善方 文太郎

The HCN4 channel is essential for heart rate regulation in vertebrates by generating pacemaker potentials in the sinoatrial node. HCN4 channel abnormality may cause bradycardia and sick sinus syndrome, making it an important target for clinical research and drug discovery. The zebrafish is a popular animal model for cardiovascular research. They are potentially suitable for studying inherited heart diseases, including cardiac arrhythmia. However, it has not been determined how similar the ion channels that underlie cardiac automaticity are in zebrafish and humans. In the case of HCN4, humans have one gene, whereas zebrafish have two ortholog genes (DrHCN4 and DrHCN4L; ‘Dr’ referring to Danio rerio). However, it is not known whether the two HCN4 channels have different physiological functions and roles in heart rate regulation. In this study, we characterized the biophysical properties of the two zebrafish HCN4 channels in Xenopus oocytes and compared them to those of the human HCN4 channel. We found that they showed different gating properties: DrHCN4L currents showed faster activation kinetics and a more positively shifted G-V curve than did DrHCN4 and human HCN4 currents. We made chimeric channels of DrHCN4 and DrHCN4L and found that cytoplasmic domains were determinants for the faster activation and the positively shifted G-V relationship in DrHCN4L. The use of a dominant-negative HCN4 mutant confirmed that DrHCN4 and DrHCN4L can form a heteromultimeric channel in Xenopus oocytes. Next, we confirmed that both are sensitive to common HCN channel inhibitors/blockers including Cs⁺, ivabradine, and ZD7288. These HCN inhibitors successfully lowered zebrafish heart rate during early embryonic stages. Finally, we knocked down the HCN4 genes using antisense morpholino and found that knocking down either or both of the HCN4 channels caused a temporal decrease in heart rate and tended to cause pericardial edema. These findings suggest that both DrHCN4 and DrHCN4L play a significant role in zebrafish heart rate regulation.

<title>Abstract</title>The mating behavior of teleost fish consists of a sequence of stereotyped actions. By observing mating of zebrafish under high-speed video, we analyzed and characterized a behavioral cascade leading to successful fertilization. When paired, a male zebrafish engages the female by oscillating his body in high frequency (<italic>quivering</italic>). In response, the female pauses swimming and bends her body (<italic>freezing</italic>). Subsequently, the male contorts his trunk to enfold the female’s trunk. This behavior is known as <italic>wrap around</italic>. Here, we found that <italic>wrap around</italic> behavior consists of two previously unidentified components. After both sexes contort their trunks, the male adjusts until his trunk compresses the female’s dorsal fin (<italic>hooking</italic>). After<italic> hooking</italic>, the male trunk slides away from the female’s dorsal fin, simultaneously sliding his pectoral fin across the female’s gravid belly, stimulating egg release (<italic>squeezing/spawning</italic>). Orchestrated coordination of <italic>spawning</italic> presumably increases fertilization success. Surgical removal of the female dorsal fin inhibited <italic>hooking</italic> and the transition to <italic>squeezing</italic>. In a neuromuscular mutant where males lack <italic>quivering</italic>, female <italic>freezing</italic> and subsequent courtship behaviors were absent. We thus identified traits of zebrafish mating behavior and clarified their roles in successful mating.

Some hypothalamic neurons expressing estrogen receptor a (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, estrogensensitive 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 nonmammalian species casts doubt on the regulation of GnRH1 neurons by kisspeptin neurons. As a step toward understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic (Tg) medaka that expresses enhanced green fluorescent protein (EGFP) specifically in esr1-expressing neurons (esr1 neurons) and analyzed their axonal projections. We found that esr1 neurons in the preoptic area (POA) project to the gnrh1 neurons. We also demonstrated by transcriptome and histological analyses that these esr1 neurons are glutamatergic or g-aminobutyric acidergic (GABAergic) but not kisspeptinergic. We therefore suggest that glutamatergic and GABAergic esr1 neurons in the POA regulate gnrh1 neurons. This hypothesis is consistent with previous studies in mice that found that glutamatergic and 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 coexpress progesterone receptors. These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone. (Endocrinology 159: 1228-1241, 2018).