Maternal High-Fat Diet Affects the Contents of Eggs and Causes Abnormal Development in the Medaka Fish.

Maternal nutritional status can affect development and metabolic phenotypes of progeny in animals. The effects of maternal diet are thought to be mediated mainly by changes inside oocytes such as organelles, maternal RNAs, and metabolites. However, to what extent each factor contributes to offspring phenotypes remains uncertain, especially in viviparous mammalian systems, where factors other than oocytes, such as placenta and milk, need to be considered. Here, using the medaka fish as an oviparous vertebrate model, we examined whether maternal high-fat diet (mHFD) feeding affects offspring development and what kind of changes occur in the contents of mature eggs. We found that mHFD caused the high frequency of embryonic deformities of offspring, accompanied by downregulation of transcription- and translation-related genes and zygotic transcripts at the blastula stage. Transcriptomic and metabolomic analyses of mature eggs suggested decreased catabolism of amino acids and glycogen, moderate upregulation of endoplasmic reticulum stress-related genes, and elevated lipid levels in mHFD eggs. Furthermore, high-fat diet females showed a higher incidence of oocyte atresia and downregulation of egg protein genes in the liver. These data suggest that attenuated amino acid catabolism triggered by decreased yolk protein load/processing, as well as elevated lipid levels inside eggs, are the prime candidates that account for the higher incidence of embryonic deformities in mHFD offspring. Our study presents a comprehensive data on the changes inside eggs in a mHFD model of nonmammalian vertebrates and provides insights into the mechanisms of parental nutritional effects on offspring.

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.

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.

Neuropeptide FF indirectly affects testicular morphogenesis and functions in medaka.

Testicular morphogenesis and functions are considered to be under the control of neural and endocrine systems. However, the available literature is mainly limited to mammals, and it remains unclear how they are regulated in teleost species. Here, we demonstrated that neuropeptide FF (NPFF) in the brain is responsible for the follicle-stimulating hormone expression in the pituitary, which facilitates the testicular morphogenesis and androgen synthesis, and subsequently contributes to successful spermatogenesis. The present findings give us important insights into the neuroendocrine regulatory mechanisms underlying the testicular morphogenesis and functions in teleosts.

In vitro and in vivo gene transfer in the cloudy catshark Scyliorhinus torazame.

Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

Molecular encoding and synaptic decoding of context during salt chemotaxis in C. elegans.

Animals navigate toward favorable locations using various environmental cues. However, the mechanism of how the goal information is encoded and decoded to generate migration toward the appropriate direction has not been clarified. Here, we describe the mechanism of migration towards a learned concentration of NaCl in Caenorhabditis elegans. In the salt-sensing neuron ASER, the difference between the experienced and currently perceived NaCl concentration is encoded as phosphorylation at Ser65 of UNC-64/Syntaxin 1 A through the protein kinase C(PKC-1) signaling pathway. The phosphorylation affects basal glutamate transmission from ASER, inducing the reversal of the postsynaptic response of reorientation-initiating neurons (i.e., from inhibitory to excitatory), guiding the animals toward the experienced concentration. This process, the decoding of the context, is achieved through the differential sensitivity of postsynaptic excitatory and inhibitory receptors. Our results reveal the mechanism of migration based on the synaptic plasticity that conceptually differs from the classical ones.

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.

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.

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.

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.

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.

GnRH suppresses excitability of visual processing neurons in the optic tectum.

Animals change their behavior in response to sensory cues in the environment as well as their physiological status. For example, it is generally accepted that their sexual behavior is modulated according to seasonal environmental changes or the individual's maturational/reproductive status, and neuropeptides have been suggested to play important roles in this process. Some behavioral modulation arises from neuropeptide modulation of sensory information processing in the central nervous system, but the neural mechanisms still remain unknown. Here we focused on the neural basis of neuropeptide modulation of visual processing in vertebrates. The terminal nerve neurons that contain gonadotropin-releasing hormone 3 (TN-GnRH3 neurons) are suggested to modulate reproductive behavior and have massive projections to the optic tectum (OT), which plays an important role in visual processing. In the present study, to examine whether GnRH3 modulates retino-tectal neurotransmission in the OT, we analyzed the effect of GnRH3 electrophysiologically and morphologically. We found that field potentials evoked by optic tract fiber stimulation, which represent retino-tectal neurotransmission, were modulated postsynaptically by GnRH3. Whole cell recording from postsynaptic neurons in the retino-tectal pathway suggested that GnRH3 activates large-conductance Ca(2+)-activated K(+) (BK) channels and thereby suppresses membrane excitability. Furthermore, our improved morphological analysis using fluorescently labeled GnRH peptides showed that GnRH receptors are localized mainly around the cell bodies of postsynaptic neurons. Our results indicate that TN-GnRH3 neurons modulate retino-tectal neurotransmission by suppressing the excitability of projection neurons in the OT, which underlies the neuromodulation of behaviorally relevant visual information processing by the neuropeptide GnRH3.

Novel middle-type Kenyon cells in the honeybee brain revealed by area-preferential gene expression analysis.

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel 'middle-type Kenyon cells' that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large- and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large- and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large- and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a 'clustering' of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.

Neuropeptide RFRP inhibits the pacemaker activity of terminal nerve GnRH neurons.

The terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons show spontaneous pacemaker activity whose firing frequency is suggested to regulate the release of GnRH peptides and control motivation for reproductive behaviors. Previous studies of the electrophysiological properties of TN-GnRH neurons reported excitatory modulation of pacemaker activity by auto/paracrine and synaptic modulations, but inhibition of pacemaker activity has not been reported to date. Our recent study suggests that neuropeptide FF, a type of Arg-Phe-amide (RFamide) peptide expressed in TN-GnRH neurons themselves, inhibits the pacemaker activity of TN-GnRH neurons in an auto- and paracrine manner. In the present study, we examined whether RFamide-related peptides (RFRPs), which are produced in the hypothalamus, modulate the pacemaker activity of TN-GnRH neurons as candidate inhibitory synaptic modulators. Bath application of RFRP2, among the three teleost RFRPs, decreased the frequency of firing of TN-GnRH neurons. This inhibition was diminished by RF9, a potent antagonist of GPR147/74, which are candidate RFRP receptors. RFRP2 changed the conductances for Na(+) and K(+). The reversal potential for RFRP2-induced current was altered by inhibitors of the transient receptor potential canonical (TRPC) channel (La(3+) and 2-aminoethoxydiphenyl borate) and by a less selective blocker of voltage-independent K(+) channels (Ba(2+)). By comparing the current-voltage relationship in artificial cerebrospinal fluid with that under each drug, the RFRP2-induced current was suggested to consist of TRPC channel-like current and voltage-independent K(+) current. Therefore, synaptic release of RFRP2 from hypothalamic neurons is suggested to inhibit the pacemaker activity of TN-GnRH neurons by closing TRPC channels and opening voltage-independent K(+) channels. This novel pathway may negatively regulate reproductive behaviors.