Peripheral ghrelin interacts with orexin neurons in glucostatic signalling

Ghrelin interactions with glycemia in appetite control as well as the potential mechanisms involving the orexin and melanin-concentrating hormone (MCH) neurons in the orexigenic ghrelin signals were investigated by using a specific anti-ghrelin antibody (AGA). Our results confirm that peripheral ghrelin is an important signal in meal initiation and appetite. Employing immunohistochemistry techniques, we found that c-fos positive neurons in the lateral hypothalamus (LH) and perifornical area (PFA) increased after insulin or 2-deoxyglucose administration. Moreover, we have also demonstrated that peripheral ghrelin blockade by the AGA, reduces the orexigenic signal induced by insulin and 2-DG administration probably partly producing a decrease of c-fos immunoreactivity in the LH and PFA as well as a lower activation of orexin neurons. In contrast, the c-fos positive MCH neurons were not apparently affected. In summary, our findings suggest that peripheral ghrelin plays an important role in regulatory "glucostatic" feeding mechanisms by means of its role as a "hunger" signal affecting the LH and PFA areas, which may contribute to energy homeostasis through orexin neurons.     

Nociceptin Induces Hypophagia in the Perifornical and Lateral Hypothalamic Area

Nociceptin/orphanin FQ (N/OFQ) is known to induce food intake when administered into the lateral ventricle or certain brain areas. This is somewhat contradictory to its reward-suppressing role, as food is a strong rewarding stimulus. This discrepancy may be due to the functional diversity of N/OFQ’s target brain areas. N/OFQ has been shown to inhibit orexin and melanin-concentrating hormone (MCH) neurons, both of which are appetite-inducing cells. As the expression of these neurons is largely confined to the lateral hypothalamus/perifornical area (LH/PFA), we hypothesized that N/OFQ inhibits food intake by acting in this area. To test this hypothesis, we examined the effect of local N/OFQ infusion within the LH/PFA on food intake in the rat and found that N/OFQ decreased sugar pellet as well as chow intake. This effect was not seen when the injection site was outside of the LH/PFA, suggesting a site-specific effect. Next, to determine a possible cellular mechanism of N/OFQ action on food intake, whole cell patch clamp recordings were performed on rat orexin neurons. As previously reported in mice, N/OFQ induced a strong and long lasting hyperpolarization. Pharmacological study indicated that N/OFQ directly inhibited orexin neurons by activating ATP-sensitive potassium (KATP) channels. This effect was partially but significantly attenuated by the inhibitors of PI3K, PKC and PKA, suggesting that the N/OFQ signaling is mediated by these protein kinases. In summary, our results demonstrate a KATP channel-dependent N/OFQ signaling and that N/OFQ is a site-specific anorexic peptide.     

Dynamic interactions between orexin and dynorphin may delay onset of functional orexin effects: a modeling study

Orexin (also known as hypocretin) neurons play a key role in regulating sleep-wake behavior, but the links between orexin neuron electrophysiology and function have not been explored. Orexin neurons are wake-active, and spiking activity in orexin neurons may anticipate transitions to wakefulness by several seconds. However, it is suggested that while the orexin system is necessary to maintain sustained wake bouts, orexin has little effect on brief wake bouts. In vitro experiments investigating the actions of orexin and dynorphin, a colocalized neuropeptide, on orexin neurons indicate that the dynamics of desensitization to dynorphin may represent a mechanism for modulating local network activity and resolving the apparent discrepancy between the onset of firing in orexin neurons and the onset of functional orexin effects. To investigate the role of dynorphin on orexin neuron activity, a Hodgkin-Huxley-type model orexin neuron was developed in which baseline electrophysiology, orexin/dynorphin action, and dynorphin desensitization were closely tied to experimental data. In this model framework, model orexin neuron responses to orexin/dynorphin action were calibrated by simulating the physiologic effects of static orexin and dynorphin bath application on orexin neurons. Then behavior in a small network of model orexin neurons was simulated with pure orexin, pure dynorphin, or combined orexin and dynorphin coupling based on the mechanisms established in the static case. It was found that the dynamics of desensitization to dynorphin can mediate a clear shift from a network in which firing is suppressed by dynorphin-mediated inhibition to a network of neurons with high firing rates sustained by orexin-mediated excitation. The findings suggest that dynamic interactions between orexin and dynorphin at transitions from sleep to wake may delay onset of functional orexin effects.     

Reverse pharmacology of orexin: from an orphan GPCR to integrative physiology

Orexins, which were initially identified as endogenous peptide ligands for two orphan G-protein coupled receptors (GPCRs), have been shown to have an important role in the regulation of energy homeostasis. Furthermore, the discovery of orexin deficiency in narcolepsy patients indicated that orexins are highly important factors for the sleep/wakefulness regulation. The efferent and afferent systems of orexin-producing neurons suggest interactions between these cells and arousal centers in the brainstem as well as important feeding centers in the hypothalamus. Electrophysiological studies have shown that orexin neurons are regulated by humoral factors, including leptin, glucose, and ghrelin as well as monoamines and acetylcholin. Thus, orexin neurons have functional interactions with hypothalamic feeding pathways and monoaminergic/cholinergic centers to provide a link between peripheral energy balance and the CNS mechanisms that coordinate sleep/wakefulness states and motivated behavior such as food seeking.     

Regulation of orexin neurons by the monoaminergic and cholinergic systems

Orexins are a pair of neuropeptides implicated in energy homeostasis and arousal. Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarization-activated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; noradrenaline and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuitries between these nuclei for either a positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.     

Interaction between sleep mechanisms and orexin neurons

Orexin is a neuropeptide that plays a highly important role in mechanisms that regulate sleep/wake states. Lack of the orexin gene or orexin-producing neurons (orexin neurons) results in narcolepsy in several mammalian species, suggesting that orexin is an important factor for the maintenance of wakefulness. Constitutive, ectopic expression of orexin in transgenic mice resulted in severe fragmentation of non–rapid eye movement sleep, along with abnormal muscle tone regulation during REM sleep, suggesting that activity of orexin neurons should be appropriately decreased during sleep to maintain consolidated sleep states. This review will discuss the mechanisms by which the orexin system is regulated during sleep.

     

Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats

Ghrelin, a gut-brain peptide, is best known for its role in the stimulation of feeding and growth hormone release. In the brain, orexin, neuropeptide Y (NPY), and ghrelin are parts of a food intake regulatory circuit. Orexin and NPY are also implicated in maintaining wakefulness. Previous experiments in our laboratory revealed that intracerebroventricular injections of ghrelin induce wakefulness in rats. To further elucidate the possible role of ghrelin in the regulation of arousal, we studied the effects of microinjections of ghrelin into hypothalamic sites, which are implicated in the regulation of feeding and sleep, such as the lateral hypothalamus (LH), medial preoptic area (MPA), and paraventricular nucleus (PVN) on sleep in rats. Sleep responses, motor activity, and food intake after central administration of 0.04, 0.2, or 1 mug (12, 60, or 300 pmol) ghrelin were recorded. Microinjections of ghrelin into the LH had strong wakefulness-promoting effects lasting for 2 h. Wakefulness was also stimulated by ghrelin injection into the MPA and PVN; the effects were confined to the first hour after the injection. Ghrelin's non-rapid-eye-movement sleep-suppressive effect was accompanied by attenuation in the electroencephalographic (EEG) slow-wave activity and changes in the EEG power spectrum. Food consumption was significantly stimulated after microinjections of ghrelin into each hypothalamic site. Together, these results are consistent with the hypothesis that forebrain ghrelinergic mechanisms play a role in the regulation of vigilance, possibly through activating the components of the food intake- and arousal-promoting network formed by orexin and NPY.     

Microdialysis perfusion of orexin-A in the basal forebrain increases wakefulness in freely behaving rats

Recent work indicates that the orexin/hypocretin-containing neurons of the lateral hypothalamus are involved in control of REM sleep phenomena, but site-specific actions in control of wakefulness have been less studied. Orexin-containing neurons project to both brainstem and forebrain regions that are known to regulate sleep and wakefulness, including the field of cholinergic neurons in the basal forebrain (BF) that is implicated in regulation of wakefulness, and includes, in the rat, the horizontal limb of the diagonal band, the substantia innominata, and the magnocellular preoptic region. The present study used microdialysis perfusion of orexin-A directly in the cholinergic BF region of rat to test the hypothesis that orexin-A enhances W via a local action in the BF. A significant dose-dependent increase in W was produced by the perfusion of three doses of orexin-A in the BF (0.1, 1.0, and 10.0 microM), with 10.0 microM producing more than a 5-fold increase in wakefulness, which occupied 44% of the light (inactive) phase recording period. Orexin-A perfusion also produced a significant dose-dependent decrease in nonREM sleep, and a trend-level decrease in REM sleep. The results clearly demonstrate a potent capacity of orexin-A to induce wakefulness via a local action in the BF, and are consistent with previous work indicating that the BF cholinergic zone neurons have a critical role in the regulation of EEG activation and W. The data suggest further that orexin-A has a significant role in the regulation of arousal/wakefulness, in addition to the previously described role of orexin in the regulation and expression of REM sleep and REM sleep-related phenomena.     

The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish.

Both glutamate and gamma-aminobutyric acid (GABA) are involved in pituitary hormone release in fish. Glutamate serves 2 purposes, both as a neurotransmitter and as a precursor for GABA synthesis. Glutamate can be catabolized to GABA by the actions of 2 distinct but related enzymes, glutamate decarboxylase 65 (GAD65) and GAD67. They derive from 2 different genes that likely arose from an early gene duplication prior to the emergence of teleosts more than 400 million years ago. There is good evidence for the involvement of GABA in luteinizing hormone (LH) release in fish. The mechanism of GABA action to stimulate LH release appears to be a combination of effects on GnRH release, potentiation of gonadotropin hormone-releasing hormone (GnRH) action, and in some cases directly at the LH cell. These actions appear to be dependent on such factors as sex or sex steroid levels, and there may also be species differences. Nevertheless, the stimulatory effects of GABA on LH are present in at least 4 fish species. In contrast, convincing data for the inhibitory effects of GABA on LH release have only been observed in 1 fish species. The sites and mechanisms of action of amino acid neurotransmitters on LH release have yet to be fully characterized. Both 130N-methyl-D-aspartic acid (NMDA) and S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors are likely to have important roles. We suggest that it is a receptor similar to the GABA(A) type which mediates the effects of GABA on LH release in fish, at least partially acting on the GnRH neuron, but likely directly acting at the gonadotroph as well. GABA may also be involved in regulating the release of other pituitary hormones in fish, namely follicle stimulating hormone (FSH = GTH-I), prolactin, and growth hormone. Based on the findings described in this review, a working model for the involvement of glutamate and GABA in the regulation of LH release in teleost fish is proposed.     

Orexin neurons receive glycinergic innervations

Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.     

Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation.

Neurons containing the neuropeptide orexin (hypocretin) are located exclusively in the lateral hypothalamus and send axons to numerous regions throughout the central nervous system, including the major nuclei implicated in sleep regulation. Here, we report that, by behavioral and electroencephalographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy patients, as well as canarc-1 mutant dogs, the only known monogenic model of narcolepsy. Moreover, modafinil, an anti-narcoleptic drug with ill-defined mechanisms of action, activates orexin-containing neurons. We propose that orexin regulates sleep/wakefulness states, and that orexin knockout mice are a model of human narcolepsy, a disorder characterized primarily by rapid eye movement (REM) sleep dysregulation.     

The ATP-cytokine-adenosine hypothesis: How the brain translates past activity into sleep

Tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1), and other cytokines are involved in non-rapid eye movement sleep (NREM) regulation under physiological and inflammatory conditions. Brain levels of IL1 and TNF increase with prolonged wakefulness. Injection of exogenous IL1 or TNF, mimicking sleep loss, induces sleepiness, excess sleep, fatigue, poor cognition, and enhanced sensitivity to pain. These symptoms characterize the syndrome associated with sleep loss. Extracellular ATP released during neuro- and glio-transmission, acting via purine P2 receptors on glia, releases IL1 and TNF. This extracellular ATP mechanism may provide an index of activity used by the brain to keep track of prior wakefulness. Prolonged wakefulness is associated with enhanced neuronal activity. TNF and IL1, in turn, act on neurons to change their intrinsic properties and sensitivities to neurotransmitters and neuromodulators such as adenosine and glutamate. Such actions change network input–output properties (i.e. state shift). State oscillations, for instance, occur within cortical columns and are responsive to TNF. Sleep is thus viewed as a local usedependent process regulated in part by cytokines. Further, state oscillations are viewed as a fundamental process of any neuronal/glia network. To investigate these hypotheses we developed an in vitro neuronal/glia culture system exhibiting field potential oscillations and have mathematically modeled the local use-dependent view of sleep initiation. These views have profound implications for sleep pathologies and function.

     

Pulsatile LH release is diminished, whereas FSH secretion is normal, in hypocretin-deficient narcoleptic men

Hypocretin (orexin) peptides are involved in the regulation of energy balance and pituitary hormone release. Narcolepsy is a sleep disorder characterized by disruption of hypocretin neurotransmission. Pituitary LH secretion is diminished in hypocretin-deficient animal models, and intracerebroventricular administration of hypocretin-1 activates the hypothalamo-pituitary-gonadal axis in rats. We evaluated whether hypocretin deficiency affects gonadotropin release in humans. To this end, we deconvolved 24-h serum concentrations of LH and FSH in seven hypocretin-deficient narcoleptic males (N) and seven controls (C) matched for age, body mass index, and sex. Basal plasma concentrations of testosterone, estradiol, and sex hormone-binding globulin were similar in both groups. Mean 24-h LH concentration was significantly lower in narcolepsy patients [3.0 +/- 0.4 (N) vs. 4.2 +/- 0.3 (C) U/l, P = 0.01], which was primarily due to a reduction of pulsatile LH secretion [23.5 +/- 1.6 (N) vs. 34.3 +/- 4.9 (C) U.l(-1).24 h(-1), P = 0.02]. The orderliness of LH and FSH secretion, quantitated by the approximate entropy statistic, was greater in patients than in controls. In contrast, all other features of FSH release were similar in narcoleptic and control groups. Also, LH and FSH secretions in response to intravenous administration of 100 microg of GnRH were similar in patients and controls. These data indicate that endogenous hypocretins are involved in the regulation of the hypothalamo-pituitary-gonadal axis activity in humans. In particular, reduced LH release in the face of normal pituitary responsivity to GnRH stimulation in narcoleptic men suggests that hypocretins promote endogenous GnRH secretion.     

Lateral hypothalamic neuropeptides in reward and drug addiction

The hypothalamus has been long considered important in feeding and other motivated behaviors. The identification of neuropeptides expressed in the hypothalamus has initiated efforts to better elucidate the underlying molecular mechanisms involved. The neuropeptides orexin and melanin-concentrating hormone (MCH) are expressed in the lateral hypothalamus (LH) and have been implicated in regulation of feeding behavior. Neurons expressing these neuropeptides have extensive projections to regions of the brain important for behavioral responses to drugs of abuse, raising the possibility that the pathways may also be important in addiction. Regulation of LH intracellular signaling pathways in response to drugs of abuse supports a role for the LH neuropeptides in addiction.     

Different neuronal phenotypes in the lateral hypothalamus and their role in sleep and wakefulness

The sleep disorder narcolepsy is now linked with a loss of neurons containing the neuropeptide hypocretin (also known as orexin). The hypocretin neurons are located exclusively in the lateral hypothalamus, a brain region that has been implicated in arousal based on observations made by von Economo during the viral encephalitic epidemic of 1916–1926. There are other neuronal phenotypes located in the lateral hypothalamus that are distinct and separate from the hypocretin neurons. Here the authors identify these neurons based on peptides and neurotransmitters that they express and review roles of these neurons in sleep. Given the heterogeneity of the neuronal phenotypes in the lateral hypothalamus, it is likely that hypocretin neurons, as well as other types of neurons in the lateral hypothalamus, influence sleep and provide state-dependent regulation of physiological functions.     

Enhanced Slow-Wave EEG Activity and Thermoregulatory Impairment following the Inhibition of the Lateral Hypothalamus in the Rat

Neurons within the lateral hypothalamus (LH) are thought to be able to evoke behavioural responses that are coordinated with an adequate level of autonomic activity. Recently, the acute pharmacological inhibition of LH has been shown to depress wakefulness and promote NREM sleep, while suppressing REM sleep. These effects have been suggested to be the consequence of the inhibition of specific neuronal populations within the LH, i.e. the orexin and the MCH neurons, respectively. However, the interpretation of these results is limited by the lack of quantitative analysis of the electroencephalographic (EEG) activity that is critical for the assessment of NREM sleep quality and the presence of aborted NREM-to-REM sleep transitions. Furthermore, the lack of evaluation of the autonomic and thermoregulatory effects of the treatment does not exclude the possibility that the wake-sleep changes are merely the consequence of the autonomic, in particular thermoregulatory, changes that may follow the inhibition of LH neurons. In the present study, the EEG and autonomic/thermoregulatory effects of a prolonged LH inhibition provoked by the repeated local delivery of the GABA_A agonist muscimol were studied in rats kept at thermoneutral (24°C) and at a low (10°C) ambient temperature (Ta), a condition which is known to depress sleep occurrence. Here we show that: 1) at both Tas, LH inhibition promoted a peculiar and sustained bout of NREM sleep characterized by an enhancement of slow-wave activity with no NREM-to-REM sleep transitions; 2) LH inhibition caused a marked transitory decrease in brain temperature at Ta 10°C, but not at Ta 24°C, suggesting that sleep changes induced by LH inhibition at thermoneutrality are not caused by a thermoregulatory impairment. These changes are far different from those observed after the short-term selective inhibition of either orexin or MCH neurons, suggesting that other LH neurons are involved in sleep-wake modulation.     

A locally generated angiotensin system in rat carotid body

Orexinergic neurons originating in the perifornical, lateral hypothalamus project to numerous brain sites including neuroendocrine centers known to be important in the physiologic response to stress. Those projections suggest an action of endogenous orexin on adrenocorticotropin (ACTH) release, either by neuromodulatory effects in the paraventricular nucleus (PVN), or by neuroendocrine actions in the pituitary gland following release into the median eminence. We sought to determine if exogenously applied orexin A might act in the brain to alter ACTH release and to determine if a site of action in the hypothalamic paraventricular nucleus could be identified. Cerebroventricular administration of orexin A in conscious male rats resulted in a dose-related elevation in circulating ACTH levels. At 30 min post-infusion, ACTH levels were elevated 2.5-fold by the low dose of orexin A (0.3 nmol), 5.7-fold by the middle dose tested (1.0 nmol), and 7.5-fold by the highest dose tested (3.0 nmol). Pretreatment with a CRH-antagonist (i.v.) blocked the ability of i.c.v. administered orexin A to activate the hypothalamo-pituitary-adrenal (HPA) axis. Bath application of orexin A in hypothalamic slice preparations resulted in depolarizations (8.0+/-0.6 mV), accompanied by increases in spike frequency in identified magno- and parvocellular neurons in the PVN. Our data suggest a potential role for endogenous orexin in the hypothalamic regulation of stress hormone secretion.     

The neuroendocrinology of human sleep

Sleep consists of several distinct patterns of CNS activation, including synchronized or slow wave of sleep and desynchronized or rapid eye movement sleep. Specific areas of the brain as well as specific biogenic amine neurotransmitters appear to be responsible for the periodic shifts between sleep stages. Hypothalamic regulation of the anterior pituitary gland also is influenced by the same biogenic amine neurotransmitters, and the episodic release patterns of the anterior pituitary hormones suggest prominent CNS influences. This review considers the relationship of these hormone release patterns to the circadian sleep-wake cycle and to sleep staging within the sleep period itself.     

A Mathematical Model for the Actions of Activin,Inhibin, and Follistatin on Pituitary Gonadotrophs

The timed secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs during the estrous cycle is crucial for normal reproductive functioning. The release of LH and FSH is stimulated by gonadotropin releasing hormone (GnRH) secreted by hypothalamic GnRH neurons. It is controlled by the frequency of the GnRH signal that varies during the estrous cycle. Curiously, the secretion of LH and FSH is differentially regulated by the frequency of GnRH pulses. LH secretion increases as the frequency increases within a physiological range, and FSH secretion shows a biphasic response, with a peak at a lower frequency. There is considerable experimental evidence that one key factor in these differential responses is the autocrine/paracrine actions of the pituitary polypeptides activin and follistatin. Based on these data, we develop a mathematical model that incorporates the dynamics of these polypeptides. We show that a model that incorporates the actions of activin and follistatin is sufficient to generate the differential responses of LH and FSH secretion to changes in the frequency of GnRH pulses. In addition, it shows that the actions of these polypeptides, along with the ovarian polypeptide inhibin and the estrogen-mediated variations in the frequency of GnRH pulses, are sufficient to account for the time courses of LH and FSH plasma levels during the rat estrous cycle. That is, a single peak of LH on the afternoon of proestrus and a double peak of FSH on proestrus and early estrus. We also use the model to identify which regulation pathways are indispensable for the differential regulation of LH and FSH and their time courses during the estrous cycle. We conclude that the actions of activin, inhibin, and follistatin are consistent with LH/FSH secretion patterns, and likely complement other factors in the production of the characteristic secretion patterns in female rats.