Recently, a novel physiologically active peptide, kisspeptin (metastin), has been reported to facilitate sexual maturation and ovulation by directly stimulating GnRH neurons in several mammalian species. Despite its importance in the neuroendocrine regulation of reproduction, kisspeptin neurons have only been studied in mammals, and there has been no report on the kisspeptin or kisspeptin neuronal systems in nonmammalian vertebrates. We used medaka for the initial identification of the KiSS-1 gene and the anatomical distribution of KiSS-1 mRNA expressing neurons (KiSS-1 neurons) in the brain of nonmammalian species. In situ hybridization for the medaka KiSS-1 gene cloned here proved that two kisspeptin neuronal populations are localized in the hypothalamic nuclei, the nucleus posterioris periventricularis and the nucleus ventral tuberis (NVT). Furthermore, NVT KiSS-1 neurons were sexually dimorphic in number (male neurons >> female neurons) under the breeding conditions. We also found that the number of KiSS-1 neurons in the NVT but not that in the nucleus posterioris periventricularis was positively regulated by ovarian estrogens. The fact that there were clear differences in the number of NVT KiSS-1 neurons between the fish under the breeding and nonbreeding conditions strongly suggests that the steroid-sensitive changes in the KiSS-1 mRNA expression in the NVT occur physiologically, according to the changes in the reproductive state. From the present results, we conclude that the medaka KiSS-1 neuronal system is involved in the central regulation of reproductive functions, and, given many experimental advantages, the medaka brain may serve as a good model system to study its physiology.

We have recently developed sensitive and specific radioimmunoassays (RIAs) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II) and have measured the contents of both GnRHs in the rainbow trout brain. Our results showed that contents of the two GnRHs are variable among different brain regions. Therefore, in order to confirm the differential distribution of the two GnRHs by a different technique, we examined the distribution of immunoreactive sGnRH and cGnRH-II in the brain of masu salmon by using immunocytochemical techniques. sGnRH immunoreactive (ir) cell bodies were scattered in the transitional areas between the olfactory nerve and the olfactory bulb, the ventral olfactory bulb, between the olfactory bulb and the telencephalon, the ventral telencephalon, and the preoptic area. These sGnRH-ir cell bodies were dispersed in a strip-like region running rostrocaudally in the most ventral part of the ventral telencephalon. sGnRH-ir fibers were distributed in the various brain regions from the olfactory bulb to the spinal cord. They were especially abundant in the olfactory bulb, ventral telencephalon, preoptic area, hypothalamus, deep layers of the optic tectum, and thalamus. sGnRH-ir fibers also innervated the pituitary directly. cGnRH-II-ir cell bodies were found in the nucleus of the medial longitudinal fasciculus (nMLF). The distribution of cGnRH-II-ir fibers was similar to that of sGnRH-ir fibers, except that cGnRH-II-ir fibers were absent in the pituitary. The number of cGnRH-II-ir fibers was much fewer than that of sGnRH-ir fibers. The results of the present immunocytochemical study are in basic agreement with those of our previous RIA study. Thus, we suggest that in masu salmon, sGnRH not only regulates gonadotropin (GTH) release from the pituitary but also functions as a neuromodulator in the brain, whereas cGnRH-II functions only as a neuromodulator.

Modulation of ionic channel properties and synaptic functions by neurotransmitters and hormones is called neuro-modulation and may be the basis for many long-lasting changes in animal behavior, for example, changes in the arousal or motivational states. We have previously shown in a teleost, the dwarf gourami, that the terminal nerve (TN) cells are a major component of the gonadotropin-releasing hormone (GnRH) system and are structurally independent from the preoptic/hypophysial-GnRH system, which projects to the pituitary and facilitates gonadotropin release from the gonadotropes. While GnRH fibers originating from TN cells are distributed widely throughout the brain, they do not project to the pituitary. Thus, TN-GnRH system does not function as a hypophysiotropic hormone but probably as a neuro-modulator, capable of affecting widespread regions of the brain. In the present study, we used a whole-brain in vitro preparation to examine the spontaneous electrical activities of TN-GnRH cells and to determine the morphology of individual cells by intracellular injections of either neurobiotin or biocytin. The recorded cells were clearly identified as GnRH-immunoreactive TN cells by using combined intracellular injection and GnRH immunocytochemistry. Most of the TN cells showed endogenous slow (1-7 Hz), regular beating discharges. The intrinsic nature of this activity was demonstrated by the voltage dependency of the beating frequency, rhythm resetting, and persistence of rhythmicity after synaptic isolation. Only a small number of TN cells showed either irregular or bursting discharge patterns. Anatomical observation of intracellularly labeled cells showed that, regardless of discharge patterns, all the TN cells had multiple axonal branches that project to those areas where we had previously demonstrated dense GnRH-immunoreactive fibers. From the present results, we propose a hypothesis that may be relevant to the peptidergic and monoaminergic neuromodulatory systems in general. The modulator neurons have endogenous rhythmic activities that vary according to the animal's hormonal or environmental conditions, and they regulate the excitability of target neurons in a wide variety of brain regions simultaneously via multiple axonal branches.

Kisspeptin, a peptide encoded by the Kiss1 gene, has been considered as a potential candidate for a factor triggering the onset of puberty, and its expression in the hypothalamus was found to increase during peripubertal period in rodent models. The present study aimed to clarify the oestrogenic regulation of peripubertal changes in Kiss1 mRNA expression in the anteroventral periventricular nucleus (AVPV) and hypothalamic arcuate nucleus (ARC), and to determine which population of kisspeptin neurones shows a change in kisspeptin expression parallel to that in luteinising hormone (LH) pulses at the peripubertal period. Quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry revealed an apparent increase in the ARC Kiss1 mRNA expression and kisspeptin immunoreactivity around the time of vaginal opening in intact female rats. The AVPV Kiss1 mRNA levels also increased at day 26, but decreased at day 31, and then increased at day 36/41. In ovariectomised (OVX) rats, ARC Kiss1 mRNA expression did not show peripubertal changes and was kept at a high level throughout peripubertal periods. Apparent LH pulses were found in these prepubertal OVX rats. Oestradiol replacement suppressed ARC Kiss1 mRNA expression in OVX prepubertal rats, but not in adults. Similarly, LH pulses were suppressed by oestradiol in the prepubertal period (days 21 and 26), but regular pulses were found in adulthood. The present study suggests that a pubertal increase of Kiss1/kisspeptin expression both in the ARC and AVPV is involved in the onset of puberty. These results also suggest that both LH pulses and ARC Kiss1 expression are more negatively regulated by oestrogen in prepubertal female rats compared to adult rats.

The present study characterizes gonadotropin-releasing hormone (GnRH) neuronal groups that are located in several different brain regions by investigating GnRH molecular species and projection patterns in an anabantid fish, Colisa lalia. First, we examined the molecular species of GnRHs in extracts of the brain and the pituitary by reverse-phase high-performance liquid chromatography followed by radioimmunoassays. We found salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and an unfamiliar GnRH-like substance. Next, to examine the distribution of each GnRH molecule in different GnRH neuronal groups, we performed immunohistochemistry using four kinds of antisera and an antibody. Furthermore, we performed brain lesioning experiments of terminal nerve (TN) cells, the most conspicuous GnRH-immunoreactive cells in Colisa lalia. Comparisons of immunoreactive structures between TN-lesioned fish and untreated fish elucidated the projection area of each neuronal group. Three major neuronal groups were observed. TN-GnRH cells, which are located in the transitional area between the olfactory bulb and the telencephalon, showed strong sGnRH and weaker cGnRH-II immunoreactivity. TN-GnRH cells projected to wide areas of the central nervous system from the olfactory bulb to the spinal cord. The second group, located in the preoptic area, showed only sGnRH immunoreactivity and projected only to the pituitary. The third one, located in the midbrain tegmentum, exhibited strong cGnRH-II and weaker sGnRH immunoreactivity. This cell group projected mainly to brain regions posterior to the hypothalamus and the spinal cord. These different projection patterns suggest functional differentiation of each GnRH neuronal group.