Akihisa Urano

Functional organization of preoptic vasotocin and isotocin neurons in the brain of rainbow trout: central and neurohypophysial projections of single neurons

Preoptic magnocellular neurosecretory cells (NSCs) in the brain of rainbow trout show synchronization of periodic Ca(2+) pulses, patterns of which differ between vasotocin (VT) and isotocin (IT) neurons. To provide neuroanatomical bases of the synchronized periodic Ca(2+) pulses and their biological implications, we examined the organization of preoptic VT and IT neurons in the brain of rainbow trout. The cytoarchitecture of the preoptic neurosecretory system was characterized by a confocal double-color immunofluorescence. Two to five VT neurons, and also IT neurons, aggregate to form cell-type specific clusters. VT clusters tend to localize medially, while IT clusters laterally. VT neurons are closely apposed at the proximal neuronal processes. A Golgi-like immunohistochemistry demonstrated that VT and IT fibers distribute widely in the brain, such as ventral telencephalon, diencephalon, and various mesencephalic structures, in addition to the neurohypophysial projections. Projections from single VT and IT neurons were examined by an intracellular staining with biocytin injection in a sagittally hemisected brain preparation, which contains the entire forebrain region. Single VT and IT neurons project toward the pituitary and the extrahypothalamic regions. Some IT neurons, but not VT neurons, were dye-coupled. These results support the idea that the same types of NSCs are connected to form cell-type-specific networks responsible for the synchronization of periodic Ca(2+) pulses. The organization of the preoptic neurosecretory system shown in the present study is suitable for the simultaneous control of neurohypophysial and extrahypothalamic outputs through the synchronization of electrical activity.

Genetic population structure of chum salmon in the Pacific Rim inferred from mitochondrial DNA sequence variation

We examined the genetic population structure of chum salmon, Oncorhynchus keta, in the Pacific Rim using mitochondrial (mt) DNA analysis. Nucleotide sequence analysis of about 500 bp in the variable portion of the 5′ end of the mtDNA control region revealed 20 variable nucleotide sites, which defined 30 haplotypes of three genealogical clades (A, B, and C), in more than 2,100 individuals of 48 populations from Japan (16), Korea (1), Russia (10), and North America (21 from Alaska, British Columbia, and Washington). The observed haplotypes were mostly associated with geographic regions, in that clade A and C haplotypes characterized Asian populations and clade B haplotypes distinguished North American populations. The haplotype diversity was highest in the Japanese populations, suggesting a greater genetic variation in the populations of Japan than those of Russia and North America. The analysis of molecular variance and contingency χ2 tests demonstrated strong structuring among the three geographic groups of populations and weak to moderate structuring within Japanese and North American populations. These results suggest that the observed geographic pattern might be influenced primarily by historic expansions or colonizations and secondarily by low or restricted gene flow between local groups within regions. In addition to the analysis of population structure, mtDNA data may be useful for constructing a baseline for stock identification of mixed populations of high seas chum salmon.

Seasonal changes of responses to gonadotropin-releasing hormone analog in expression of growth hormone/prolactin/somatolactin genes in the pituitary of masu salmon

Gonadotropin-releasing hormone (GnRH) is considered to stimulate secretion of growth hormone (GH), prolactin (PRL), and somatolactin (SL) at particular stages of growth and sexual maturation in teleost fishes. We therefore examined seasonal variation in the pituitary levels of GH/PRL/SL mRNAs, and tried to clarify seasonal changes of responses to GnRH in expression of GH/PRL/SL genes, in the pituitaries of growing and maturing masu salmon (Oncorhynchus masou). Pituitary samples were monthly collected one week after implantation with GnRH analog (GnRHa). The levels of mRNAs encoding GH, PRL, and SL precursors in single pituitaries were determined by a real-time polymerase chain reaction method. The fork lengths and body weights of control and GnRHa-implanted fish of both sexes gradually increased and peaked out in September of 2-year-old (2+) when fish spawned. GnRHa implantation did not stimulate somatic growth, nor elevate gonadosomatic index (GSI) of 1+ and 2+ males, whereas it significantly increased GSI of 2+ females in late August to early September. The GnRHa-implanted 1+ males had higher levels of GH and PRL mRNAs in July, and SL mRNA from June to August than the control males. The levels of GH, PRL, and SL mRNAs in the control and GnRHa-implanted 1+ females, however, did not show any significant changes. Afterward, the PRL mRNA levels elevated in the control 2+ fish of both sexes in spring. GnRHa elevated the GH mRNA levels in both males and females in 2+ winter, and the PRL mRNA levels in females in early spring. Regardless of sex and GnRHa-implantation, the SL mRNA levels increased during sexual maturation. In growing and maturing masu salmon, expression of genes encoding GH, PRL, and SL in the pituitary is thus sensitive to GnRH in particular seasons probably in relation to physiological roles of the hormones.

Gonadotropin-releasing hormones modulate electrical activity of vasotocin and isotocin neurons in the brain of rainbow trout

Gonadotropin-releasing hormone (GnRH) is widely distributed in the vertebrate brains; however, its significance in the brain function is poorly understood. Both GnRH and vasopressin-family hormones are involved in control of reproductive behavior. Anatomical evidence indicated the possible action of GnRH on classical neurosecretory neurons. In the present study, we examined whether GnRH modulates electrical activity of vasotocin (VT) and isotocin (IT) neurons in the brain of rainbow trout (Oncorhynchus mykiss). Two forms of GnRH, salmon GnRH and chicken GnRH II, are present in the rainbow trout brain, and their fibers are localized in the close vicinity of VT and IT neurons. Applications of both GnRH forms elevated the frequency of cell-type-specific synchronous Ca(2+) pulses in VT and IT neurons that are blocked by a GnRH-receptor antagonist. Our results showed facilitatory actions of GnRHs on VT and IT neurons, suggesting that GnRH neurons modulate classical neurosecretory neurons to control reproductive behavior.

Five Different Types of Putative GnRH Receptor Gene are Expressed in the Brain of Masu Salmon (Oncorhynchus masou)

Abstract Recent studies have shown that there are multiple genes encoding gonadotropin-releasing hormone receptor (GnRH-R) in single species. In salmonids, however, only a single gene has been identified in the rainbow trout. We therefore isolated partial cDNAs from the brain and the pituitary of masu salmon Oncorhynchus masou by reverse transcription-polymerase chain reaction and 5′-rapid amplification of cDNA ends, using primers corresponding to conserved transmembrane domains (TMs). Five different partial cDNAs were isolated from an individual and termed as msGnRH-R1, R2, R3, R4 and R5. They are divided into two groups, msGnRH-R1, R2, R3 and msGnRH-R4, R5. Two groups share 59-71% nucleotide sequence identities. Phylogenetic analysis showed that the former group is closely related to the goldfish GnRH-R GfA, and the latter to GfB. All five msGnRH-R genes were expressed in the brain and msGnRHR1, R3 and R5 were expressed in the pituitary. In addition, we found mRNA for msGnRH-R1 in the kidney and ovary, and R2 in the ovary, whereas msGnRH-R5 gene was widely expressed in the muscle, heart, kidney and testis. Differences in the expression of msGnRH-R genes between maturing and spawning fish were observed in the brain and pituitary, except for the constantly expressed msGnRH-R5. A splicing variant of msGnRH-R1 mRNA that is capable of generating a truncated GnRH-R that consists of 5TMs was also expressed in the brain, pituitary and kidney. These results indicate that five different types of putative GnRH-R gene are present and expressed in the brain of masu salmon.

Year-to-year differences in plasma levels of steroid hormones in pre-spawning chum salmon.

Changes in plasma levels of steroid hormones in pre-spawning chum salmon (Oncorhynchus keta) were examined for 6 years in association with sexual maturation. Fish were sampled along their homing pathway from the coastal sea to the spawning ground from 1995 to 2000. Plasma levels of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), and cortisol were determined by enzyme immunoassays. Sexual maturity was comprehensively estimated by gonadosomatic indices, histology of gonads, nuptial color, spermiation or ovulation ratio. Since the plasma levels of steroid hormones and sexual maturation differed from year to year, they were compared with year-to-year variation of sea surface temperature (SST) of coastal sea to study influence of oceanographic environment on these physiological data. The SST of the migratory route varied among the years, so that we classified the 6 years into cool, intermediate, and warm years. Concerning maturity, the males that returned to the natal hatchery in the warm years were sexually more advanced than those in the cool years. Furthermore, histological data suggested that final oocyte maturation occurred before arrival at the hatchery in one of the warm years, i.e., 1999, while it occurred at the hatchery in one of the intermediate years, i.e., 2000. In the males, T and 11KT levels increased significantly on midway of the homing route in the warm years, whereas they did not show any noticeable changes in the cool years. Furthermore, the levels of T and 11KT on midway of the homing route in the warm years, i.e., 1998 and 1999, were significantly higher than those in one of the cool years, i.e., 1995, in both sexes. In the females, the levels of E2 decreased during upstream migration. Conversely, those of DHP considerably elevated at spawning ground in all years examined. The levels of cortisol were different from year to year regardless of the SST. The present results showed that there were year-to-year differences in plasma levels of steroid hormones and maturity, and some of them may be influenced by the year-to-year variation of SST.

Retinotectal transmission in the optic tectum of rainbow trout

Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N‐methyl‐D‐aspartic acid (NMDA) receptors and the GluR2 subunit of (±)‐α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors. J. Comp. Neurol. 484:249–259, 2005.

Localization of mRNAs encoding α and β subunits of soluble guanylyl cyclase in the brain of rainbow trout: comparison with the distribution of neuronal nitric oxide synthase

Abstract Detailed distribution of mRNAs encoding α and β subunits of soluble guanylyl cyclase (sGC) was examined in the brain of rainbow trout by in situ hybridization. In addition, distribution of nitric oxide synthase (NOS) was mapped in adjacent parallel sections by neuronal NOS (nNOS) immunocytochemistry and NADPH-diaphorase (NADPHd) histochemistry. Following application of digoxigenin-labeled riboprobes for sGC α and β subunit mRNAs, we found comparatively intense hybridization signals in the telencephalon, preoptic area, thalamus, hypothalamus, pretectum and tegmentum. Both nNOS immunocytochemistry and NADPHd histochemistry showed extensive distribution of nitroxergic neurons in various brain areas, although various degrees of dissociation of nNOS immunoreactivity (ir) and NADPHd staining were detected. In comparison with sGC subunit mRNAs, nNOS signals were more widely distributed in many neurons, including parvocellular neurons in the preoptic area, nucleus anterior tuberis in the hypothalamus, periventricular neurons in the optic tectum, most of the rhombencephalic neurons and pituitary cells. However, wide overlaps of sGC mRNA-containing neurons and nNOS-positive neurons were observed in the olfactory bulb, telencephalon, preoptic area, thalamus, hypothalamus, pretectum, optic tectum, tegmentum and cerebellum. The widespread overlapping in sGC subunit mRNAs and nNOS distribution suggests a role for sGC in various neuronal functions, such as processing of olfactory and visual signals and neuroendocrine function, possibly via NO/cGMP signaling in the brain of rainbow trout.

Prepubertal increases in the levels of two salmon gonadotropin-releasing hormone mRNAs in the ventral telencephalon and preoptic area of masu salmon

Ontogenic changes in the expression levels of two salmon gonadotropin-releasing hormone genes (sGnRH-I and -II) were examined in the forebrain region including the ventral telencephalon and preoptic area of masu salmon by competitive reverse transcription-polymerase chain reaction (RT-PCR). Two genes showed similar expression patterns throughout the lifetime in both sexes, although the levels of sGnRH-II mRNA were about 20 times higher than those of sGnRH-I mRNA. In males, the levels of sGnRH mRNAs increased at the beginning of the second year and reached their maximum in the autumn. The levels decreased gradually until the autumn of the third year when fish sexually matured. In females, the levels reached their maximum in the first autumn and fluctuated considerably along with the seasons in the third year. These results suggest that, in the salmon brain, sGnRH genes are activated long before the sexual maturation under sexually different control mechanisms.

Multiple-site optical recording for characterization of functional synaptic organization of the optic tectum of rainbow trout

To map the functional synaptic organization over a wide area in the optic tectum, we directly monitored two‐dimensional propagation of postsynaptic depolarization evoked by firing of retinotectal afferents in optic tectum slices prepared from rainbow trout (Oncorhynchus mykiss), using a voltage‐sensitive dye and a photodiode array system. The postsynaptic responses to afferent stimulation first propagated in the stratum opticum and stratum fibrosum et griseum superficiale in an anterograde fashion in the afferents and then expanded vertically into the deep layers. This vertical propagation appeared to occur along a bundle‐like structure that corresponded well with a cluster of neurons whose somata are located in the stratum periventriculare. Pharmacological studies showed that these postsynaptic responses were mediated by ionotropic glutamate receptors. On the other hand, the optical signals appeared to consist of at least two components (a transient signal and a slow signal). The second transient signal summated with the first slow signal by paired stimulation, suggesting that the transient and slow signals originated from different cell types. Taken together, these results showed that the functional synaptic organization of the teleost optic tectum comprises of two depolarization‐signal propagating paths along a horizontal layer structure and a vertical bundle‐like structure and that these synaptic responses occur via glutamatergic transmission.