Expression of orexin receptors in the brain and peripheral tissues of the male sheep

Orexins exert their effects through two specific receptors (OX1R and OX2R) that have been found mainly in the brain and also in peripheral tissues of rats and humans. Here, we demonstrate expression of mRNA encoding for ovine OX1R and OX2R in central and peripheral tissues of sheep. Gene expression for orexin receptors in the hypothalamus and the preoptic area was localised by in situ hybridisation. OX1R was detected in arcuate nuclei (ARC), median eminence (ME), the lateral hypothalamic nuclei and preoptic area (POA) and it was scattered along the third ventricle from the paraventricular (PVN) to the ventromedial hypothalamic nuclei (VMH). OX2R was localised in the PVN, ARC, ME, ventral VMH and a small region of the ventral POA. Gene expression for OX1R and OX2R in central and peripheral tissues was analysed using quantitative real time RT-PCR. Both orexin receptor genes were expressed in the hypothalamus, POA, hippocampus, amygdala, olfactory bulb, pineal gland and recess and pituitary gland, whereas only OX1R mRNA was detected in the testis, kidney and adrenal gland. The expression of the genes for orexin receptors in this range of ovine tissues suggests roles for orexins in multiple physiological functions, with actions at both central and peripheral levels.     

Expressions of the prepro-orexin and orexin type 2 receptor genes in obese rat

We examined the expressions of the prepro-orexin gene in the lateral hypothalamic area (LHA), the genes of the neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the arcuate nucleus (ARC), the orexin type 1 receptor (OX1R) gene in the ventromedial hypothalamic nucleus (VMH) and the orexin type 2 receptor (OX2R) gene in the paraventricular nucleus (PVN) in 6-, 12- and 18-week-old male lean (Fa/?) and obese (fa/fa) Zucker rats, using in situ hybridization histochemistry. The fa/fa rats showed hyperglycemia at 12- and 18-week-old. The prepro-orexin mRNA level in fa/fa rats at 18-week-old and the OX2R mRNA level in fa/fa rats at 12- and 18-week-old were significantly decreased compared to controls. The NPY mRNA levels in fa/fa rats at each time point were significantly increased compared to controls, but the POMC mRNA levels were decreased. Prepro-orexin and OX2R mRNA levels in fa/fa rats pretreated with insulin normalized to the levels found in Fa/? rats. These results suggest that the regulation of prepro-orexin gene expression might be independent of the regulation of the NPY and POMC genes in the ARC in fa/fa rats.     

Effects of restricted feeding on the activity of hypothalamic Orexin (OX)-A containing neurons and OX2 receptor mRNA level in the paraventricular nucleus of rats

We have examined the effects of 3 weeks of food restriction on both the activity of neurons containing hypothalamic orexin (OX)-A and the level of OX receptor type 2 (OX2R) mRNA in the paraventricular nucleus (PVN) of rats. Double immunohistochemistry was used to examine the expression of OX-A and Fos in the lateral hypothalamic area (LHA), and in situ hybridization histochemistry was used to measure levels of OX2R mRNA in the PVN. After the period of restricted feeding, 20-30% of OX-A-containing neurons exhibited Fos-like immunoreactivity (LI). The distribution of OX-A-LI/Fos-LI cells in the food-restricted rats was similar to that observed in glucose-deprived rats after intracerebroventricular (icv) administration of 2-deoxy-D-glucose (2-DG). In addition, 3 weeks of food restriction caused a significant decrease in the expression of the OX2R gene in the parvocellular division of the PVN. These results suggest that the activation of OX-A-containing neurons induced by restricted feeding may be involved in neuroendocrine responses to food restriction.     

Hypocretin/orexin type 1 receptor in brain: role in cardiovascular control and the neuroendocrine response to immobilization stress

Hypocretin/orexin acts pharmacologically in the hypothalamus to stimulate stress hormone secretion at least in part by an action in the hypothalamic paraventricular nucleus, where the peptide's receptors have been localized. In addition, orexin acts in the brain to increase sympathetic tone and, therefore, mean arterial pressure and heart rate. We provide evidence for the role of endogenously produced hypocretin/orexin in the physiological response to immobilization stress and identify the receptor subtype responsible for this action of the peptide. Antagonism of the orexin type 1 receptor (OX(1)R) in the brain prevented the ACTH-stimulating effect of centrally administered hypocretin/orexin. Furthermore, pretreatment of animals with the OX(1)R antagonist blocked the ACTH response to immobilization/restraint stress. The OX(1)R antagonist did not, however, block the pharmacological or physiological release of prolactin in these two models. Antagonism of the OX(1)R also blocked the central action of orexin to elevate mean arterial pressures and heart rates in conscious rats. These data suggest receptor subtype-selective responses to hypocretin/orexin and provide further evidence for the importance of endogenously produced peptide in the physiological control of stress hormone secretion.     

Neural basis of orexigenic effects of ghrelin acting within lateral hypothalamus

Ghrelin stimulates feeding when administered centrally and peripherally. The lateral hypothalamus (LH) is thought to mediate ghrelin-induced hyperphagia. Thus, we examined central mechanisms underlying feeding generated by LH ghrelin. We determined that 0.3nmol of LH-injected ghrelin was the lowest dose increasing food consumption and it induced Fos immunoreactivity (IR; a marker of neuronal activation) in feeding-related brain areas, including the hypothalamic paraventricular, arcuate, and dorsomedial nuclei, amygdala, and nucleus of the solitary tract. Also, LH ghrelin induced Fos IR in LH orexin neurons. We conclude that the LH, as part of larger central circuitry, integrates orexigenic properties of ghrelin.     

Up-regulation of galanin and corticotropin-releasing hormone mRNAs in the key hypothalamic and amygdaloid nuclei in a mouse model of visceral pain

Cyclophosphamide (CP)-induced cystitis is often used as an animal model of visceral pain. Various neuropeptides in the hypothalamic and amygdaloid nuclei are implicated in pain-induced responses. However, little information is available regarding the regulation of the neuropeptides in response to visceral pain. In the present study, we examined the effects of CP-induced cystitis on the levels of mRNAs encoding galanin, corticotropin-releasing hormone (CRH), substance P, and enkephalins in the hypothalamic and limbic nuclei using in situ hybridization histochemistry in mouse. Galanin mRNA levels in CP-treated group increased significantly in the arcuate nucleus and the paraventricular nucleus (PVN) but not in the medial preoptic area after the intraperitoneal administration of CP (200 mg/kg body weight) in comparison to those in saline-treated group. CRH mRNA levels in CP-treated group also increased significantly in the central amygdala as well as the PVN after the CP administration. In contrast, CP-induced cystitis failed to upregulate the preprotachykinin-A and preproenkephalin genes which encode substance P and enkephalins, respectively in the hypothalamic and limbic nuclei at any of the time points examined. These results suggest that visceral nociception may upregulate both galanin and CRH gene expression in the hypothalamic and limbic nuclei.     

Ascending projections to the hypothalamus and limbic nuclei from the dorsolateral pontine tegmentum: a biochemical and electron microscopic study.

Axons arising from the dorsolateral pontine tegmentum of the rat were traced in various hypothalamic and limbic nuclei by the electron microscopic degeneration method (0.5-8 day survival times) and by measuring regional norepinephrine (NE) concentrations after 12 days of survival using a radioenzymatic method. Significant reductions (41-85%) in NE contents were observed in the supraoptic, arcuate, basal and lateral amygdaloid nuclei and in the hippocampus 12 days after the bilateral electrolytic lesions of the locus coeruleus. No changes in NE concentrations were observed in the ventromedial, septal, central amygdaloid nuclei, in the median eminence and olfactory tubercle. Parabrachial lesions resulted in a decrease of NE content only in the olfactory tubercle. By means of electron microscopy terminal degeneration was found in the hypothalamic paraventricular, dorsomedial nuclei, in the median eminence, in the bed nucleus of the stria terminalis, in the central, lateral and basal amygdaloid nuclei, in the hippocampus and in the anterior ventral thalamic nucleus.     

Orexin/Hypocretin Activates mTOR Complex 1 (mTORC1) via an Erk/Akt-independent and Calcium-stimulated Lysosome v-ATPase Pathway

The lack of the neuropeptide orexin, also known as hypocretin, results in narcolepsy, a chronic sleep disorder characterized by frequent sleep/cataplexy attacks and rapid eye movement sleep abnormalities. However, the downstream pathways of orexin signaling are not clearly understood. Here, we show that orexin activates the mTOR pathway, a central regulator of cell growth and metabolism, in the mouse brain and multiple recombinant cell lines that express the G protein-coupled receptors (GPCRs), orexin 1 receptor (OX1R) or orexin 2 receptor (OX2R). This orexin/GPCR-stimulated mTOR activation is sensitive to rapamycin, an inhibitor of mTOR complex 1 (mTORC1) but is independent of two well known mTORC1 activators, Erk and Akt. Rather, our studies indicate that orexin activates mTORC1 via extracellular calcium influx and the lysosome pathway involving v-ATPase and Rag GTPases. Moreover, a cytoplasmic calcium transient is sufficient to mimic orexin/GPCR signaling to mTORC1 activation in a v-ATPase-dependent manner. Together, our studies suggest that the mTORC1 pathway functions downstream of orexin/GPCR signaling, which plays a crucial role in many physiological and metabolic processes.     

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response.     

Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity

The paraventricular hypothalamic nucleus (PVN) appears to integrate orexigenic properties of a novel peptide, ghrelin. Thus, we examined central mechanisms underlying feeding generated by intra-PVN ghrelin. We established that 0.03 nmol of PVN-injected ghrelin was the lowest dose increasing food consumption and it induced c-Fos immunoreactivity (a marker of neuronal activation) in the PVN itself, as well as in other feeding-related brain areas, including the hypothalamic arcuate and dorsomedial nuclei, central nucleus of the amygdala, and nucleus of the solitary tract. We conclude that the PVN, as part of larger central circuitry, mediates orexigenic properties of ghrelin.     

Peripheral ghrelin participates in the glucostatic signaling mediated by the ventromedial and lateral hypothalamus neurons

Employing immunohistochemistry techniques, we examined the c-fos expression in different hypothalamic areas, when plasma glucose levels were modified by the administration of insulin and 2-deoxyglucose (2-DG) respectively. Subsequently, the hypoglycemia produced by an injection of insulin significantly increased feeding concomitant to higher c-fos expression in the arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and lateral hypothalamus (LH), while no statistical changes in the ventromedial hypothalamus (VMH) were found. Also, the glucopenia induced by 2-DG administration produced similar stimulatory effects on appetite and the neuronal activity affecting all the hypothalamic areas studied, including the VMH. The peripheral blockade of the orexigenic hormone ghrelin with a specific antibody (AGA) significantly decreased food intake as induced from acute hypoglycemia and glucopenia. Curiously, the conjoint AGA and insulin or 2-DG administration produced a differential effect on the hypothalamic neurons analyzed, by increasing the number of c-fos positive neurons in the ARC, PVN and DMH, but not in the VMH and LH. This outcome suggests an interactive effect of the glucostatic pathways involving these two areas with the ghrelin signaling.     

Orexins/hypocretins in the ob/ob mouse: hypothalamic gene expression, peptide content and metabolic effects

Orexins (forms A and B) belong to a new family of peptides that, as neuropeptide Y (NPY), stimulate food intake when centrally injected. The ob/ob mouse is a well-characterized model of hyperphagia and obesity associated with strong metabolic disturbances and a central dysregulation of peptides involved in the control of feeding. In the present report, we investigated the hypocretin (Hcrt)/orexin (OX) peptide pathway in lean and ob/ob mice. Prepro-Hcrt/OX mRNA expression, measured by in situ hybridization was restricted to the lateral hypothalamus area. It was significantly decreased in ob/ob mice (-18%; p<0.01). When estimated by real time RT-PCR in the whole hypothalamus, this decrease amounted to 65% (p<0.001). Hcrt-1/OX-A peptide concentrations, measured by RIA in microdissected hypothalamic nuclei were high in the lateral hypothalamus (LH) and lower in the arcuate (ARC) and paraventricular nuclei (PVN). In ob/ob mice, OX-A levels were significantly lower than in lean mice in the LH (-34%; p<0.02) and in the PVN (-72%; p<0.005). Acute intracerebroventricular injection of Hcrt-1/OX-A (1-10 nmol) stimulated feeding in lean, but not in ob/ob mice, whereas Hcrt-2/OX-B (1-10 nmol) had the opposite effect. Acute third ventricle (i3vt) injections of Hcrt/OX peptides in ob/ob mice transiently increased their metabolic rate and stimulated lipid substrate utilization. These findings provide direct evidence that Hcrt/OX peptides are down-regulated in the hypothalamus of ob/ob mice, contrary to the NPY system. The present data argues that Hcrt/OX peptides are not primarily responsible for the metabolic syndrome of the ob/ob mice. The diminution in the OX tone might participate in a counterregulatory system necessary to limit the adverse effects of NPY on food intake and body weight.     

Elevated hypothalamic orexin signaling, sensitivity to orexin A, and spontaneous physical activity in obesity-resistant rats

Selectively-bred obesity-resistant [diet resistant (DR)] rats weigh less than obesity-prone [diet-induced obese (DIO)] rats, despite comparable daily caloric intake, suggesting phenotypic energy expenditure differences. Human data suggest that obesity is maintained by reduced ambulatory or spontaneous physical activity (SPA). The neuropeptide orexin A robustly stimulates SPA. We hypothesized that DR rats have greater: 1) basal SPA, 2) orexin A-induced SPA, and 3) preproorexin, orexin 1 and 2 receptor (OX1R and OX2R) mRNA, compared with DIO rats. A group of age-matched out-bred Sprague-Dawley rats were used as additional controls for the behavioral studies. DIO, DR, and Sprague-Dawley rats with dorsal-rostral lateral hypothalamic (rLHa) cannulas were injected with orexin A (0, 31.25, 62.5, 125, 250, and 500 pmol/0.5 microl). SPA and food intake were measured for 2 h after injection. Preproorexin, OX1R and OX2R mRNA in the rLHa, and whole hypothalamus were measured by real-time RT-PCR. Orexin A significantly stimulated feeding in all rats. Orexin A-induced SPA was significantly greater in DR and Sprague-Dawley rats than in DIO rats. Two-mo-old DR rats had significantly greater rLHa OX1R and OX2R mRNA than DIO rats but comparable preproorexin levels. Eight-mo-old DR rats had elevated OX1R and OX2R mRNA compared with DIO rats, although this increase was significant for OX2R only at this age. Thus DR rats show elevated basal and orexin A-induced SPA associated with increased OX1R and OX2R gene expression, suggesting that differences in orexin A signaling through OX1R and OX2R may mediate DIO and DR phenotypes.     

Heat-shock protein 90 complexes in resting and thrombin-activated platelets

The orexins are peptides which were recently isolated from the rat hypothlamus. They play a role in energy homeostasis and regulation of feeding as well as in other functions such as the sleep-wake cycle. The involvement of glucocorticoids in stress processes as well as in body weight regulation is well known. In the present paper, we investigated the role of glucocorticoids on hypocretin (Hcrt)/orexin (OX) pathway in Sprague-Dawley rats. We confirmed by in situ hybridization that prepro-Hcrt/OX mRNA expression is restricted to the lateral hypothalamus area with extension to the perifornical nucleus and the posterior hypothalamic area. Lateral hypothalamic prepro-Hcrt/OX mRNA expression was decreased by 50% after adrenalectomy (99.8+/-5.0 vs 49.2+/-4.4 nCi/g, p<0.01). Peripheral glucocorticoid treatment (dexamethasone) restored its expression to normal levels (105.4+/-6.1 nCi/g). The present data provide direct evidence that Hcrt/OX expression in the lateral hypothalamus is modulated by the glucocorticoids status. As the Hcrt/Ox system is closely interactive with the corticotropin-releasing hormone and neuropeptide Y systems, we propose that hypocretin/orexins peptides constitute a very sensitive key relay for mediating both stress and feeding behavior.     

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine pituitary during the oestrous cycle

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2–3 relative to days 10–12, 14–16 and 17–19 (p < 0.05). In NP the OX1R mRNA level was elevated on days 10–12 compared to the remaining stages (p < 0.05). OX2R gene expression in AP was the lowest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19) and the expression peak occurred on days 17–19 (p < 0.05 vs. the all studied stages). In NP the highest (p < 0.05) expression of OX2R mRNA was noted on days 17–19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10–12 (p < 0.05), whereas in NP it was greatest on days 2–3 and 14–16 (p < 0.05 vs. days 10–12 and 17–19). In both cases the lowest OX1R protein expression was observed during follicular phase (p < 0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p < 0.05) on days 2–3 and 14–16 compared to days 10–12 and 17–19. In NP the lowest (p < 0.05) expression of this protein was on days 17–19 and the highest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.     

Distribution of somatostatinlike immunoreactivity in the brain of the little brown bat (Myotis lucifugus)

The distribution of somatostatinlike immunoreactive (SLI) perikarya, axons, and terminals was mapped in subcortical areas of the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry. A preponderance of immunoreactivity was localized in reticular, limbic, and hypothalamic areas including: 1) in the forebrain: the bed nucleus of the stria terminalis; lateral preoptic, dorsal, anterior, lateral and posterior hypothalamic areas; amygdaloid, periventricular, arcuate, supraoptic, suprachiasmatic, ventromedial, dorsomedial, paraventricular, lateral and medial mammillary, and lateral septal nuclei; the nucleus of the diagonal band of Broca and nucleus accumbens septi; 2) in the midbrain: the periaqueductal gray, interpeduncular, dorsal and ventral tegmental, pretectal, and Edinger-Westphal nuclei; and 3) in the hindbrain: the superior central and parabrachial nuclei, nucleus incertus, locus coeruleus, and nucleus reticularis gigantocellularis. Other areas containing SLI included the striatum (caudate nucleus and putamen), zona incerta, infundibulum, supramammillary and premammillary nuclei, medial and dorsal lateral geniculate nuclei, entopeduncular nucleus, lateral habenular nucleus, central medial thalamic nucleus, central tegmental field, linear and dorsal raphe nuclei, nucleus of Darkschewitsch, superior and inferior colliculi, nucleus ruber, substantia nigra, mesencephalic nucleus of V, inferior olivary nucleus, inferior central nucleus, nucleus prepositus, and deep cerebellar nuclei. While these results were similar in some respects to those previously reported in rodents, they also provided interesting contrasts.     

Expression of neuropeptide Y and agouti-related peptide in the hypothalamic arcuate nucleus of newborn neurogenin3 null mutant mice

Mice deficient in neurogenin 3 (Ngn3) fail to generate pancreatic endocrine cells and intestinal endocrine cells. Hypothalamic neuropeptides implicated in the control of energy homeostasis might also be affected in Ngn3 homozygous null mutant mice. We investigated the expression of two prominent orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), in the hypothalamic arcuate nucleus of newborn wild-type and Ngn3 null mutant mice. Immunohistochemical analysis demonstrated that, in Ngn3 null mutants, the number of NPY-immunoreactive neurons and nerve fibers was markedly increased in the arcuate nucleus, and the nerve fibers were widely distributed in the hypothalamic area, including the paraventricular and dorsomedial nuclei. Little increase of AgRP immunoreactivity was detected in the arcuate nucleus of mutant mice. In situ hybridization analysis confirmed the increased population of the NPY neurons in the arcuate nucleus of the mutants. The NPY mRNA level, as estimated by laser capture microdissection and real-time quantitative polymerase chain reaction, was 371% higher in Ngn3 null mutants than in wild-type mice. AgRP mRNA levels did not differ significantly between the null mutants and wild-type mice. Thus, up-regulation of the hypothalamic NPY system is probably a feature characteristic of Ngn3 null mice.