB (AXB, Eclipse) and 0.11 MeV (7.7%) in collapsed cone convolution (CCC, RayStation). The differences in the VMAT calculated dose distributions varied for each dose region, plan, X-ray energy, and dose calculation algorithm. The ranges of the differences in the dose constraints were -5.6% to 3.0% for AXB and -24.1% to 2.8% for CCC. In several VMAT plans, the calculated dose distributions of GBD's beam model tended to be lower in high-dose regions and higher in low-dose regions than those of the MBDs' beam models. CONCLUSIONS: We found that small differences in beam data have large impacts on the beam models, and on calculated dose distributions in clinical VMAT plan, even if beam data correspond within ±1%. GBD's beam model was not a representative beam model. The beam models of GBD and MBDs and their calculated dose distributions under clinical conditions were significantly different. These differences are most likely due to the extensive variation in the beam models, reflecting the characteristics of beam data. The energy spectrum and radial energy in the beam model varied in a wide range, even if the differences in the beam data were <±1%. To minimize the uncertainty of the calculated dose distributions in clinical plans, it was best to use the institutional MBD for beam modeling, or the beam model that ensures the accuracy of calculated dose distributions.

Examination of methods for manipulating serum 17ß-Estradiol (E2) levels by analysis of blood E2 concentration in medaka (Oryzias latipes).

It is widely known that reproduction in vertebrates is regulated by the hypothalamus-pituitary-gonadal (HPG) axis. Although the mechanism of the HPG axis has been well documented in mammals, it cannot be always applied to that in non-mammalian species, which is a great disadvantage in understanding reproduction of vertebrates in general. Recently, transgenic and genome editing tools have rapidly been developed in small teleosts, and thus these species are expected to be useful for the understanding of general mechanism of reproduction in vertebrates. One of the major sex steroid hormones in female vertebrates 17ß-Estradiol (E2) plays crucial roles in the formation of sexual dimorphism and the HPG axis regulation. In spite of the importance of E2 in reproductive regulation, only a few studies have analyzed blood E2 levels in small teleosts that are easily amenable to genetic manipulation. In the present study, we analyzed blood E2 concentration in medaka and demonstrated that female medaka show diurnal changes in blood E2 concentration. We then examined the best method for manipulating the circulating E2. First, we found that ovariectomy (OVX) drastically removes endogenous E2 in a day in female medaka. We examined different methods for E2 administration and revealed that feeding administration of E2-containing food is the most convenient and physiological method for mimicking the diurnal E2 changes of female medaka. On the other hand, the medaka exposed to E2 containing water showed high blood E2 concentrations, which exceeds those of environmental water, suggesting that E2 may cause bioconcentration.

Multiple functions of non-hypophysiotropic gonadotropin releasing hormone neurons in vertebrates.

Gonadotropin releasing hormone (GnRH) is a hypophysiotropic hormone that is generally thought to be important for reproduction. This hormone is produced by hypothalamic GnRH neurons and stimulates the secretion of gonadotropins. On the other hand, vertebrates also have non-hypophysiotropic GnRH peptides, which are produced by extrahypothalamic GnRH neurons. They are mainly located in the terminal nerve, midbrain tegmentum, trigeminal nerve, and spinal cord (sympathetic preganglionic nerves). In vertebrates, there are typically three gnrh paralogues (gnrh1, gnrh2, gnrh3). GnRH-expression in the non-hypophysiotropic neurons (gnrh1 or gnrh3 in the terminal nerve and the trigeminal nerve, gnrh2 in the midbrain tegmentum) occurs from the early developmental stages. Recent studies have suggested that non-hypophysiotropic GnRH neurons play various functional roles. Here, we summarize their anatomical/physiological properties and discuss their possible functions, focusing on studies in vertebrates. GnRH neurons in the terminal nerve show different spontaneous firing properties during the developmental stages. These neurons in adulthood show regular pacemaker firing, and it has been suggested that these neurons show neuromodulatory function related to the regulation of behavioral motivation, etc. In addition to their recognized role in neuromodulation in adult, in juvenile fish, these neurons, which show more frequent burst firing than in adults, are suggested to have novel functions. GnRH neurons in the midbrain tegmentum show regular pacemaker firing similar to that of the adult terminal nerve and are suggested to be involved in modulations of feeding (teleosts) or nutrition-related sexual behaviors (musk shrew). GnRH neurons in the trigeminal nerve are suggested to be involved in nociception and chemosensory avoidance, although the literature on their electrophysiological properties is limited. Sympathetic preganglionic cells in the spinal cord were first reported as peptidergic modulatory neurons releasing GnRH with a putative function in coordinating interaction between vasomotor and exocrine outflow in the sympathetic nervous system. The functional role of non-hypophysiotropic GnRH neurons may thus be in the global modulation of neural circuits in a manner dependent on internal conditions or the external environment.

Gene knockout analysis reveals essentiality of estrogen receptor ß1 (Esr2a) for female reproduction in medaka.

In vertebrates, sex steroids play crucial roles in multiple systems related to reproduction. In females, estrogens and their receptor estrogen receptor (ER or Esr) play indispensable roles in the negative sex steroid feedback regulation of pituitary gonadotropin secretion, which prevents excessive development of ovarian follicles. However, the mechanism of this feedback regulation of a gonadotropin, follicle stimulating hormone (FSH), which is essential for folliculogenesis throughout vertebrates, is poorly understood. In the present study, we generated knockouts of all subtypes of nuclear estrogen receptors in a model teleost medaka, which is suitable for the study of endocrine control and behavioral assays, and analyzed fertility, behavior and functionality of estrogen feedback in each knockout line. Among the estrogen receptors, we revealed that an estrogen receptor Esr2a plays an essential role in this feedback regulation. In addition to this, we also found that esr2a-/- females showed oviduct atresia, which causes complete infertility. Interestingly, esr2a-/- females showed apparently normal sexual behavior but without oviposition in response to male courtship. This phenotype indicates that physical readiness and motivation of sexual behavior is independently controlled.

Sexually Dimorphic Neuropeptide B Neurons in Medaka Exhibit Activated Cellular Phenotypes Dependent on Estrogen.

Brain and behavior of teleosts are highly sexually plastic throughout life, yet the underlying neural mechanisms are largely unknown. On examining brain morphology in the teleost medaka (Oryzias latipes), we identified distinctively large neurons in the magnocellular preoptic nucleus that occurred much more abundantly in females than in males. Examination of sex-reversed medaka showed that the sexually dimorphic abundance of these neurons is dependent on gonadal phenotype, but independent of sex chromosome complement. Most of these neurons in females, but none in males, produced neuropeptide B (Npb), whose expression is known to be estrogen-dependent and associated with female sexual receptivity. In phenotypic analysis, the female-specific Npb neurons had a large euchromatic nucleus with an abundant cytoplasm containing plentiful rough endoplasmic reticulum, exhibited increased overall transcriptional activity, and typically displayed a spontaneous regular firing pattern. These phenotypes, which are probably indicative of cellular activation, were attenuated by ovariectomy and restored by estrogen replacement. Furthermore, the population of Npb-expressing neurons emerged in adult males treated with estrogen, not through frequently occurring neurogenesis in the adult teleost brain, but through the activation of preexisting, quiescent male counterpart neurons. Collectively, our results demonstrate that the morphological, transcriptional, and electrophysiological phenotypes of sexually dimorphic preoptic Npb neurons are highly dependent on estrogen and can be switched between female and male patterns. These properties of the preoptic Npb neurons presumably underpin the neural mechanism for sexual differentiation and plasticity of brain and behavior in teleosts.

Juvenile-Specific Burst Firing of Terminal Nerve GnRH3 Neurons Suggests Novel Functions in Addition to Neuromodulation.

Peptidergic neurons are suggested to play a key role in neuromodulation of animal behaviors in response to sensory cues in the environment. Terminal nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons are thought to be one of the peptidergic neurons important for such neuromodulation in adult vertebrates. On the other hand, it has been reported that TN-GnRH3 neurons are labeled by a specific GnRH3 antibody from early developmental stages to adulthood and are thus suggested to produce mature GnRH3 peptide even in the early developmental stages. However, it remains unknown when TN-GnRH3 neurons show spontaneous burst firing, which is suggested to be involved in neuropeptide release. Using a whole-brain in vitro preparation of gnrh3:enhanced green fluorescent protein (EGFP) medaka fish, we first recorded spontaneous firings of TN-GnRH3 neurons after hatching to adulthood. Contrary to what one would expect from their neuromodulatory functions-that TN-GnRH3 neurons are more active in adulthood-TN-GnRH3 neurons in juveniles showed spontaneous burst firing more frequently than in adulthood (juvenile-specific burst firing). Ca2+ imaging of TN-GnRH3 neurons in juveniles may further suggest that juvenile-specific burst firing triggers neuropeptide release. Furthermore, juvenile-specific burst firing was suggested to be induced by blocking persistent GABAergic inhibition to the glutamatergic neurons, which leads to an increase in glutamatergic synaptic inputs to TN-GnRH3 neurons. The present study reports that peptidergic neurons show juvenile-specific burst firing involved in triggering peptide release and suggests that juvenile TN-GnRH3 neurons have novel functions, in addition to neuromodulation.

Genome-wide association study identifies seven novel susceptibility loci for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide for which 15 disease-associated loci had been discovered. Among them, only 5 loci have been associated with POAG in Asians. We carried out a genome-wide association study and a replication study that included a total of 7378 POAG cases and 36 385 controls from a Japanese population. After combining the genome-wide association study and the two replication sets, we identified 11 POAG-associated loci, including 4 known (CDKN2B-AS1, ABCA1, SIX6 and AFAP1) and 7 novel loci (FNDC3B, ANKRD55-MAP3K1, LMX1B, LHPP, HMGA2, MEIS2 and LOXL1) at a genome-wide significance level (P < 5.0×10-8), bringing the total number of POAG-susceptibility loci to 22. The 7 novel variants were subsequently evaluated in a multiethnic population comprising non-Japanese East Asians (1008 cases, 591 controls), Europeans (5008 cases, 35 472 controls) and Africans (2341 cases, 2037 controls). The candidate genes located within the new loci were related to ocular development (LMX1B, HMGA2 and MAP3K1) and glaucoma-related phenotypes (FNDC3B, LMX1B and LOXL1). Pathway analysis suggested epidermal growth factor receptor signaling might be involved in POAG pathogenesis. Genetic correlation analysis revealed the relationships between POAG and systemic diseases, including type 2 diabetes and cardiovascular diseases. These results improve our understanding of the genetic factors that affect the risk of developing POAG and provide new insight into the genetic architecture of POAG in Asians.