Evidence that Orexin-Containing Neurones Provide Direct Input to Gonadotropin-Releasing Hormone Neurones in the Ovine Hypothalamus

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.     

Glucokinase inhibitor glucosamine stimulates feeding and activates hypothalamic neuropeptide Y and orexin neurons

Maintaining glucose levels within the appropriate physiological range is necessary for survival. The identification of specific neuronal populations, within discreet brain regions, sensitive to changes in glucose concentration has led to the hypothesis of a central glucose-sensing system capable of directly modulating feeding behaviour. Glucokinase (GK) has been identified as a glucose-sensor responsible for detecting such changes both within the brain and the periphery. We previously reported that antagonism of centrally expressed GK by administration of glucosamine (GSN) was sufficient to induce protective glucoprivic feeding in rats. Here we examine a neurochemical mechanism underlying this effect and report that GSN stimulated food intake is highly correlated with the induction of the neuronal activation marker cFOS within two nuclei with a demonstrated role in central glucose sensing and appetite, the arcuate nucleus of the hypothalamus (ARC) and lateral hypothalamic area (LHA). Furthermore, GSN stimulated cFOS within the ARC was observed in orexigenic neurons expressing the endogenous melanocortin receptor antagonist agouti-related peptide (AgRP) and neuropeptide Y (NPY), but not those expressing the anorectic endogenous melanocortin receptor agonist alpha-melanocyte stimulating hormone (α-MSH). In the LHA, GSN stimulated cFOS was found within arousal and feeding associated orexin/hypocretin (ORX), but not orexigenic melanin-concentrating hormone (MCH) expressing neurons. Our data suggest that GK within these specific feeding and arousal related populations of AgRP/NPY and ORX neurons may play a modulatory role in the sensing of and appetitive response to hypoglycaemia.     

Differential expression of orexin receptors 1 and 2 in the rat brain

Orexins (hypocretins) are neuropeptides synthesized in the central nervous system exclusively by neurons of the lateral hypothalamus. Orexin-containing neurons have widespread projections and have been implicated in complex physiological functions including feeding behavior, sleep states, neuroendocrine function, and autonomic control. Two orexin receptors (OX(1)R and OX(2)R) have been identified, with distinct expression patterns throughout the brain, but a systematic examination of orexin receptor expression in the brain has not appeared. We used in situ hybridization histochemistry to examine the patterns of expression of mRNA for both orexin receptors throughout the brain. OX(1)R mRNA was observed in many brain regions including the prefrontal and infralimbic cortex, hippocampus, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus. OX(2)R mRNA was prominent in a complementary distribution including the cerebral cortex, septal nuclei, hippocampus, medial thalamic groups, raphe nuclei, and many hypothalamic nuclei including the tuberomammillary nucleus, dorsomedial nucleus, paraventricular nucleus, and ventral premammillary nucleus. The differential distribution of orexin receptors is consistent with the proposed multifaceted roles of orexin in regulating homeostasis and may explain the unique role of the OX(2)R receptor in regulating sleep state stability.     

Hypothalamic neuropeptides implicated in the regulation of sleep/wakefulness states

Several neuropeptides, including galanin, orexin, melanin-concentrating hormone (MCH), urocortin-2, pituitary adenylate cyclase activating protein, and vasoactive intestinal peptide, have been implicated in the regulation of sleep/wakefulness states. In particular, neuropeptides produced in the hypothalamus, including galanin, orexin, and MCH, have been shown to play crucial roles. Galanin is localized to the prepotic area of the hypothalamus and is likely to be involved in the promotion and maintenance of sleep. MCH, which is expressed by neurons in the lateral hypothalamic area (LHA), seems to be implicated in rapid eye movement sleep regulation. Orexins are also localized in the LHA and have been established as one of the most important factors in the regulation of sleep/wakefulness states. A series of studies have suggested that orexin deficiency causes narcolepsy in humans and other mammalian species, highlighting the roles of this hypothalamic neuropeptide in the regulation of sleep and wakefulness. Studies of efferent and afferent systems of orexin-producing neurons have shown that the orexin neuronal system has close interactions with the systems that regulate emotion, energy homeostasis, reward, and arousal. These observations suggest that orexin neurons are involved in sensing the body's external and internal environments and regulate vigilance states accordingly.     

Direct pyrogenic input from prostaglandin EP3 receptor-expressing preoptic neurons to the dorsomedial hypothalamus

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.     

Expression patterns of corticotropin-releasing factor, arginine vasopressin, histidine decarboxylase, melanin-concentrating hormone, and orexin genes in the human hypothalamus

The hypothalamus regulates numerous autonomic responses and behaviors. The neuroactive substances corticotropin-releasing factor (CRF), arginine-vasopressin (AVP), histidine decarboxylase (HDC), melanin-concentrating hormone (MCH), and orexin/hypocretins (ORX) produced in the hypothalamus mediate a subset of these processes. Although the expression patterns of these genes have been well studied in rodents, less is known about them in humans. We combined classical histological techniques with in situ hybridization histochemistry to produce both 2D and 3D images and to visually align and quantify expression of the genes for these substances in nuclei of the human hypothalamus. The hypothalamus was arbitrarily divided into rostral, intermediate, and caudal regions. The rostral region, containing the paraventricular nucleus (PVN), was defined by discrete localization of CRF- and AVP-expressing neurons, whereas distinct relationships between HDC, MCH, and ORX mRNA-expressing neurons delineated specific levels within the intermediate and caudal regions. Quantitative mRNA signal intensity measurements revealed no significant differences in overall CRF or AVP expression at any rostrocaudal level of the PVN. HDC mRNA expression was highest at the level of the premammillary area, which included the dorsomedial and tuberomammillary nuclei as well as the dorsolateral hypothalamic area. In addition, the overall intensity of hybridization signal exhibited by both MCH and ORX mRNA-expressing neurons peaked in distinct intermediate and caudal hypothalamic regions. These results suggest that human hypothalamic neurons involved in the regulation of the HPA axis display distinct neurochemical patterns that may encompass multiple local nuclei.     

Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain

Using radioactive in situ hybridisation, the distribution of prodynorphin mRNA in the brains of C57Bl/6 mice was systemically investigated, and double-labelling in situ hybridisation was used to determine the extent to which neuropeptide Y (NPY) and prodynorphin mRNAs were co-expressed. Our results demonstrate that prodynorphin mRNA expression in the mouse brain is localised at specific subregions of the olfactory bulb, cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, mesencephalon and myelencephalon. Among the regions displaying the most intense labelling were the olfactory tubercle, lateral septum (LS), caudate putamen (Cpu), central amygdaloid nucleus (Ce), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), lateral reticular nucleus (LRt) and solitary tract nucleus (NTS). In the arcuate nucleus of the hypothalamus (Arc), double-labelling in situ hybridisation revealed that prodynorphin expressing neurons also contained NPY mRNA, with a co-localisation rate of approximately 88% in the lateral part of the Arc, and 79% in the dorsal part of the Arc, respectively, suggesting potential overlapping functions of these two neurotransmitters in feeding type behaviour.