Orexin receptor subtype activation and locomotor behaviour in the rat

Aim: Orexin-producing neurones, located primarily in the perifornical region of the lateral hypothalamus, project to a wide spectrum of brain sites where they influence numerous behaviours as well as modulating the neuroendocrine and autonomic responses to stress. While some of the actions of orexin appear to be mediated via the type 1 receptor, some are not, including its action on the release of one stress hormone, prolactin. We describe here the ability of orexin to increase locomotor behaviours and identify the importance of both receptor subtypes in these actions. Methods: Rats were tested for their behavioural responses to the central activation of both the type 1 (OX₁R) and type 2 (OX₂R) receptor (ICV orexin A), compared to OX₂R activation using a relatively selective OX₂R agonist in the absence or presence of an orexin receptor antagonist that possesses highest affinity for OX₁R. Results: Increases in locomotor activity were observed, effects which were expressed by not only orexin A, which binds to both the OX₁R and the OX₂R receptors, but also by the relatively selective OX₂R agonist [(Ala¹¹, Leu¹⁵)-orexin B]. Furthermore, the OX₁R selective antagonist only partially blocked the action of orexin A on most locomotor behaviours and did not block the actions of [(Ala¹¹, Leu¹⁵)-orexin B]. Conclusion: We conclude that orexin A exerts its effects on locomotor behaviour via both the OX₁R and OX₂R and that agonism or antagonism of only one of these receptors for therapeutic purposes (i.e. sleep disorders) would not provide selectivity in terms of associated behavioural side effects.     

Merkel cells, a new localization of prepro-orexin and orexin receptors

Orexins (OXA and OXB) are peptides derived from a common precursor called prepro-orexin. They act through G-protein receptors named orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R). Orexins were first demonstrated in neurons of the lateral hypothalamus and found to be related to the control of food intake. However, it has been shown that they are widely distributed in both the nervous system and peripheral tissues, including endocrine organs such as the pituitary and adrenal glands. Merkel cells are neuroendocrine cells situated in the epidermis, tactile hairs and oral mucosa, and act as mechanoreceptors. Up to the present, various neuropeptides have been detected in these cells. The aim of the present study was to detect the presence of prepro-orexin and orexin receptors (OX(1)R and OX(2)R) in porcine Merkel cells using immunohistochemistry. Prepro-orexin was expressed in the cytoplasm of Merkel cells in the skin of the pig snout. Immunoreactivity for prepro-orexin was more intense in the mature side of the cell, where the dense-cored granules are accumulated. Epidermal nerve terminals associated with Merkel cells and dermal nerve fibres showed no immunostaining. Both orexin receptors (OX(1)R and OX(2)R) were also demonstrated in the cytoplasm of Merkel cells of pig snout skin. The finding of orexins and their receptors in Merkel cells suggests that they have an autocrine function. Further studies are needed to ascertain the significance of this function.     

Functions of orexins in peripheral tissues

Orexin A (OXA) and orexin B were originally isolated as hypothalamic peptides regulating sleep, wakefulness and feeding. However, growing evidence suggests that orexins have major functions also in the peripheral tissues. Central orexigenic pathways originating from medulla activate the hypothalamus-pituitary axis and can influence the sympathetic tone. Orexins and their receptors are widely dispersed throughout the intestine, where orexin receptors are regulated by the nutritional status, affect insulin secretion and intestinal motility. Although the primary source of the peptide has not been elucidated, OXA is detected in plasma and its level varies in response to the metabolic state. In this review, we focus on the current knowledge on peripheral functions of orexins and discuss possible endocrine, paracrine and neurocrine roles.     

Activation of the basal forebrain by the orexin/hypocretin neurones

The orexin neurones play an essential role in driving arousal and in maintaining normal wakefulness. Lack of orexin neurotransmission produces a chronic state of hypoarousal characterized by excessive sleepiness, frequent transitions between wake and sleep, and episodes of cataplexy. A growing body of research now suggests that the basal forebrain (BF) may be a key site through which the orexin-producing neurones promote arousal. Here we review anatomical, pharmacological and electrophysiological studies on how the orexin neurones may promote arousal by exciting cortically projecting neurones of the BF. Orexin fibres synapse on BF cholinergic neurones and orexin-A is released in the BF during waking. Local application of orexins excites BF cholinergic neurones, induces cortical release of acetylcholine and promotes wakefulness. The orexin neurones also contain and probably co-release the inhibitory neuropeptide dynorphin. We found that orexin-A and dynorphin have specific effects on different classes of BF neurones that project to the cortex. Cholinergic neurones were directly excited by orexin-A, but did not respond to dynorphin. Non-cholinergic BF neurones that project to the cortex seem to comprise at least two populations with some directly excited by orexin-A that may represent wake-active, GABAergic neurones, whereas others did not respond to orexin-A but were inhibited by dynorphin and may be sleep-active, GABAergic neurones. This evidence suggests that the BF is a key site through which orexins activate the cortex and promote behavioural arousal. In addition, orexins and dynorphin may act synergistically in the BF to promote arousal and improve cognitive performance.     

Rapid changes in the sensitivity of arcuate nucleus neurons to central ghrelin in relation to feeding status

Ghrelin, the endogenous ligand for the growth hormone secretagogue (GHS) receptor, stimulates feeding and increases body weight. Systemic ghrelin administration induces the immediate-early gene protein product, c-Fos, in the arcuate nucleus of the hypothalamus (ARC) of satiated rats and this increase is potentiated in fasted rats. The aim of this study was to determine whether potentiation was seen in fasted animals after intracerebroventricular (i.c.v) administration of ghrelin and to identify the hypothalamic nuclei activated by this peptide. In addition we investigated if allowing fasted animals to re-feed for 1 h prior to i.c.v. ghrelin injection affected the c-Fos response. Using c-Fos immunocytochemistry, we demonstrated that i.c.v. ghrelin activated several hypothalamic nuclei, including the ARC, paraventricular nucleus (PVH) and the lateral hypothalamus (LH). The c-Fos response was greater in fasted animals compared with satiated animals. Fasted rats allowed access to food for 1 h prior to central ghrelin administration showed an attenuated response in the ARC, similar to the response seen in fed animals. However, the response in the LH (including in the orexin neurons) was further potentiated. The latter may reflect a connection between the hypothalamus and regions of the brain responding to the reward value of the meal.     

Individual differences in wheel-running rhythms are related to temporal and spatial patterns of activation of orexin A and B cells in a diurnal rodent (Arvicanthis niloticus)

This study investigated the relationship between the orexins and patterns of activity in the diurnal Nile grass rat, Arvicanthis niloticus. Some individuals of this species switch to a more nocturnal pattern when given access to a running wheel, while others continue to be most active during the day. In both day- and night-active grass rats, the percentages of orexin A (OXA) and orexin B (OXB) cells expressing Fos were highest when animals were actively running in wheels. In night-active animals, removal of the running wheel significantly decreased OXA and OXB cell Fos expression. Additionally, in night-active animals, clear regional differences were apparent. In these animals the presence of a wheel induced higher percentages of Fos in both OXA and OXB cells in medial regions of the lateral hypothalamus than in lateral regions. In night-active animals without access to wheels, this medial-lateral gradient was present only in OXA cells. No regional differences were observed in day-active animals. This study demonstrates that individual differences in the patterns of activation of OXA and OXB cell populations are related to differences in the temporal pattern of wheel running. We also present evidence that orexin cells have projections to the intergeniculate leaflet that appear to make contact with neuropeptide-Y cells. We discuss the possibility that these fibers may be involved in relaying feedback regarding the activity state of the animal to the circadian system through these projections.     

Sex differences in the responses of orexin neurons in the lateral hypothalamic area and feeding behavior to fasting

Because there are sex differences in feeding-related behavior and orexin neurons are involved in feeding, we looked for a possible sex difference in the response of orexin neurons in the lateral hypothalamic area to fasting, using the phosphorylated cyclic AMP response element-binding protein (pCREB) as a marker of neural activity. Intact male and female rats at proestrus, estrus, or diestrus, were fed normally or fasted for 48 h. After fasting, they were intravenously injected with saline or glucose and subjected to immunohistochemical processing for the detection of orexin and pCREB. In the rats fed normally and injected with saline, only a small population of orexin neurons expressed pCREB in both male and female rats. However, fasting increased the number of orexin neurons with pCREB (double-stained cells) in female rats regardless of the estrous day but not in male rats, revealing a significant sex difference in the response of orexin neurons to fasting. Glucose injection in fasted rats decreased the number of double-stained cells in female rats, and the magnitude of glucose-dependent decrease was greater at proestrus and estrus than at diestrus 2. We also found that female rats, but not male rats, showed an increase in total food intake after fasting (rebound feeding). We speculate that the demonstrated sex differences in the response of orexin neurons to fasting reflect the vulnerability of feeding mechanisms in females.     

大鼠腹侧被盖区多巴胺能神经元介导食欲素促进全麻后觉醒效应

目的:研究大鼠腹侧被盖区(VTA)多巴胺能神经元是否介导食欲素(orexin)的促进全麻后觉醒效应。方法:将兴奋性光遗传病毒注射至Hcrt-cre大鼠的orexin阳性神经元所在的下丘脑外侧穹隆周区(PeFLH),同时在VTA区注射抑制性化学遗传病毒及带有酪氨酸羟化酶(TH)-cre的混合病毒抑制多巴胺能神经元,并在VTA区植入光纤。观察通过化学遗传抑制VTA区多巴胺能神经元后,光遗传兴奋VTA区的orexin阳性神经投射终末是否仍能引起促进异氟醚麻醉后觉醒的效应。结果:与对照组相比,用化学遗传技术预先抑制VTA区多巴胺能神经元,阻断了光遗传兴奋VTA区orexin阳性神经投射终末所产生的缩短大鼠1.4%异氟醚麻醉后觉醒时间的作用;并阻断了光遗传兴奋VTA区orexin阳性神经投射终末所产生的降低大鼠1.4%异氟醚麻醉中脑电图爆发抑制率(BSR)效应。用Fos染色法验证了化学遗传法抑制多巴胺能神经元的可靠性。结论:抑制VTA区的多巴胺能神经元能够阻断兴奋大鼠VTA区orexin阳性神经末梢所产生的促进大鼠异氟醚后觉醒的效应,提示VTA区的多巴胺神经神经元介导了orexin调控VTA区产生的促进觉醒效应。     

Feeding-elicited cataplexy in orexin knockout mice

Mice lacking orexin/hypocretin signaling have sudden episodes of atonia and paralysis during active wakefulness. These events strongly resemble cataplexy, episodes of sudden muscle weakness triggered by strong positive emotions in people with narcolepsy, but it remains unknown whether murine cataplexy is triggered by positive emotions. To determine whether positive emotions elicit murine cataplexy, we placed orexin knockout (KO) mice on a scheduled feeding protocol with regular or highly palatable food. Baseline sleep/wake behavior was recorded with ad libitum regular chow. Mice were then placed on a scheduled feeding protocol in which they received 60% of their normal amount of chow 3 h after dark onset for the next 10 days. Wild-type and KO mice rapidly entrained to scheduled feeding with regular chow, with more wake and locomotor activity prior to the feeding time. On day 10 of scheduled feeding, orexin KO mice had slightly more cataplexy during the food-anticipation period and more cataplexy in the second half of the dark period, when they may have been foraging for residual food. To test whether more palatable food increases cataplexy, mice were then switched to scheduled feeding with an isocaloric amount of Froot Loops, a food often used as a reward in behavioral studies. With this highly palatable food, orexin KO mice had much more cataplexy during the food-anticipation period and throughout the dark period. The increase in cataplexy with scheduled feeding, especially with highly palatable food, suggests that positive emotions may trigger cataplexy in mice, just as in people with narcolepsy. Establishing this connection helps validate orexin KO mice as an excellent model of human narcolepsy and provides an opportunity to better understand the mechanisms that trigger cataplexy.     

The aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), blocks testosterone-induced olfactory behaviour in the hamster

The effect of ATD on olfactory investigation in intact and in castrated, testosterone-treated male hamsters was studied using subcutaneous silastic implants. In intact males, there was a dose-dependent action of ATD in reducing sniffing towards novel females and in eliminating the discrimination between females after pre-exposure to vaginal odour. Both sniffing and olfactory discrimination reappeared after removal of ATD implants. Neither the weight nor the general behavioural activity of treated males was affected, indicating a specific behavioural affect. Testosterone (T) maintained olfactory behaviour in castrated males. Untreated castrates and castrates with ATD + T implants showed reduced sniffing and showed no discrimination between females after exposure to female odour. We conclude that conversion of T to oestrogen plays an essential role in the control of male olfactory behaviour.     

Promotion of Sleep by Suvorexant—A Novel Dual Orexin Receptor Antagonist

Abstract: Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX₁R) and orexin 2 receptor (OX₂R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX₁R and OX₂R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX₂R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 μM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.     

Role of orexin/hypocretin in reward-seeking and addiction: Implications for obesity

Orexins (also named hypocretins) are recently discovered neuropeptides made exclusively in the hypothalamus. Recent studies have shown that orexin cells located specifically in lateral hypothalamus (LH) are involved in motivated behavior for drugs of abuse as well as natural rewards. Administration of orexin has been shown to stimulate food consumption, and orexin signaling in VTA has been implicated in intake of high-fat food. In self-administration studies, the orexin 1 receptor antagonist SB-334867 (SB) attenuated operant responding for high-fat pellets, sucrose pellets and ethanol, but not cocaine, demonstrating that signaling at orexin receptors is necessary for reinforcement of specific rewards. The orexin system is also implicated in associations between rewards and relevant stimuli. For example, Fos expression in LH orexin neurons varied in proportion to conditioned place preference (CPP) for food, morphine, or cocaine. This Fos expression was altered accordingly for CPP administered during protracted abstinence from morphine or cocaine, when preference for natural rewards was decreased and drug preference was increased. Additionally, orexin has been shown to be involved in reward-stimulus associations in the self-administration paradigm, where SB attenuated cue-induced reinstatement of extinguished sucrose- or cocaine-seeking. Although the specific circuitry mediating the effects of orexin on food reward remains unknown, VTA seems likely to be a critical target for at least some of these orexin actions. Thus, recent studies have established a role for orexin in reward-based feeding, and further investigation is warranted for determining whether function/dysfunction of the orexin system may contribute to the overeating associated with obesity.The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009.     

Orexins promote survival of rat cortical neurons

Orexin A and B (hypocretin-1 and -2) are hypothalamic peptides that exert their biological functions by stimulation of two specific, membrane-bound receptors, OX₁R and OX₂R. Recently, we have demonstrated the expression of both types of orexin receptors in rat cortical neurons, with the OX₂R level being markedly higher compared to OX₁R. In the present study we investigated the receptor-mediated effects of orexin A, an agonist of OX₁R and OX₂ R, orexin B and [Ala¹¹-D-Leu¹⁵]orexin B, preferential agonists of OX₂R, on survival of cultured neurons derived from rat cerebral cortex. The three tested peptides markedly increased neuronal viability in a concentration-dependent manner. The pro-survival properties of orexins were associated with an attenuation of caspase-3 activity. Comparable potency of orexin A, orexin B and [Ala¹¹-D-Leu¹⁵]orexin B suggests a predominant role of OX₂R in the studied phenomenon. Our findings provide new insights into the role of orexins in CNS as potential neuroprotective factors.     

Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity.     

Interaction between the noradrenergic and serotonergic systems in locomotor hyperactivity and striatal expression of Fos induced by amphetamine in rats

It is classically considered that Amphetamine acts by increasing extracellular dopamine levels. However, some data suggest a relevant role of other neurochemical systems. The striatum is of particular interest to the study of this question. We have investigated the involvement of the noradrenergic and serotonergic systems and their possible interaction in the striatal responses to Amphetamine using a double behavioral and immunohistochemical approach (i.e., changes in locomotor activity and striatal expression of Fos). In normal rats, Amphetamine induced locomotor hyperactivity and striatal expression of Fos. Pretreatment with the ₁-adrenergic-receptor antagonist Prazosin or lesion of the serotonergic system significantly reduced the locomotor hyperactivity and striatal Fos expression induced by Amphetamine. Administration of Prazosin to rats with serotonergic denervation did not produce any further reduction in the Amphetamine-induced locomotor hyperactivity or striatal Fos expression compared with that observed in rats with serotonergic denervation only. Amphetamine did not induce a detectable increase in Fos expression in dopamine-denervated striata, and elicited intense rotation towards the dopamine-denervated side. This suggests that striatal dopamine release is essential in the Amphetamine-induced effects on striatal neurons. However, the noradrenergic system plays an important role, and the serotonergic system is necessary for mediating the effects of the Amphetamine-induced noradrenergic stimulation. Concurrent stimulation of dopaminergic and serotonergic receptors appears necessary to regulate Amphetamine-induced responses in the striatal neurons.