Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

The cooperative induction of macrophage activation by fucoidan derived from Cladosiphon okamuranus and ß-glucan derived from Saccharomyces cerevisiae.

The present study investigated immune stimulatory effects of Cladosiphon okamuranus-derived fucoidan to activate murine macrophage-like cell line RAW264, and the functional relationship with zymosan, a Saccharomyces cerevisiae-derived ß-glucan. The production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in RAW264 cells were remarkably enhanced in the presence of 10 µg/mL fucoidan, and the stimulatory effects of fucoidan were maximally augmented in combinational treatment with 500 ng/mL zymosan, whereas any TLR ligands had no those effects. Confocal microscopic analyses suggested that fucoidan bound on plasma membrane, and it was estimated that some cell surface molecules acted as receptor for fucoidan because cytochalasin D, an inhibitor of phagocytosis, did not affect the immune enhancing activities, whereas methyl-ß-cyclodextrin (MßCD), a general agent for disruption of lipid rafts, diminished that. Furthermore, it was revealed that the additive effects of zymosan on the immune activation with fucoidan was thought to be mediated by dectin-1 based on the results with dectin-1-knockdown RAW264 cells. All of results suggested that fucoidan and some kinds of ß-glucan would cooperatively reinforce the activity of innate immune cells via interactive receptor crosstalk.

Molecular basis of social competence in medaka fish.

Oryzias latipes (Medaka) is an established vertebrate model for studying developmental genetics, genomics, and evolutionary biology. The physiology, embryology, and genetics of this species have been extensively investigated for centuries. Medaka fish recently attracted attention in the field of social neuroscience. This review introduces recent advances in medaka behavioral studies, focusing on female mating preferences and male mate-guarding behaviors. The medaka female has the ability to discriminate male individuals and prefers to mate with socially familiar males (female mating preference). In triadic relationships (two males and one female), the dominant male remains closer to the female and repels the other male (mate-guarding). Interestingly, mate-guarding blocks female social familiarization of the rival male, which can increase the mating success of the dominant male. Importantly, behavioral analyses using a series of medaka mutants revealed critical roles of neuropeptide neuromodulatory systems in regulating their social behaviors. The extra-hypothalamic gonadotropin-releasing hormone system has a central role in activating female mating preference. The arginine-vasotocin system is required for the emergence of mate-guarding behavior.

Analysis of the Differentiation of Kenyon Cell Subtypes Using Three Mushroom Body-Preferential Genes during Metamorphosis in the Honeybee (Apis mellifera L.).

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages.

A neural mechanism underlying mating preferences for familiar individuals in medaka fish.

Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes).

Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

Induction of c-fos transcription in the medaka brain (Oryzias latipes) in response to mating stimuli.

Immediate-early genes (IEGs) are useful for mapping active brain regions in various vertebrates. Here we identified a c-fos homologue gene in medaka and demonstrated that the amounts of c-fos transcripts and proteins in the medaka brain increased in relation to an artificially evoked seizure, suggesting that the homologue gene has the characteristics of IEGs, which are used as markers of neural activity. Next, quantitative reverse-transcription-polymerase chain reaction revealed that female mating behaviors upregulated c-fos transcription in some brain regions including the telencephalon, optic tectum, and cerebellum. In addition, we performed in situ hybridization with a c-fos intron probe to detect the de novo synthesis of c-fos transcripts and confirmed induction of c-fos transcription in these brain regions after mating. This is the first report of IEG induction in response to mating stimuli in teleost fish. Our results indicated that c-fos expression was induced in response to behavioral stimuli in the medaka brain and that medaka c-fos could be a useful marker of neural activity.

Transient and permanent gene transfer into the brain of the teleost fish medaka (Oryzias latipes) using human adenovirus and the Cre-loxP system.

In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the beta-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae.

Novel insect picorna-like virus identified in the brains of aggressive worker honeybees.

To identify candidate genes involved in the aggressive behavior of worker honeybees, we used the differential display method to search for RNAs exclusively detected in the brains of aggressive workers that had attacked a hornet. We identified a novel, 10,152-nucleotide RNA, termed Kakugo RNA. Kakugo RNA encodes a protein of 2,893 amino acid residues that shares structural features and sequence similarities with various picorna-like virus polyproteins, especially those from sacbrood virus, which infects honeybees. The Kakugo protein contains several domains that correspond to the virion protein, helicase, protease, and RNA-dependent RNA polymerase domains of various picorna-like virus polyproteins. When the worker bee tissue lysate was subjected to sucrose density gradient centrifugation, Kakugo RNA, except for the material at the bottom, was separated into two major peaks. One of the peaks corresponded to the position of Kakugo mRNA, and the other corresponded to the position of the poliovirus virion. These results suggest that the Kakugo RNA exists as an mRNA-like free RNA and virion RNA in the honeybee. Furthermore, injection of the lysate supernatant from the attacker heads into the heads of noninfected bees resulted in a marked increase in Kakugo RNA. These results demonstrate that Kakugo RNA is a plus-strand RNA of a novel picorna-like virus and that the brains of aggressive workers are infected by this novel virus. Kakugo RNA was detected in aggressive workers but not in nurse bees or foragers. In aggressive workers, Kakugo RNA was detected in the brain but not in the thorax or abdomen, indicating a close relation between viral infection in the brain and aggressive worker behaviors.

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray.

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.