RF-amide related peptide-3 (RFRP-3): a novel neuroendocrine regulator of energy homeostasis, metabolism, and reproduction.

A hypothalamic neuropeptide, RF-amide related peptide-3 (RFRP-3), the mammalian ortholog of the avian gonadotropin-inhibitory hormone (GnIH) has inhibitory signals for reproductive axis via G-protein coupled receptor 147 in mammals. Moreover, RFRP-3 has orexigenic action but the mechanism involved in energy homeostasis and glucose metabolism is not yet known. Though, the RFRP-3 modulates orexigenic action in co-operation with other neuropeptides, which regulates metabolic cues in the hypothalamus. Administration of GnIH/RFRP-3 suppresses plasma luteinizing hormone, at the same time stimulates feeding behavior in birds and mammals. Likewise, in the metabolically deficient conditions, its expression is up-regulated suggests that RFRP-3 contributes to the integration of energy balance and reproduction. However, in many other metabolic conditions like induced diabetes and high-fat diet obesity, etc. its role is still not clear while, RFRP-3 induces the glucose homeostasis by adipocytes is reported. The physiological role of RFRP-3 in metabolic homeostasis and the metabolic effects of RFRP-3 signaling in pharmacological studies need a detailed discussion. Further studies are required to find out whether RFRP-3 is associated with restricted neuroendocrine function observed in type II diabetes mellitus, aging, or sub-fertility. In this context, the current review is focused on the role of RFRP-3 in the above-mentioned mechanisms. Studies from search engines including PubMed, Google Scholar, and science.gov are included after following set inclusion/exclusion criteria. As a developing field few mechanisms are still inconclusive, however, based on the available information RFRP-3 seems to be a putative tool in future treatment strategies towards metabolic disease.

Thyroid hormone receptors are required for the melatonin-dependent control of Rfrp gene expression in mice.

Mammals adapt to seasons using a neuroendocrine calendar defined by the photoperiodic change in the nighttime melatonin production. Under short photoperiod, melatonin inhibits the pars tuberalis production of TSHß, which, in turn, acts on tanycytes to regulate the deiodinase 2/3 balance resulting in a finely tuned seasonal control of the intra-hypothalamic thyroid hormone T3. Despite the pivotal role of this T3 signaling for synchronizing reproduction with the seasons, T3 cellular targets remain unknown. One candidate is a population of hypothalamic neurons expressing Rfrp, the gene encoding the RFRP-3 peptide, thought to be integral for modulating rodent's seasonal reproduction. Here we show that nighttime melatonin supplementation in the drinking water of melatonin-deficient C57BL/6J mice mimics photoperiodic variations in the expression of the genes Tshb, Dio2, Dio3, and Rfrp, as observed in melatonin-proficient mammals. Notably, we report that this melatonin regulation of Rfrp expression is no longer observed in mice carrying a global mutation of the T3 receptor, TRα, but is conserved in mice with a selective neuronal mutation of TRα. In line with this observation, we find that TRα is widely expressed in the tanycytes. Altogether, our data demonstrate that the melatonin-driven T3 signal regulates RFRP-3 neurons through non-neuronal, possibly tanycytic, TRα.

Molecular characterization of putative neuropeptide, amine, diffusible gas and small molecule transmitter biosynthetic enzymes in the eyestalk ganglia of the American lobster, Homarus americanus.

The American lobster, Homarus americanus, is a model for investigating the neuromodulatory control of physiology and behavior. Prior studies have shown that multiple classes of chemicals serve as locally released/circulating neuromodulators/neurotransmitters in this species. Interestingly, while many neuroactive compounds are known from Homarus, little work has focused on identifying/characterizing the enzymes responsible for their biosynthesis, despite the fact that these enzymes are key components for regulating neuromodulation/neurotransmission. Here, an eyestalk ganglia-specific transcriptome was mined for transcripts encoding enzymes involved in neuropeptide, amine, diffusible gas and small molecule transmitter biosynthesis. Using known Drosophila melanogaster proteins as templates, transcripts encoding putative Homarus homologs of peptide precursor processing (signal peptide peptidase, prohormone processing protease and carboxypeptidase) and immature peptide modifying (glutaminyl cyclase, tyrosylprotein sulfotransferase, protein disulfide isomerase, peptidylglycine-α-hydroxylating monooxygenase and peptidyl-α-hydroxyglycine-α-amidating lyase) enzymes were identified in the eyestalk assembly. Similarly, transcripts encoding full complements of the enzymes responsible for dopamine [tryptophan-phenylalanine hydroxylase (TPH), tyrosine hydroxylase and DOPA decarboxylase (DDC)], octopamine (TPH, tyrosine decarboxylase and tyramine ß-hydroxylase), serotonin (TPH or tryptophan hydroxylase and DDC) and histamine (histidine decarboxylase) biosynthesis were identified from the eyestalk ganglia, as were those responsible for the generation of the gases nitric oxide (nitric oxide synthase) and carbon monoxide (heme oxygenase), and the small molecule transmitters acetylcholine (choline acetyltransferase), glutamate (glutaminase) and GABA (glutamic acid decarboxylase). The presence and identity of the transcriptome-derived transcripts were confirmed using RT-PCR. The data presented here provide a foundation for future gene-based studies of neuromodulatory control at the level of neurotransmitter/modulator biosynthesis in Homarus.

Effects of feeding time on daily rhythms of neuropeptide and clock gene expression in the rat hypothalamus.

Shiftworkers are exposed to several adverse health conditions, one being eating at night. Food consumption at an unnatural time-of-day is thought to be one of the main factors responsible for the increased risk of developing metabolic diseases, such as obesity and diabetes mellitus. The underlying mechanism is considered to include disruption of the circadian organization of physiology, leading to disruption of metabolism. When food is consumed at night, the hypothalamus, a brain region central to homeostasis, receives contradicting input from the central clock and the systemic circulation. This study investigated how timing of feeding affects hypothalamic function by studying, in different hypothalamic nuclei, expression of clock genes and key neuropeptide genes involved in energy metabolism, including orexin, melanin-concentrating hormone (MCH) and neuropeptide Y. Animals with food available ad libitum showed diurnal variation in the expression of clock genes Per1 and Per2 in the perifornical area and arcuate nucleus. Clock gene rhythms were lost in both nuclei when food was restricted to the light (i.e., sleep) period. Neuropeptide genes did not display significant daily variation in either feeding groups, except for orexin-receptor 1 in ad libitum animals. Analysis of genes involved in glutamatergic and GABAergic signaling did not reveal diurnal variation in expression, nor effects of feeding time. In conclusion, feeding at the 'wrong' time-of-day not only induces desynchronization between brain and body clocks but also within the hypothalamus, which may contribute further to the underlying pathology of metabolic dysregulation.

Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain.

VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well as in the adult.

Suppression of neuropeptide production by quercetin in allergic rhinitis model rats.

BACKGROUND: Quercetin, a dietary flavonoid found in many fruits, red wine and onion, among others, has been reported to have potent anti-oxidant, anti-viral and anti-cancer effects. Although quercetin is also reported to have anti-inflammatory and anti-allergic effects, the precise mechanisms by which quercetin favorably modify the clinical conditions of allergic diseases such as allergic rhinitis (AR). The present study was designed to examine the influence of quercetin on the development of AR by using AR model rats. METHODS: Sprague-Dawley (SD) rats were sensitized with toluene 2,4-diisocyanate (TDI) by intranasal instillation of a 10 % TDI in ethyl acetate in a volume of 5 µl once a day for 5 consecutive days. This sensitization procedure was repeated after a 2-day interval. After 5 days of the second sensitization, rats were treated with various doses of quercetin once a day for 2 to 7 days. Nasal allergy-like symptoms, which were induced by bilateral application of 5 µl of 10 % TDI in ethyl acetate, were assessed by counting sneezing and nasal rubbing behaviors for 10 min just after TDI nasal challenge. The levels of substance P (SP), calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) in nasal lavage fluids obtained 6 h after TDI nasal challenge was examined by ELISA. RESULTS: Oral administration of quercetin for 5 and 7 days, but not 2 and 3 days, could inhibit sneezing and nasal rubbing movements, which were increased by TDI nasal challenge. The minimum dose that caused significant inhibition was 25 mg/kg. Oral administration of quercetin at more than 25 mg/kg for 5 days significantly inhibited the increase in SP, CGRP and NGF contents in nasal lavage fluids induced by TDI nasal challenge. CONCLUSION: The present results strongly suggested that quercetin will be a good candidate for the supplement on the management and treatment of allergic diseases, especially AR.

Reduced glutathione biosynthesis in Drosophila melanogaster causes neuronal defects linked to copper deficiency.

Glutathione (GSH) is a tripeptide often considered to be the master antioxidant in cells. GSH plays an integral role in cellular redox regulation and is also known to have a role in mammalian copper homeostasis. In vitro evidence suggests that GSH is involved in copper uptake, sequestration and efflux. This study was undertaken to further investigate the roles that GSH plays in neuronal copper homeostasis in vivo, using the model organism Drosophila melanogaster. RNA interference-mediated knockdown of the Glutamate-cysteine ligase catalytic subunit gene (Gclc) that encodes the rate-limiting enzyme in GSH biosynthesis was utilised to genetically deplete GSH levels. When Gclc was knocked down in all neurons, this caused lethality, which was partially rescued by copper supplementation and was exacerbated by additional knockdown of the copper uptake transporter Ctr1A, or over-expression of the copper efflux transporter ATP7. Furthermore, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. In these cells, Gclc suppression caused a decrease in axon branching, a phenotype further enhanced by ATP7 over-expression. Therefore, we conclude that GSH may play an important role in regulating neuronal copper levels and that reduction in GSH may lead to functional copper deficiency in neurons in vivo. We provide genetic evidence that glutathione (GSH) levels influence Cu content or distribution in vivo, in Drosophila neurons. GSH could be required for binding Cu imported by Ctr1A and distributing it to chaperones, such as Mtn, CCS and Atox1. Alternatively, GSH could modify the copper-binding and transport activities of Atox1 and the ATP7 efflux protein via glutathionylation of copper-binding cysteines.

Neuroleptics Affect Neuropeptide S and NPSR mRNA Levels in the Rat Brain.

Neuropeptide S (NPS) has a multidirectional regulatory activity, especially when considered as a potent endogenous anxiolytic factor. Accumulating data suggests that neuroleptics affect peptidergic signaling in various brain structures. However, there is no information regarding the influence of treatment with antipsychotics on brain NPS expression. In the current study, we assessed the NPS and NPS receptor (NPSR) mRNA levels in the brains of rats shortly and chronically treated with chlorpromazine and olanzapine using quantitative real-time PCR. Both single-dose and long-term (4 months) olanzapine treatment led to the upregulation of NPS expression in the rat hypothalamus. It supports the hypothesis that NPS is involved in the dopamine-dependent anxiolytic actions of selected neuroleptics and possibly also in the pathophysiology of mental disorders. On the other hand, NPSR expression decreased after single-dose and chronic chlorpromazine administration in the hypothalamus, as well as after chronic olanzapine and chlorpromazine administration in the striatum and hippocampus. These results cast a new light on the pharmacology of antipsychotics and contribute to a better understanding of the mechanisms responsible for their action. Furthermore, our findings underline the complex nature of potential interactions between dopamine receptors and brain peptidergic pathways, which has potential clinical applications.

Distinct functions of neuromedin u and neuromedin s in orange-spotted grouper.

Neuromedin U (NMU) and neuromedin S (NMS) play inhibitory roles in the regulation of food intake and energy homeostasis in mammals. However, their functions are not clearly established in teleost fish. In the present study, nmu and nms homologs were identified in several fish species. Subsequently, their cDNA sequences were cloned from the orange-spotted grouper (Epinephelus coioides). Sequence analysis showed that the orange-spotted grouper Nmu proprotein contains a 21-amino acid mature Nmu peptide (Nmu-21). The Nms proprotein lost the typical mature Nms peptide, but it retains a putative 34-amino acid peptide (Nmsrp). In situ hybridization revealed that nmu- and nms-expressing cells are mainly localized in the hypothalamic regions associated with appetite regulation. Food deprivation decreased the hypothalamic nmu mRNA levels but induced an increase of nms mRNA levels. Periprandial expression analysis showed that hypothalamic expression of nmu increased significantly at 3 h post-feeding, while nms expression was elevated at the normal feeding time. I.p. injection of synthetic Nmu-21 peptide suppressed the hypothalamic neuropeptide y (npy) expression, while Nmsrp administration significantly increased the expression of npy and orexin in orange-spotted grouper. Furthermore, the mRNA levels of LH beta subunit (lhß) and gh in the pituitary were significantly down-regulated after Nmu-21 peptide administration, while Nmsrp was able to significantly stimulate the expression of FSH beta subunit (fshß), prolactin (prl), and somatolaction (sl). Our results indicate that nmu and nms possess distinct neuroendocrine functions and pituitary functions in the orange spotted grouper.

Re-purposing of histological tissue sections for corroborative western blot analysis of hypothalamic metabolic neuropeptide expression following delineation of transactivated structures by Fos immuno-mapping.

Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow.

Antidepressant Effect of Crocus sativus Aqueous Extract and its Effect on CREB, BDNF, and VGF Transcript and Protein Levels in Rat Hippocampus.

Crocus sativus L., commonly known as saffron, is a perennial stemless herb in Iridaceae family. It has been used in traditional medicine as well as in modern pharmacological studies for variety of conditions including depression. Recent studies have suggested brain-derived neurotrophic factor (BDNF), VGF Neuropeptide, Cyclic-AMP Response Element Binding Protein (CREB) and phospho-CREB (p-CREB) may play roles in depression. In this research the molecular mechanism of antidepressant effect of aqueous extract of saffron and its effect on the levels of BDNF, VGF, CREB and p-CREB in rat hippocampus, were investigated. The aqueous extract of saffron (40, 80 and 160 mg/kg/day) and imipramine 10 mg/kg/day were injected intraperitoneally (i.p.) for 21 days to rats. The FST (forced swimming test) was performed on the days 1(st) and 21(st). The protein expression and transcript levels of BDNF, VGF CREB and phospho-CREB in rat hippocampus, were evaluated using western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results of FST showed that saffron reduced the immobility time. The protein levels of BDNF, CREB and p-CREB were significantly increased in saffron treated rats. VGF protein expression was also increased, but not significantly. The transcript levels of BDNF significantly increased. No significant changes in CREB and VGF transcript levels were observed. It was concluded that aqueous extract of saffron has antidepressant effects and the mechanism of its antidepressant effect may be due to increasing the levels of BDNF, VGF, CREB and P-CREB in rat hippocampus.

Hypothalamic Kiss1 and RFRP gene expressions are changed by a high dose of lipopolysaccharide in female rats.

Reproductive function is suppressed by several types of stress. Hypothalamic kisspeptin, which is a product of the Kiss1 gene, and GnIH/RFRP have pivotal roles in the regulation of GnRH and gonadotropins through their receptors Kiss1r and GPR147 in many species. However, alterations of these factors under stress conditions have not been fully evaluated. This study investigated the mechanisms of immune stress-induced reproductive dysfunction, especially focusing on the changes of Kiss1 and RFRP gene expression. Serum LH levels and hypothalamic Kiss1 and GnRH mRNA levels were decreased, while hypothalamic RFRP and GPR147 mRNA levels were increased by administration of a high dose of LPS (5mg/kg) in both ovariectomized and gonadal intact female rats. In this condition, Kiss1 and/or RFRP mRNA levels were positively and negatively correlated with GnRH expression, respectively. In contrast, hypothalamic Kiss1, RFRP, and GPR147 mRNA levels were not changed by administration of a moderate dose of LPS (500µg/kg) in ovariectomized rats. Rats with high-dose LPS injection showed more prolonged fever responses and severe anorexia compared with rats with moderate-dose LPS injection, indicating that more energy was used for the immune response in the former. These results suggest that the underlying mechanisms of dysfunction of gonadotropin secretion are changed according to the severity of immune stress, and that changes of some reserved factors, such as kisspeptin and RFRP, begin to participate in the suppression of GnRH and gonadotropin in severe conditions. As reproduction needs a large amount of energy, dysfunction of gonadotropin secretion under immune stress may be a biophylatic mechanism by which more energy is saved for the immune response.

Expression profile of hypothalamic neuropeptides in chicken lines selected for high or low residual feed intake.

The R(+) and R(-) chicken lines have been divergently selected for high (R(+)) or low (R(-)) residual feed intake. For the same body weight and egg production, the R(+) chickens consume 40% more food than their counterparts R(-) lines. In the present study we sought to determine the hypothalamic expression profile of feeding-related neuropeptides in these lines maintained under fed or food-deprived conditions. In the fed condition, the suppressor of cytokine signaling 3 (SOCS3) was 17-fold lower (P<0.05) and the ghrelin receptor was 7-fold higher (P<0.05) in R(+) compared to R(-) chicken lines. The hypothalamic expression of the other studied genes remained unchanged between the two lines. In the fasted state, orexigenic neuropeptide Y and agouti-related peptide were more responsive, with higher significant levels in the R(+) compared to R(-) chickens, while no significant differences were seen for the anorexigenic neuropeptides pro-opiomelanocortin and corticotropin releasing hormone. Interestingly, C-reactive protein, adiponectin receptor 1 and ghrelin receptor gene expression were significantly higher (12-, 2- and 3-folds, respectively), however ghrelin and melanocortin 5 receptor mRNA levels were lower (4- and 2-folds, P=0.05 and P=0.03, respectively) in R(+) compared to R(-) animals. We identified several key feeding-related genes that are differently expressed in the hypothalamus of R(+) and R(-) chickens and that might explain the difference in feed intake observed between the two lines.

Effects of sex and reproductive experience on the number of orexin A-immunoreactive cells in the prairie vole brain.

Large populations of cells synthesizing the neuropeptide orexin (OX) exist in the caudal hypothalamus of all species examined and are implicated in physiological and behavioral processes including arousal, stress, anxiety and depression, reproduction, and goal-directed behaviors. Hypothalamic OX expression is sexually dimorphic in different directions in laboratory rats (F>M) and mice (M>F), suggesting different roles in male and female physiology and behavior that are species-specific. We here examined if the number of hypothalamic cells immunoreactive for orexin A (OXA) differs between male and female prairie voles (Microtus ochrogaster), a socially monogamous species that pairbonds after mating and in which both sexes care for offspring, and if reproductive experience influences their number of OXA-immunoreactive (OXA-ir) cells. It was found that the total number of OXA-ir cells did not differ between the sexes, but females had more OXA-ir cells than males in anterior levels of the caudal hypothalamus, while males had more OXA-ir cells posteriorly. Sexually experienced females sacrificed 12 days after the birth of their first litter, or one day after birth of a second litter, had more OXA-ir cells in anterior levels but not posterior levels of the caudal hypothalamus compared to females housed with a brother (incest avoidance prevents sibling mating). Male prairie voles showed no effect of reproductive experience but showed an unexpected effect of cohabitation duration regardless of mating. The sex difference in the distribution of OXA-ir cells, and their increased number in anterior levels of the caudal hypothalamus of reproductively experienced female prairie voles, may reflect a sex-specific mechanism involved in pairbonding, parenting, or lactation in this species.

Molecular evolution of GPCRs: 26Rfa/GPR103.

Neuropeptides possessing the Arg-Phe-NH2 (RFamide) motif at their C-termini (designated as RFamide peptides) have been characterized in a variety of animals. Among these, neuropeptide 26RFa (also termed QRFP) is the latest member of the RFamide peptide family to be discovered in the hypothalamus of vertebrates. The neuropeptide 26RFa/QRFP is a 26-amino acid residue peptide that was originally identified in the frog brain. It has been shown to exert orexigenic activity in mammals and to be a ligand for the previously identified orphan G protein-coupled receptor, GPR103 (QRFPR). The cDNAs encoding 26RFa/QRFP and QRFPR have now been characterized in representative species of mammals, birds, and fish. Functional studies have shown that, in mammals, the 26RFa/QRFP-QRFPR system may regulate various functions, including food intake, energy homeostasis, bone formation, pituitary hormone secretion, steroidogenesis, nociceptive transmission, and blood pressure. Several biological actions have also been reported in birds and fish. This review summarizes the current state of identification, localization, and understanding of the functions of 26RFaQRFP and its cognate receptor, QRFPR, in vertebrates.

DCX-expressing cells in the vicinity of the hypothalamic neurogenic niche: a comparative study between mouse, sheep, and human tissues.

Neural stem and precursor cells persist postnatally throughout adulthood and are capable of responding to numerous endogenous and exogenous signals by modifying their proliferation and differentiation. Whereas adult neurogenesis has been extensively studied in the dentate gyrus of the hippocampal formation and in the subventricular zone adjacent to the wall of the lateral ventricles, we and others have recently reported constitutive adult neurogenesis in other brain structures, including the hypothalamus. In this study, we used immunohistochemistry to study the expression of the neuroblast marker doublecortin (DCX), and compared its expression pattern in adult ovine, mouse, and human hypothalamic tissues. Our results indicate that DCX-positive cells resembling immature and developing neurons occur in a wide range of hypothalamic nuclei in all three species, although with different distribution patterns. In addition, the morphology of DCX-positive cells varied depending on their location. DCX-positive cells near the third ventricle had the morphology of very immature neuroblasts, a round shape with no processes, whereas those located deeper in the parenchyma such as in the ventromedial nucleus were fusiform and showed a bipolar morphology. Extending this observation, we showed that among the cohort of immature neurons entering the ventromedial nucleus, some appeared to undergo maturation, as revealed by the partial colocalization of DCX with markers of more mature neurons, e.g., human neuronal protein C and D (HuC/D). This study provides further confirmation of the existence of an adult hypothalamic neurogenic niche and argues for the potential existence of a migratory path within the hypothalamus.

Orexin and neuropeptide Y: tissue specific expression and immunoreactivity in the hypothalamus and preoptic area of the cichlid fish Cichlasoma dimerus.

Neuropeptide Y (NPY) and orexin are neuropeptides involved in the regulation of feeding in vertebrates. In this study we determined the NPY and orexin mRNA tissue expression and their immunoreactivity distribution in both preoptic area and hypothalamus, regions involved in the regulation of feeding behavior. Both peptides presented a wide expression in all tissues examined. The NPY-immunoreactive (ir) cells were localized in the ventral nucleus posterioris periventricularis (NPPv) and numerous ir-NPY fibers were found in the nucleus lateralis tuberis (NLT), the nucleus recess lateralis (NRL) and the neurohypophysis. Ir-orexin cells were observed in the NPPv, dorsal NLT, ventral NLT, lateral NLT (NLTl) and the lateral NRL. Ir-orexin fibers were widespread distributed along all the hypothalamus, especially in the NLTl. Additionally, we observed the presence of ir-orexin immunostaining in adenohypophyseal cells, especially in somatotroph cells and the presence of a few ir-orexin-A fibers in the neurohypophysis. In conclusion, both peptides have an ubiquitous mRNA tissue expression and are similarly distributed in the hypothalamus and preoptic area of Cichlasoma dimerus. The presence of ir-orexin in adenohypohyseal cells and the presence of ir-orexin and NPY fibers in the neurohypophysis suggest that both peptides may play an important neuroendocrine role in anterior pituitary.

Orexin gene therapy restores the timing and maintenance of wakefulness in narcoleptic mice.

STUDY OBJECTIVES: Narcolepsy is caused by selective loss of the orexin/hypocretin-producing neurons of the hypothalamus. For patients with narcolepsy, chronic sleepiness is often the most disabling symptom, but current therapies rarely normalize alertness and do not address the underlying orexin deficiency. We hypothesized that the sleepiness of narcolepsy would substantially improve if orexin signaling were restored in specific brain regions at appropriate times of day. DESIGN: We used gene therapy to restore orexin signaling in a mouse model of narcolepsy. In these Atx mice, expression of a toxic protein (ataxin-3) selectively kills the orexin neurons. INTERVENTIONS: To induce ectopic expression of the orexin neuropeptides, we microinjected an adeno-associated viral vector coding for prepro-orexin plus a red fluorescence protein (AAV-orexin) into the mediobasal hypothalamus of Atx and wild-type mice. Control mice received an AAV coding only for red fluorescence protein. Two weeks later, we recorded sleep/wake behavior, locomotor activity, and body temperature and examined the patterns of orexin expression. MEASUREMENTS AND RESULTS: Atx mice rescued with AAV-orexin produced long bouts of wakefulness and had a normal diurnal pattern of arousal, with the longest bouts of wake and the highest amounts of locomotor activity in the first hours of the night. In addition, AAV-orexin improved the timing of rapid eye movement sleep and the consolidation of nonrapid eye movement sleep in Atx mice. CONCLUSIONS: These substantial improvements in sleepiness and other symptoms of narcolepsy demonstrate the effectiveness of orexin gene therapy in a mouse model of narcolepsy. Additional work is needed to optimize this approach, but in time, AAV-orexin could become a useful therapeutic option for patients with narcolepsy.

Expression of orexin A and B in the porcine hypothalamus during the oestrous cycle.

Orexins A and B are hypothalamic peptides engaged in a variety of physiological functions related to the control of energy homeostasis, sleep and wakefulness. The presence of orexin receptors in the tissues of the hypothalamus-pituitary-gonadal axis indicates that these hormones are also involved in the control of the reproductive system. The aim of this study was to compare the expression levels of prepro-orexin (a precursor of orexins A and B) mRNA in the porcine hypothalamic structures involved in reproductive processes - mediobasal hypothalamus (MBH), preoptic area (POA) and stalk median eminence (SME), during four stages (days 2-3, 10-12, 14-16, 17-19) of the oestrous cycle. In MBH, lower concentrations of PPO mRNA were observed on days 2-3 than in the remaining stages. In POA, the highest mRNA expression of PPO was noted on days 17-19. In SME, the highest concentrations of PPO was observed on days 2-3, and the lowest on days 14-16. We also investigated the intensity of OXA and OXB immunoreactivity and detected both peptides in all examined structures. In MBH, signal intensity for OXA was highest on days 14-16 and lowest on days 17-19. The highest levels of immunoreactivity were noted on days 2-3 and 10-12 in POA, and in SME additionally on days 17-19. OXB immunoreactivity in hypothalamic tissues also changed during the cycle, and the highest signal intensity was reported on days 10-12 in MBH, on days 14-16 in POA, and on days 14-16 and 17-19 in SME. The results of our study indicate that orexins A and B are produced in the porcine hypothalamus and that their concentrations vary subject to the pig's hormonal status. Our findings also suggest that orexins may affect reproductive functions at the highest level of the hypothalamus-pituitary-gonadal axis.

Hypoglycemia differentially regulates hypothalamic glucoregulatory neurotransmitter gene and protein expression: role of caudal dorsomedial hindbrain catecholaminergic input.

The hypothalamic neurochemicals neuropeptide Y (NPY), orexin-A (ORX), and oxytocin (OXY) exert glucoregulatory effects upon intracerebral administration, findings that support their potential function within neural pathways that maintain glucostasis. Current understanding of how these neurotransmitter systems respond to the diabetes mellitus complication, insulin-induced hypoglycemia, is limited to knowledge of neuropeptide gene transcriptional reactivity. We investigated the hypothesis that hypoglycemia elicits hypothalamic site-specific alterations in levels of these neurochemicals, and that adjustments in local neurotransmitter availability may be regulated by catecholaminergic (CA) input from the caudal dorsomedial hindbrain. The arcuate (ARH) and paraventricular (PVH) hypothalamic nuclei and lateral hypothalamic area (LHA) were each microdissected from adult male rats pretreated by caudal fourth ventricular administration of the selective CA neurotoxin, 6-hydroxydopamine (6-OHDA), or vehicle prior to insulin (INS)-induced hypoglycemia. Hypoglycemia stimulated ARH NPY gene expression and NPY accumulation in the ARH and LHA, but not PVH. 6-OHDA pretreatment did not modify the positive NPY mRNA response to INS, but blunted hypoglycemic augmentation of ARH and LHA NPY content while increasing PVH NPY levels in response to hypoglycemia. INS-treated rats exhibited diminished LHA ORX gene expression and increased [ARH; LHA] or decreased [PVH] tissue ORX protein levels. 6-OHDA+INS animals showed a comparable decline in ORX transcripts, but attenuated augmentation of ARH and LHA ORX content and elevated PVH ORX levels. OT mRNA and protein were respectively decreased or unchanged during hypoglycemia, responses that were uninfluenced by hindbrain CA nerve cell destruction. These results illustrate divergent adjustments in glucoregulatory neurotransmitter gene expression and site-specific protein accumulation in the hypothalamus during hypoglycemia. Evidence that 6-OHDA pretreatment does not modify NPY or ORX transcriptional reactivity to hypoglycemia, but alters hypoglycemic patterns of NPY and ORX accretion implicates dorsomedial hindbrain CA neurons in regulation of translation/post-translational processing and site-specific availability of these neurotransmitters in the hypothalamus during hypoglycemia.