The increase in the number of amylin neurons in the medial preoptic area throughout the lactational period and its relationship with melanin-concentrating hormone.

The amylin and the melanin-concentrating hormone [MCH] are two peptides related to energetic homeostasis. During lactation, it is possible to locate neurons expressing these peptides in the preoptic area of rat dams. In addition, it was demonstrated that the number of MCH neurons in this region is modulated by litter size. Taken together, the aims of this work were (1) to verify the time course of amylin immunoreactivity during lactation; (2) to verify whether litter size modulates the number of amylin-ir neurons (3) to verify whether there is colocalization between the amylin-ir and MCH-ir neurons. Our results show that (1) there is an increase in the number of amylin-ir neurons during lactation, which reaches a peak at postpartum day 19 and drastically reduces after weaning; (2) there is no correlation between litter size and the number of amylin-ir neurons; and (3) there is minimal overlap between amylin-ir and MCH-ir neurons.

Melanin-concentrating hormone regulation by estradiol and progesterone in the incerto-hypothalamic area.

Melanin-concentrating hormone (MCH) is a peptide related to the reproductive function by interacting with the hypothalamus-pituitary-gonadal axis. In addition to the MCH central production, it is also found in the blood with a putative role as a neurohormone. Thereby, our focus is on steroid hormones' role in regulating centrally produced MCH in the incerto-hypothalamic area (IHy) and the peripheral MCH in the serum. For this, we investigated the effect of estradiol and/or progesterone injection on the number of MCH immunoreactive (MCH-ir) neurons at the IHy and serum levels. For further study of the role of progesterone, we analyzed the effect of blockade of progesterone receptors by its antagonist on MCH-ir neurons at the IHy and serum. To identify whether such regulation over MCH is established before sexual maturation, we assessed the effect of peripubertal removal of steroid hormones on MCH-ir neurons at the IHy and serum levels at adult age. Our results show that injecting estradiol in ovariectomized female rats reduces the number of MCH-ir neurons in the IHy, in addition to its serum levels. Blockade of progesterone receptors in intact females increases the number of MCH-ir neurons in the IHy and its serum concentration. The regulation of these hormones over the MCH peptidergic system is established before sexual maturation, once the peripubertal removal of the ovaries changes the serum levels of MCH and the number of MCH-ir neurons in the IHy of adult females. Such results support the inhibitory role of steroid hormones over the MCH system.

The effect of estrogen and progesterone on melanin-concentrating hormone producing-neurons in brain areas related to reproductive behavior in lactating dams.

The hypothalamus plays a role in reproductive cycle control, and it is a site of action of steroid hormones. Throughout the production of melanin-concentrating hormone (MCH), the hypothalamus shows adaptive changes during lactation. Therefore, in this work, we aimed to test the effects of estrogen and progesterone manipulation on MCH-immunoreactive (ir) neurons in hypothalamic brain areas related to reproductive behavior and on the MCH serum concentration. Our results show that the removal of steroid hormones by ovariectomy increases the number of MCH-ir neurons in the medial preoptic area (MPOA) and incerto-hypothalamic area (IHy) but not in the anterior part of the paraventricular nucleus of the hypothalamus (PVHa). The MCH in the serum levels also increases. In accordance, the injection of estradiol alone or estradiol and progesterone decreased the number of MCH-ir neurons in the MPOA and IHy, as well as its serum levels. The MPOA and IHy are the brain areas targeted by the steroid hormone inhibitory effect of the MCH system during lactation. This effect is also reflected in the MCH serum levels.

Melanin-concentrating hormone regulates the hypercapnic chemoreflex by acting in the lateral hypothalamic area.

NEW FINDINGS: What is the central question of this study? Melanin-concentrating hormone (MCH) suppresses the hypercapnic chemoreflex: what is the mechanism by which this effect is produced? What is the main finding and its importance? MCH acting in the lateral hypothalamic area but not in the locus coeruleus in rats, in the light period, attenuates the hypercapnic chemoreflex. The data provide new insight into the role of MCH in the modulation of the hypercapnic ventilatory response. ABSTRACT: Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide involved in a broad range of homeostatic functions including regulation of the hypercapnic chemoreflex. We evaluated whether MCH modulates the hypercapnic ventilatory response by acting in the lateral hypothalamic area (LHA) and/or in the locus coeruleus (LC). Here, we measured pulmonary ventilation ( V ̇ E ${\dot V_{\rm{E}}}$ ), body temperature, electroencephalogram (EEG) and electromyogram (EMG) of unanaesthetized adult male Wistar rats before and after microinjection of MCH (0.4 mM) or MCH receptor 1 (MCH1-R) antagonist (SNAP-94847; 63 mM) into the LHA and LC, in room air and 7% CO2 conditions during wakefulness and sleep in the dark and light periods. MCH intra-LHA caused a decreased CO2 ventilatory response during wakefulness and sleep in the light period, while SNAP-94847 intra-LHA increased this response, during wakefulness in the light period. In the LC, MCH or the MCH1-R antagonist caused no change in the hypercapnic ventilatory response. Our results suggest that MCH, in the LHA, exerts an inhibitory modulation of the hypercapnic ventilatory response during the light-inactive period in rats.

In vivo uptake of a fluorescent conjugate of melanin-concentrating hormone in the rat brain.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide synthesized by posterior hypothalamic and incerto-hypothalamic neurons that project throughout the central nervous system. The MCHergic system modulates several important functions such as feeding behavior, mood and sleep. MCH exerts its biological functions through interaction with the MCHR-1 receptor, the only functional receptor present in rodents. The internalization process of MCHR-1 triggered by MCH binding was described in vitro in non-neuronal heterologous systems with over-expression of MCHR-1. Reports of in vivo MCHR-1 internalization dynamics are scarce, however, this is an important process to explore based on the critical functions of the MCHergic system. We had previously determined that 60 min after intracerebroventricular (i.c.v.) microinjections of MCH conjugated with fluorophore rhodamine (R-MCH), the dorsal and median raphe nucleus presented R-MCH positive labeled neurons. In the present work, we further studied the in vivo uptake process focusing on the distribution and time-dependent pattern of R-MCH positive cells 10, 20 and 60 min (T10, T20 and T60, respectively) after i.c.v. microinjection of R-MCH. We also explored this uptake process to see whether it was receptor- and clathrin-dependent and examined the phenotype of R-MCH positive cells and their proximity to MCHergic fibers. We found a great number of R-MCH positive cells with high fluorescence intensity in the lateral septum, nucleus accumbens and hippocampus at T20 and T60 (but not at T10), while a lower number with low intensity was observed in the dorsal raphe nucleus. At T20, in rats pre-treated with a MCHR-1 antagonist (ATC-0175) or with phenylarsine oxide (PAO), a clathrin endocytosis inhibitor, a robust decrease (> 50 %) of R-MCH uptake occurred in these structures. The R-MCH positive cells were identified as neurons (NeuN positive, GFAP negative) and some MCHergic fibers run in the vicinities of them. We concluded that neurons localized at structures that were close to the ventricular surfaces could uptake R-MCH in vivo through a receptor-dependent and clathrin-mediated process. Our results support volume transmission of MCH through the cerebrospinal fluid to reach distant targets. Finally, we propose that R-MCH would be an effective tool to study MCH-uptake in vivo.

Gonadotropin-inhibitory hormone (GnIH) distribution in the brain of the ancient fish Atractosteus tropicus (Holostei, Lepisosteiformes).

The Holostei group occupies a critical phylogenetic position as the sister group of the Teleostei. However, little is known about holostean pituitary anatomy or brain distribution of important reproductive neuropeptides, such as the gonadotropin-inhibitory hormone (GnIH). Thus, the present study set out to characterize the structure of the pituitary and to localize GnIH-immunoreactive cells in the brain of Atractosteus tropicus from the viewpoint of comparative neuroanatomy. Juveniles of both sexes were processed for general histology and immunohistochemistry. Based on the differences in cell organization, morphology, and staining properties, the neurohypophysis and three regions in the adenohypophysis were identified: the rostral and proximal pars distalis (PPD) and the pars intermedia. This last region was found to be innervated by the neurohypophysis. This organization, together with the presence of a saccus vasculosus, resembles the general teleost pituitary organization. A vast number of blood vessels were also recognized between the infundibulum floor of the hypothalamus and the PPD, evidencing the characteristic presence of a median eminence and a portal system. However, this well-developed pituitary portal system resembles that of tetrapods. As regards the immunohistochemical localization of GnIH, we found four GnIH-immunoreactive (GnIH-ir) populations in three hypothalamic nuclei (suprachiasmatic, retrotuberal, and tuberal nuclei) and one in the diencephalon (prethalamic nucleus), as well as a few scattered neurons throughout the olfactory bulbs, the telencephalon, and the intersection between them. GnIH-ir fibers showed a widespread distribution over almost all brain regions, suggesting that GnIH function is not restricted to reproduction only. In conclusion, the present study describes, for the first time, the pituitary of A. tropicus and the neuroanatomical localization of GnIH in a holostean fish that exhibits a similar distribution pattern to that of teleosts and other vertebrates, suggesting a high degree of phylogenetic conservation of this system.

Regulation and neurochemical identity of melanin-concentrating hormone neurones in the preoptic area of lactating mice.

Neurones expressing the melanin-concentrating hormone (MCH) can be found in the medial preoptic area (mPOA) and ventral aspects of the periventricular preoptic nucleus of rats by mid-to-late lactation and this expression disappears after weaning. The transitory expression of MCH in the preoptic area suggests a role for these neurones in the control of the end of lactation. However, the neurochemical identity of mPOA MCH neurones and the regulatory factors that control the transient MCH expression remain largely unknown, especially in the mouse. In the present study, we showed that mice also present the transitory expression of MCH in the mPOA at late lactation. mPOA MCH cells did not colocalise significantly with markers of GABAergic (VGAT), glutamatergic (VGLUT2 and VGLUT3) or dopaminergic (tyrosine hydroxylase) neurones. mPOA MCH cells also did not express Kiss1 or oxytocin. By contrast, approximately 70% and 90% of mPOA MCH neurones colocalised with oestrogen receptor α and prolactin-induced phosphorylated signal transducer and activator of transcription 5 (STAT5), respectively. Finally, we demonstrated that the number of MCH neurones in the mPOA is significantly higher in females during the first lactation, compared to mice on the second lactation or pregnant mice during the first lactation or brain-specific STAT5 knockout mice during the first lactation. In summary, our findings indicate that MCH neurones in the mPOA of lactating mice are sensitive to oestrogens and prolactin. Thus, mPOA MCH expression is possibly influenced by hormonal variations. Furthermore, the STAT5 signalling pathway is likely involved in the regulation of MCH expression in the mPOA of lactating mice.

Melanin-concentrating hormone in the Locus Coeruleus aggravates helpless behavior in stressed rats.

Animal studies have shown that antagonists of receptor 1 of Melanin-Concentrating Hormone (MCH-R1) elicit antidepressive-like behavior, suggesting that MCH-R1 might be a novel target for the treatment of depression and supports the hypothesis that MCHergic signaling regulates depressive-like behaviors. Consistent with the evidence that MCHergic neurons send projections to dorsal and median raphe nuclei, we have previously demonstrated that MCH microinjections in both nuclei induced a depressive-like behavior. Even though MCH neurons also project to Locus Coeruleus (LC), only a few studies have reported the behavioral and neurochemical effect of MCH into the LC. We studied the effects of MCH (100 and 200 ng) into the LC on coping-stress related behaviors associated with depression, using two different behavioral tests: the forced swimming test (FST) and the learned helplessness (LH). To characterize the functional interaction between MCH and the noradrenergic LC system, we also evaluated the neurochemical effects of MCH (100 ng) on the extracellular levels of noradrenaline (NA) in the medial prefrontal cortex (mPFC), an important LC terminal region involved in emotional processing. MCH administration into the LC elicited a depressive-like behavior evidenced in both paradigms. Interestingly, in the LH, MCH (100) elicited a significant increase in escape failures only in stressed animals. A significant decrease in prefrontal levels of NA was observed after MCH microinjection into the LC. Our results demonstrate that increased MCH signaling into the LC triggers depressive-like behaviors, especially in stressed animals. These data further corroborate the important role of MCH in the neurobiology of depression.

Melanin-concentrating hormone peptidergic system: Comparative morphology between muroid species.

Melanin-concentrating hormone (MCH) is a conserved neuropeptide, predominantly located in the diencephalon of vertebrates, and associated with a wide range of functions. While functional studies have focused on the use of the traditional mouse laboratory model, critical gaps exist in our understanding of the morphology of the MCH system in this species. Even less is known about the nontraditional animal model Neotomodon alstoni (Mexican volcano mouse). A comparative morphological study among these rodents may, therefore, contribute to a better understanding of the evolution of the MCH peptidergic system. To this end, we employed diverse immunohistochemical protocols to identify key aspects of the MCH system, including its spatial relationship to another neurochemical population of the tuberal hypothalamus, the orexins. Three-dimensional (3D) reconstructions were also employed to convey a better sense of spatial distribution to these neurons. Our results show that the distribution of MCH neurons in all rodents studied follows a basic plan, but individual characteristics are found for each species, such as the preeminence of a periventricular group only in the rat, the lack of posterior groups in the mouse, and the extensive presence of MCH neurons in the anterior hypothalamic area of Neotomodon. Taken together, these data suggest a strong anatomical substrate for previously described functions of the MCH system, and that particular neurochemical and morphological features may have been determinant to species-specific phenotypes in rodent evolution.

Characterisation and origins of melanin-concentrating hormone immunoreactive fibres of the posterior lobe of the pituitary and median eminence during lactation in the Long-Evans rat.

Although the melanin-concentrating hormone (MCH) and its coding mRNA are predominantly found in the tuberal hypothalamus, there is detectable synthesis of MCH in the preoptic hypothalamus exclusively in lactating dams, suggesting a participation of MCH in the alterations that take place after parturition. Also implicated in the dam physiology is oxytocin, a neurohormone released from the posterior pituitary that is necessary for milk ejection. Because the projection fields from oxytocin-immunoreactive (-IR) neurones and the mediobasal preoptic hypothalamus overlap and MCH-IR neurones are found in proximity to oxytocin neurones, we investigated the spatial relationship between MCH and oxytocin fibres. Accordingly, we employed multiple immunohistochemistry labelling for MCH and oxytocin for light and electron microscopy techniques, in addition to i.v. tracer injection combined with in situ hybridisation to identify MCH neurones that project to neurosecretory areas. As described for other strains, lactating Long-Evans dams also display immunoreactivity for MCH in the preoptic hypothalamus on days 12 and 19 of lactation. The appearance of these neurones is contemporaneous with an increase in MCH-IR fibres in both the internal layer of the median eminence and the posterior pituitary. In both regions, MCH- and oxytocin-IR fibres were found in great proximity, although there was no evidence for synaptic interaction between these two populations at the ultrastructural level. The tracer injection revealed that only mediobasal preoptic MCH neurones project to the posterior pituitary, suggesting a neuroendocrine-modulatory role for this population. When taken together, the results obtained in the present study indicate that neuroplasticity events at the mediobasal preoptic hypothalamus that occur during late lactation may be part of a neuroendocrinology control loop involving both MCH and oxytocin.

Melanin-concentrating hormone does not modulate serotonin release in primary cultures of fetal raphe nucleus neurons.

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ±â€¯0.7% of serotonergic and 48.9 ±â€¯5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.

Role of RFRP-3 in the development of cold stress-induced polycystic ovary phenotype in rats

RFamide-related peptide (RFRP-3) is a regulator of GnRH secretion from the brain, but it can also act in human ovary to influence steroidogenesis. We aimed to study the putative local role of RFRP-3 in the ovary and its potential participation in the development of a polycystic ovary phenotype induced by chronic sympathetic stress (cold stress). We used adult Sprague­Dawley rats divided into control and stressed groups. In both groups, we studied the effect of intraovarian exposure to RFRP-3 on follicular development and plasma ovarian steroid concentrations. We also tested the effect of RFRP-3 on ovarian steroid production in vitro. Chronic in vivo intraovarian exposure to RFRP-3 decreased basal testosterone concentrations and cold stress-induced progesterone production by the ovary. In vitro, RFRP-3 decreased hCG-induced ovarian progesterone and testosterone secretion. Immunohistochemistry and mRNA expression analysis showed a decrease in Rfrp and expression of its receptor in the ovary of stressed rats, a result which is in line with the increased testosterone levels found in stressed rats. In vivo application of RFRP-3 recovered the low levels of secondary and healthy antral follicles found in stressed rats. Taken together, our data indicate a previously unknown response of hypothalamic and ovarian RFRP-3 to chronic cold stress, influencing ovarian steroidogenesis and follicular dynamics. Thus, it is likely that RFRP-3 modulation in the ovary is a key component of development of the polycystic ovary phenotype.

Distribution of MCH-containing fibers in the feline brainstem: Relevance for REM sleep regulation.

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized in the postero-lateral hypothalamus and incerto-hypothalamic area. These neurons project diffusely throughout the central nervous system and have been implicated in critical physiological processes, such as sleep. Unlike rodents, in the order carnivora as well as in humans, MCH exerts its biological functions through two receptors: MCHR-1 and MCHR-2. Hence, the cat is an optimal animal to model MCHergic functions in humans. In the present study, we examined the distribution of MCH-positive fibers in the brainstem of the cat. MCHergic axons with distinctive varicosities and boutons were heterogeneously distributed, exhibiting different densities in distinct regions of the brainstem. High density of MCHergic fibers was found in the dorsal raphe nucleus, the laterodorsal tegmental nucleus, the periaqueductal gray, the pendunculopontine tegmental nucleus, the locus coeruleus and the prepositus hypoglossi. Because these areas are involved in the control of REM sleep, the present anatomical data support the role of this neuropeptidergic system in the control of this behavioral state.

Does MCH play a role on establishment or maintenance of social hierarchy in Nile tilapia?

Body coloration has a fundamental role in animal communication by signaling sex, age, reproductive behavior, aggression, etc. Nile-tilapia exhibits dominance hierarchy and the dominants are paler than subordinates. During social interactions in these animals, these color changes occur rapidly, and normally the subordinates become dark. In teleosteans, from the great number of hormones and neurotransmitters involved in color changes, melanocyte hormone stimulates (α-MSH) and melanin concentrates hormone (MCH) are the most remarkable. The aim of this project was to investigate the role of MCH in the establishment of hierarchical dominance of the Nile-tilapia. We analyzed the effect of background coloration in the dominance hierarchy. It was then compared to the melanophore sensibility of dominants and subordinates' fishes to MCH; finally, it was checked if the social rank affects the number of these pigment cells in dominants and subordinated fishes. Fishes which have a social hierarchy established and adjusted individually to the background exhibits paler body coloration when a visual contact was possible, independently of previous social rank and background color. Probably, even recognizing each other, fishes could be defending their new territory. Melanophores of the subordinate fishes were more sensible to MCH than dominants. It suggests that dominants fishes, which are paler than subordinates, could be under a chronic effect of MCH, which could be due a desensitization of melanophores to this hormone. The opposite effect seems to be occurring on subordinate fishes. It was not observed a significant change in the number of melanophores when the fishes were exposed to a prolonged period of agonistic interaction. It is possible that the exposure time for this interaction might not have been sufficient to have any change in the number of these cells of dominants and subordinate fishes.

Participation of hypothalamic CB1 receptors in reproductive axis disruption during immune challenge.

Immune challenge inhibits reproductive function and endocannabinoids (eCB) modulate sexual hormones. However, no studies have been performed to assess whether the eCB system mediates the inhibition of hormones that control reproduction as a result of immune system activation during systemic infections. For that reason, we evaluated the participation of the hypothalamic cannabinoid receptor CB1 on the hypothalamic-pituitary-gonadal (HPG) axis activity in rats submitted to immune challenge. Male adult rats were treated i.c.v. administration with a CB1 antagonist/inverse agonist (AM251) (500 ng/5 µL), followed by an i.p. injection of lipopolysaccharide (LPS) (5 mg/kg) 15 minutes later. Plasmatic, hypothalamic and adenohypophyseal pro-inflammatory cytokines, hormones and neuropeptides were assessed 90 or 180 minutes post-LPS. The plasma concentration of tumour necrosis factor α and adenohypophyseal mRNA expression of Tnfα and Il1ß increased 90 and 180 minutes post i.p. administration of LPS. However, cytokine mRNA expression in the hypothalamus increased only 180 minutes post-LPS, suggesting an inflammatory delay in this organ. CB1 receptor blockade with AM251 increased LPS inflammatory effects, particularly in the hypothalamus. LPS also inhibited the HPG axis by decreasing gonadotrophin-releasing hormone hypothalamic content and plasma levels of luteinising hormone and testosterone. These disruptor effects were accompanied by decreased hypothalamic Kiss1 mRNA expression and prostaglandin E2 content, as well as by increased gonadotrophin-inhibitory hormone (Rfrp3) mRNA expression. All these disruptive effects were prevented by the presence of AM251. In summary, our results suggest that, in male rats, eCB mediate immune challenge-inhibitory effects on reproductive axis at least partially via hypothalamic CB1 activation. In addition, this receptor also participates in homeostasis recovery by modulating the inflammatory process taking place after LPS administration.

Feeding during the rest phase promotes circadian conflict in nuclei that control energy homeostasis and sleep-wake cycle in rats.

Food intake during the rest phase promotes circadian desynchrony, which has been associated with metabolic diseases. However, the link between circadian rhythm and metabolic alterations is not well understood. To investigate this issue, we explored the circadian rhythm of c-Fos immunoreactivity (IR) in rats fed during the day, during the night or with free access to food for 3 weeks. The analysis was focused on the hypothalamic nuclei, which are interconnected and involved in the control of energy homeostasis and/or arousal: lateral hypothalamus (LH), perifornical area, arcuate, ventrolateral pre-optic (VLPO) and tuberomammillary nuclei. The results show that food intake during the rest phase flattened the circadian c-Fos expression in the LH and perifornical area, and induced a phase shift in the VLPO area. In addition, c-Fos expression was analyzed in the orexin and melanin-concentrating hormone (MCH) neurons of the LH, which are involved in the control of food intake and arousal, and in α-melanin-stimulating hormone and neuropeptide Y (NPY) cells in the arcuate nucleus, all of which are involved in feeding-fasting cycles, energy homeostasis and sending projections to the LH. The results indicate that feeding during the rest phase decreased orexin neuron activation in the light in comparison with the other groups. Feeding during this phase also flattened the activity rhythm of MCH and α-melanin-stimulating hormone neurons and increased NPY IR when the light was turned on. This evidence indicates that mealtime differentially affected the hypothalamic nuclei under investigation leading to a circadian conflict that might account for metabolic impairment.

Characterization of Gnrh/Gnih elements in the olfacto-retinal system and ovary during zebrafish ovarian maturation.

Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and named as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.

A screen for modulators reveals that orexin-A rapidly stimulates thyrotropin releasing hormone expression and release in hypothalamic cell culture.

In the paraventricular nucleus of the mammalian hypothalamus, hypophysiotropic thyrotropin releasing hormone (TRH) neurons integrate metabolic information and control the activity of the thyroid axis. Additional populations of TRH neurons reside in various hypothalamic areas, with poorly defined connections and functions, albeit there is evidence that some may be related to energy balance. To establish extracellular modulators of TRH hypothalamic neurons activity, we performed a screen of neurotransmitters effects in hypothalamic cultures. Cell culture conditions were chosen to facilitate the full differentiation of the TRH neurons; these conditions had permitted the characterization of the effects of known modulators of hypophysiotropic TRH neurons. The major end-point of the screen was Trh mRNA levels, since they are generally rapidly (0.5-3h) modified by synaptic inputs onto TRH neurons; in some experiments, TRH cell content or release was also analyzed. Various modulators, including histamine, serotonin, ß-endorphin, met-enkephalin, and melanin concentrating hormone, had no effect. Glutamate, as well as ionotropic agonists (kainate and N-Methyl-d-aspartic acid), increased Trh mRNA levels. Baclofen, a GABAB receptor agonist, and dopamine enhanced Trh mRNA levels. An endocannabinoid receptor 1 inverse agonist promoted TRH release. Somatostatin increased Trh mRNA levels and TRH cell content. Orexin-A rapidly increased Trh mRNA levels, TRH cell content and release, while orexin-B decreased Trh mRNA levels. These data reveal unaccounted regulators, which exert potent effects on hypothalamic TRH neurons in vitro.

Environmental obesogen tributyltin chloride leads to abnormal hypothalamic-pituitary-gonadal axis function by disruption in kisspeptin/leptin signaling in female rats.

Tributyltin chloride (TBT) is a xenobiotic used as a biocide in antifouling paints that has been demonstrated to induce endocrine-disrupting effects, such as obesity and reproductive abnormalities. An integrative metabolic control in the hypothalamus-pituitary-gonadal (HPG) axis was exerted by leptin. However, studies that have investigated the obesogenic TBT effects on the HPG axis are especially rare. We investigated whether metabolic disorders as a result of TBT are correlated with abnormal hypothalamus-pituitary-gonadal (HPG) axis function, as well as kisspeptin (Kiss) action. Female Wistar rats were administered vehicle and TBT (100ng/kg/day) for 15days via gavage. We analyzed their effects on the tin serum and ovary accumulation (as biomarker of TBT exposure), estrous cyclicity, surge LH levels, GnRH expression, Kiss action, fertility, testosterone levels, ovarian apoptosis, uterine inflammation, fibrosis, estrogen negative feedback, body weight gain, insulin, leptin, adiponectin levels, as well as the glucose tolerance (GTT) and insulin sensitivity tests (IST). TBT led to increased serum and ovary tin levels, irregular estrous cyclicity, and decreased surge LH levels, GnRH expression and Kiss responsiveness. A strong negative correlation between the serum and ovary tin levels with lower Kiss responsiveness and GnRH mRNA expression was observed in TBT rats. An increase in the testosterone levels, ovarian and uterine fibrosis, ovarian apoptosis, and uterine inflammation and a decrease in fertility and estrogen negative feedback were demonstrated in the TBT rats. We also identified an increase in the body weight gain and abnormal GTT and IST tests, which were associated with hyperinsulinemia, hyperleptinemia and hypoadiponectinemia, in the TBT rats. TBT disrupted proper functioning of the HPG axis as a result of abnormal Kiss action. The metabolic dysfunctions co-occur with the HPG axis abnormalities. Hyperleptinemia as a result of obesity induced by TBT may be associated with abnormal HPG function. A strong negative correlation between the hyperleptinemia and lower Kiss responsiveness was observed in the TBT rats. These findings provide evidence that TBT leads to toxic effects direct on the HPG axis and/or indirectly by abnormal metabolic regulation of the HPG axis.

Gonadotrophin-Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure, Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone.

The role of gonadotrophin-inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well-established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH-immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto-retinalis, with no differences between the sexes. GnIH-immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed in vitro studies on intact pituitary cultures to evaluate the effect of two doses (10(-6)  m and 10(-8)  m) of synthetic C. dimerus (cd-) LPQRFa-1 and LPQRFa-2 on the release of gonadotrophins and GH. We observed that cd-LPQRFa-1 decreased ß-luteinising hormone (LH) and ß-follicle-stimulating hormone (FSH) and also increased GH release to the culture medium. The release of ß-FSH was increased only when it was stimulated with the higher cd-LPQRFa-2 dose. The results of the present study indicate that cd-LPQRFa-1, the cichlid fish GnIH, inhibits ß-LH and ß-FSH release and stimulates GH release in intact pituitary cultures of C. dimerus. The results also show that cd-LPQRF-2 could act as an ß-FSH-releasing factor in this fish species.