Gonadotropin-inhibitory hormone (GnIH) neurons project to GnRH neurons to negatively regulate reproductive function. To fully explore the projections of the GnIH neurons, we created transgenic rats carrying an enhanced green fluorescent protein (EGFP) tagged to the GnIH promoter. With these animals, we show that EGFP-GnIH neurons are localized mainly in the dorsomedial hypothalamic nucleus (DMN) and project to the hypothalamus, telencephalon, and diencephalic thalamus, which parallels and confirms immunocytochemical and gene expression studies. We observed an age-related reduction in c-Fos-positive GnIH cell numbers in female rats. Furthermore, GnIH fiber appositions to GnRH neurons in the preoptic area were lessened in middle-aged females (70 weeks old) compared with their younger counterparts (9-12 weeks old). The fiber density in other brain areas was also reduced in middle-aged female rats. The expression of estrogen and progesterone receptors mRNA in subsets of EGFP-GnIH neurons was shown in laser-dissected single EGFP-GnIH neurons. We then examined estradiol-17β and progesterone regulation of GnIH neurons, using c-Fos presence as a marker. Estradiol-17β treatment reduced c-Fos labeling in EGFP-GnIH neurons in the DMN of young ovariectomized adult females but had no effect in middle-aged females. Progesterone had no effect on the number of GnIH cells positive for c-Fos. We conclude that there is an age-related decline in GnIH neuron number and GnIH inputs to GnRH neurons. We also conclude that the response of GnIH neurons to estrogen diminishes with reproductive aging.
In order to understand the biological processes underlying dopaminergic neurons (DpN) regeneration in a 6-hydroxydopamine(6-OHDA)-induced adult zebrafish-based Parkinson's disease model, this study investigated the specific phases of neuroregeneration in a time-based manner. Bromodeoxyuridine (BrdU) was administered 24 h before the harvest of brain tissues at day three, five, seven, nine, 12 and 14 postlesion. Potential migration of proliferative cells was tracked over 14 days postlesion through double-pulse tracking [BrdU and 5-ethynyl-2'-deoxyuridine (EdU)] of cells and immunohistostaining of astrocytes [glial fibrillary acidic protein (GFAP)]. Gene expression of foxa2 and nurr1 (nr4a2a) at day three, nine, 14, 18, 22 and 30 postlesion was quantified using qPCR. Protein expression of foxa2 at day three, seven, 14 and 22 postlesion was validated using the western blot technique. Double labelling [EdU and tyrosine hydroxylase (TH)] of proliferative cells was performed to ascertain their fate after the neuroregeneration processes. It was found that whilst cell proliferation remained unchanged in the area of substantial DpN loss, the ventral diencephalon (vDn), there was a transient increase of cell proliferation in the olfactory bulb (OB) and telencephalon (Tel) seven days postlesion. BrdU-immunoreactive (ir)/ EdU-ir cells and activated astrocytes were later found to be significantly increased in the vDn and its nearby area (Tel) 14 days postlesion. There was a significant but transient downregulation of foxa2 at day three and nine postlesion, and nr4a2a at day three, nine and 14 postlesion. The expression of both genes remained unchanged in the OB and Tel. There was a transient downregulation of foxa2 protein expression at day three and seven postlesion. The significant increase of EdU-ir/ TH-ir cells in the vDn 30 days postlesion indicates maturation of proliferative cells (formed between day five-seven postlesion) into DpN. The present findings warrant future investigation of critical factors that govern the distinctive phases of DpN regeneration.
The habenula is a phylogenetically conserved epithalamic structure, which conveys negative information via inhibition of mesolimbic dopamine neurons. We have previously shown the expression of kisspeptin (Kiss1) in the habenula and its role in the modulation of fear responses in the zebrafish. In this study, to investigate whether habenular Kiss1 regulates fear responses via dopamine neurons in the zebrafish, Kiss1 peptides were intracranially administered close to the habenula, and the expression of dopamine-related genes (th1, th2 and dat) were examined in the brain using real-time PCR and dopamine levels using LC-MS/MS. th1 mRNA levels and dopamine levels were significantly increased in the telencephalon 24-h and 30-min after Kiss1 administration, respectively. In fish administered with Kiss1, expression of neural activity marker gene, npas4a and kiss1 gene were significantly decreased in the ventral habenula. Application of neural tracer into the median raphe, site of habenular Kiss1 neural terminal projections showed tracer-labelled projections in the medial forebrain bundle towards the telencephalon where dopamine neurons reside. These results suggest that Kiss1 negatively regulates its own neuronal activity in the ventral habenula via autocrine action. This, in turn affects neurons of the median raphe via interneurons, which project to the telencephalic dopaminergic neurons.
Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3'-, 5'- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.
Glial cell line-derived neurotrophic factor (GDNF) was initially described as important for dopaminergic neuronal survival and is involved in many other essential functions in the central nervous system. Characterization of GDNF phenotype in mammals is well described; however, studies in non-mammalian vertebrate models are scarce. Here, we characterized the anatomical distribution of gdnf-expressing cells in adult zebrafish brain by means of combined in situ hybridization (ISH) and immunohistochemistry. Our results revealed that gdnf was widely dispersed in the brain. gdnf transcripts were co-localized with radial glial cells along the ventricular area of the telencephalon and in the hypothalamus. Interestingly, Sox2 positive cells expressed gdnf in the neuronal layer but not in the ventricular zone of the telencephalon. A subset of GABAergic precursor cells labeled with dlx6a-1.4kbdlx5a/6a: green fluorescence protein (GFP) in the pallium, parvocellular preoptic nucleus, and the anterior and dorsal zones of the periventricular hypothalamus also showed expression with gdnf mRNA. In addition, gdnf signals were detected in subsets of dopaminergic neurons, including those in the ventral diencephalon, similar to what is seen in mammalian brain. Our work extends our knowledge of gdnf action sites and suggests a potential role for gdnf in adult brain neurogenesis and regeneration.