Changes in sensorimotor cortex oscillatory activity by orexin-A in the ventrolateral preoptic area of the hypothalamus reflect increased muscle tone.

Orexin-A (OXA) is a hypothalamic neuropeptide implicated in the regulation of wakefulness, appetite, reward processing, muscle tone, motor activity, and other physiological processes. The broad range of systems affected stems from the widespread projections of orexin neurons toward multiple brain regions regulating numerous physiological processes. Orexin neurons integrate nutritional, energetic, and behavioral cues and modulate the functions of target structures. Orexin promotes spontaneous physical activity (SPA), and we recently showed that orexin injected into the ventrolateral preoptic area (VLPO) of the hypothalamus increases behavioral arousal and SPA in rats. However, the specific mechanisms underlying the role of orexin in physical activity are unknown. Here we tested the hypothesis that OXA injected into the VLPO alters the oscillatory activity in the electroencephalogram (EEG) to reflect an increased excitability of the sensorimotor cortex, which may explain the associated increase in SPA. The results showed that OXA increased wakefulness following injections into the VLPO. In addition, OXA altered the power spectrum of the EEG during the awake state by decreasing the power of 5-19 Hz oscillations and increasing the power of >35 Hz oscillations, which are markers of increased sensorimotor excitability. Consistently, we found that OXA induced greater muscle activity. Furthermore, we found a similar change in power spectrum during slow-wave sleep, which suggests that OXA altered the EEG activity in a fundamental way, even in the absence of physical activity. These results support the idea that OXA increases the excitability of the sensorimotor system, which may explain the corresponding increase in awake time, muscle tone, and SPA.

Orexin enhances neuronal synchronization in adult rat hypothalamic culture: a model to study hypothalamic function.

The regulation of sleep/wake behavior and energy homeostasis is maintained in part by the hypothalamic neuropeptide orexin A (OXA, hypocretin). Reduction in orexin signaling is associated with sleep disorders and obesity, whereas higher lateral hypothalamic (LH) orexin signaling and sensitivity promotes obesity resistance. Similarly, dysregulation of hypothalamic neural networks is associated with onset of age-related diseases, including obesity and several neurological diseases. Despite the association of obesity and aging, and that adult populations are the target for the majority of pharmaceutical and obesity studies, conventional models for neuronal networks utilize embryonic neural cultures rather than adult neurons. Synchronous activity describes correlated changes in neuronal activity between neurons and is a feature of normal brain function, and is a measure of functional connectivity and final output from a given neural structure. Earlier studies show alterations in hypothalamic synchronicity following behavioral perturbations in embryonic neurons obtained from obesity-resistant rats and following application of orexin onto embryonic hypothalamic cultures. Synchronous network dynamics in adult hypothalamic neurons remain largely undescribed. To address this, we established an adult rat hypothalamic culture in multi-electrode-array (MEA) dishes and recorded the field potentials. Then we studied the effect of exogenous orexin on network synchronization of these adult hypothalamic cultures. In addition, we studied the wake promoting effects of orexin in vivo when directly injected into the lateral hypothalamus (LH). Our results showed that the adult hypothalamic cultures are viable for nearly 3 mo in vitro, good quality MEA recordings can be obtained from these cultures in vitro, and finally, that cultured adult hypothalamus is responsive to orexin. These results support that adult rat hypothalamic cultures could be used as a model to study the neural mechanisms underlying obesity. In addition, LH administration of OXA enhanced wakefulness in rats, indicating that OXA enhances wakefulness partly by promoting neural synchrony in the hypothalamus.NEW & NOTEWORTHY This study, for the first time, demonstrates that adult hypothalamic cultures are viable in vitro for a prolonged duration and are electrophysiologically active. In addition, the study shows that orexin enhances neural synchronization in adult hypothalamic cultures.

Orexin, serotonin, and energy balance.

The lateral hypothalamus is critical for the control of ingestive behavior and spontaneous physical activity (SPA), as lesion or stimulation of this region alters these behaviors. Evidence points to lateral hypothalamic orexin neurons as modulators of feeding and SPA. These neurons affect a broad range of systems, and project to multiple brain regions such as the dorsal raphe nucleus, which contains serotoninergic neurons (DRN) important to energy homeostasis. Physical activity is comprised of intentional exercise and SPA. These are opposite ends of a continuum of physical activity intensity and structure. Non-goal-oriented behaviors, such as fidgeting, standing, and ambulating, constitute SPA in humans, and reflect a propensity for activity separate from intentional activity, such as high-intensity voluntary exercise. In animals, SPA is activity not influenced by rewards such as food or a running wheel. Spontaneous physical activity in humans and animals burns calories and could theoretically be manipulated pharmacologically to expend calories and protect against obesity. The DRN neurons receive orexin inputs, and project heavily onto cortical and subcortical areas involved in movement, feeding and energy expenditure (EE). This review discusses the function of hypothalamic orexin in energy-homeostasis, the interaction with DRN serotonin neurons, and the role of this orexin-serotonin axis in regulating food intake, SPA, and EE. In addition, we discuss possible brain areas involved in orexin-serotonin cross-talk; the role of serotonin receptors, transporters and uptake-inhibitors in the pathogenesis and treatment of obesity; animal models of obesity with impaired serotonin-function; single-nucleotide polymorphisms in the serotonin system and obesity; and future directions in the orexin-serotonin field. This article is categorized under: Metabolic Diseases > Molecular and Cellular Physiology.

Synchronous neuronal interactions in rat hypothalamic culture: a novel model for the study of network dynamics in metabolic disorders.

Synchronous neural activity is a feature of normal brain function, and altered synchronization is observed in several neurological diseases. Dysfunction in hypothalamic pathways leads to obesity, suggesting that hypothalamic neural synchrony is critical for energy homeostasis. The lateral hypothalamic orexin neurons are extensively interconnected with other brain structures and are important for energy balance. Earlier studies show that rats with higher orexin sensitivity are obesity resistant. Similarly, topiramate, an anti-epileptic drug, has been shown to reduce weight in humans. Since orexin enhances neuronal excitation, we hypothesized that obesity-resistant rats with higher orexin sensitivity may exhibit enhanced hypothalamic synchronization. We further hypothesized that anti-obesity agents such as orexin and topiramate will enhance hypothalamic synchronization. To test this, we examined neural synchronicity in primary embryonic hypothalamic cell cultures, obtained from embryonic day 18 (E18) obesity-susceptible Sprague-Dawley (SD) and obesity-resistant rats. Hypothalamic tissue was cultured in multielectrode array (MEA), and recordings were performed twice weekly, from 4th to 32nd day in vitro (DIV). Next, we tested the effects of orexin and topiramate application on neural synchronicity of hypothalamic cultures obtained from SD rat embryos. Signals were analyzed for synchronization using cross correlation. Our results showed that (1) obesity-resistant hypothalamus exhibits significantly higher synchronization compared to obesity-sensitive hypothalamus; and (2) orexin and topiramate enhance hypothalamic synchronization. These results support that enhanced orexin sensitivity is associated with greater neural synchronization, and that anti-obesity treatments enhance network synchronization, thus constrain variability in hypothalamic output signals, to extrahypothalamic structures involved in energy homeostasis.

Role of orexin A signaling in dietary palmitic acid-activated microglial cells.

Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment.

Sleep disorders, obesity, and aging: the role of orexin.

The hypothalamic neuropeptides orexin A and B (hypocretin 1 and 2) are important homeostatic mediators of central control of energy metabolism and maintenance of sleep/wake states. Dysregulation or loss of orexin signaling has been linked to narcolepsy, obesity, and age-related disorders. In this review, we present an overview of our current understanding of orexin function, focusing on sleep disorders, energy balance, and aging, in both rodents and humans. We first discuss animal models used in studies of obesity and sleep, including loss of function using transgenic or viral-mediated approaches, gain of function models using exogenous delivery of orexin receptor agonist, and naturally-occurring models in which orexin responsiveness varies by individual. We next explore rodent models of orexin in aging, presenting evidence that orexin loss contributes to age-related changes in sleep and energy balance. In the next section, we focus on clinical importance of orexin in human obesity, sleep, and aging. We include discussion of orexin loss in narcolepsy and potential importance of orexin in insomnia, correlations between animal and human studies of age-related decline, and evidence for orexin involvement in age-related changes in cognitive performance. Finally, we present a summary of recent studies of orexin in neurodegenerative disease. We conclude that orexin acts as an integrative homeostatic signal influencing numerous brain regions, and that this pivotal role results in potential dysregulation of multiple physiological processes when orexin signaling is disrupted or lost.

Use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model.

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.

Neuropeptides controlling energy balance: orexins and neuromedins.

In this chapter, we review the feeding and energy expenditure effects of orexin (also known as hypocretin) and neuromedin. Orexins are multifunctional neuropeptides that affect energy balance by participating in regulation of appetite, arousal, and spontaneous physical activity. Central orexin signaling for all functions originates in the lateral hypothalamus-perifornical area and is likely functionally differentiated based on site of action and on interacting neural influences. The effect of orexin on feeding is likely related to arousal in some ways but is nonetheless a separate neural process that depends on interactions with other feeding-related neuropeptides. In a pattern distinct from other neuropeptides, orexin stimulates both feeding and energy expenditure. Orexin increases in energy expenditure are mainly by increasing spontaneous physical activity, and this energy expenditure effect is more potent than the effect on feeding. Global orexin manipulations, such as in transgenic models, produce energy balance changes consistent with a dominant energy expenditure effect of orexin. Neuromedins are gut-brain peptides that reduce appetite. There are gut sources of neuromedin, but likely the key appetite-related neuromedin-producing neurons are in the hypothalamus and parallel other key anorectic neuropeptide expression in the arcuate to paraventricular hypothalamic projection. As with other hypothalamic feeding-related peptides, hindbrain sites are likely also important sources and targets of neuromedin anorectic action. Neuromedin increases physical activity in addition to reducing appetite, thus producing a consistent negative energy balance effect. Together with the other various neuropeptides, neurotransmitters, neuromodulators, and neurohormones, neuromedin and orexin act in the appetite network to produce changes in food intake and energy expenditure, which ultimately influences the regulation of body weight.

Nicotine self-administration in the rat: effects of hypocretin antagonists and changes in hypocretin mRNA.

RATIONALE: The hypocretin (hcrt) system has been implicated in addiction-relevant effects of several drugs, but its role in nicotine dependence has been little studied. OBJECTIVES: These experiments examined the role of the hcrt system in nicotine reinforcement. METHODS: Rats were trained for nicotine self-administration (NSA) on fixed-ratio schedules. The effects of acute, presession treatments with the hcrtR1 antagonist SB334867 and the hcrtR1/2 antagonist almorexant were examined on NSA maintained on a fixed-ratio (FR) 5 schedule. Gene expression for the hcrt system (mRNA for hcrt, hcrtR1, and hcrtR2) was measured in animals following NSA on a FR 1 schedule for a 19-day period. RESULTS: The hcrtR1 antagonist SB334867 and the hcrtR1/2 antagonist almorexant both reduced NSA dose-dependently (significantly at doses of 30 and 300 mg/kg, respectively); SB334867 did not affect food-maintained responding whereas almorexant (at the 300 mg/kg) did. Tissue from animals collected 5 h after self-administration showed an increase in hcrtR1 mRNA in the arcuate nucleus compared to control subjects. In tissue collected immediately after a similar 19-day self-administration period, mRNA for hcrtR1 was decreased in the rostral lateral hypothalamus compared to controls. CONCLUSIONS: These data confirm a previous report (Hollander et al., Proc Natl Acad Sci U S A 105:19480-19485, 2008) that the hypocretin receptor hcrtR1 is activated in nicotine reinforcement and in addition show that both the arcuate nucleus and lateral hypothalamus are sites at which hcrt receptor mechanisms may influence reinforcement. Different patterns of mRNA expression at different times after NSA suggest that changes in the hcrt system may be labile with time.

Predisposition to late-onset obesity in GIRK4 knockout mice.

G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels mediate the inhibitory effects of many neurotransmitters on excitable cells. Four Girk genes have been identified (Girk1-4). Whereas GIRK4 is associated with the cardiac GIRK channel, Girk4 expression has been detected in a few neuron populations. Here, we used a transgenic mouse expressing enhanced green fluorescent protein (EGFP) under the control of the Girk4 gene promoter to clarify the expression pattern of Girk4 in the brain. Although small subsets of EGFP-positive neurons were evident in some areas, prominent labeling was seen in the hypothalamus. EGFP expression was most pronounced in the ventromedial, paraventricular, and arcuate nuclei, neuron populations implicated in energy homeostasis. Consistent with a contribution of GIRK4-containing channels to energy balance, Girk4 knockout -/- mice were predisposed to late-onset obesity. By 9 months, Girk4-/- mice were approximately 25% heavier than wild-type controls, a difference attributed to greater body fat. Before the development of overweight, Girk4-/- mice exhibited a tendency toward greater food intake and an increased propensity to work for food in an operant task. Girk4-/- mice also exhibited reduced net energy expenditure, despite displaying elevated resting heart rates and core body temperatures. These data implicate GIRK4-containing channels in signaling crucial to energy homeostasis and body weight.

Integration of feeding and spontaneous physical activity: role for orexin.

Spontaneous physical activity is activity that is non-volitional, or subconscious, such as fidgeting and shifting in one's seat, and time spent moving (standing and ambulating). Recent evidence indicates that spontaneous physical activity, and the resulting thermogenesis (non-exercise activity thermogenesis) may be regulated by brain systems. A large number of brain areas, with their associated neurotransmitter populations and connectivity, participate in the regulation of feeding behavior by acting as energy sensing and modulating centers. Although less well characterized, it is likely that a multitude of neurotransmitters and brain areas act to mediate spontaneous physical activity. These two behaviors, feeding and spontaneous physical activity, affect energy intake and expenditure and thus are important to body weight. Interestingly, often the two behaviors are affected simultaneously; when feeding is affected, so too is spontaneous physical activity, and both food intake and physical activity (whether spontaneous or volitional) influence activity of brain areas important to both. Several brain areas and neuropeptides are important to feeding and spontaneous physical activity. The lateral hypothalamus is one area that appears important to both behaviors, as stimulation or lesion of this region produces alterations in feeding behavior and spontaneous physical activity. Orexin neurons, with their central location in the lateral hypothalamus, widespread projections and connectivity to other brain areas important to energy homeostasis, are well situated to perform an integrative function. This review focuses on how hypothalamic orexins participate in both feeding and spontaneous physical activity, and provides potential models for the integration of signals important to both.

Effect of opioid receptor ligands injected into the rostral lateral hypothalamus on c-fos and feeding behavior.

The lateral hypothalamic area (LHa) is an important brain site for the regulation of food intake. Central injection of opioids increases food intake, and the LHa contains mu and kappa opioid receptors, both of which are involved in feeding behavior. It is unclear whether opioids impact feeding when injected directly into the rostral portion of the LHa (rLHa) in rats. We performed a series of studies in which free-feeding rLHa-cannulated rats were injected with opioid agonists (DAMGO, morphine, dynorphin, U-50488H) followed by the measurement of food intake at 1, 2, and 4 h postinjection. To determine whether opioid receptor ligands administered into the rLHa affect neuronal activation in this brain site, we studied cFos immunoreactivity (cFos IR) in response to rLHa stimulation with naltrexone. We found that the only compound that stimulated feeding behavior was morphine. The other agonists had no effect on food consumption. Naltrexone injection into the rLHa increased neural activation in the LHa, indicating the presence of functional opioid receptors in this region. These data suggest that although neuronal activity is affected by opioid agents acting in the rLHa, administration of selective mu and kappa opioid ligands in this subdivision of the LHa does not have a reliable effect on feeding behavior.

Central orexin sensitivity, physical activity, and obesity in diet-induced obese and diet-resistant rats.

Nonexercise activity thermogenesis (NEAT), the most variable component of energy expenditure, can account for differential capacities for human weight gain. Also highly variable, spontaneous physical activity (SPA) may similarly affect weight balance in animals. In the following study, we utilized the rat model of obesity, the diet-induced obese (DIO) rat, as well as the diet-resistant (DR) rat strain, to investigate how access to a high-fat diet alters SPA and the associated energy expenditure (i.e., NEAT). DIO and DR rats showed no differences in the amount of SPA before access to the high-fat diet. After 29 days on a high-fat diet, the DIO rats showed significant decreases in SPA, whereas the DR rats did not. Next, we wanted to determine whether the DIO and DR rats showed differential sensitivity to microinjections of orexin into the paraventricular nucleus of the hypothalamus (PVN). Unilateral guide cannulae were implanted, aimed at the PVN. Orexin A (0, 0.125, 0.25, and 1.0 nmol in 500 nl) was microinjected through the guide cannula into the PVN, then SPA and energy expenditure were measured for 2 h. Using the response to vehicle as a baseline, the DR rats showed significantly greater increase in NEAT compared with the DIO rats. These data indicate that diet-induced obesity is associated with decreases in SPA and a lack of increase in NEAT. A putative mechanism for changes in NEAT that accompany obesity is a decreased sensitivity to the NEAT-activating effects of neuropeptides such as orexin.

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.

Urocortin in the lateral septal area modulates feeding induced by orexin A in the lateral hypothalamus.

The intermediate portion of the lateral septum (LSi) contains high levels of urocortin (UCN) peptide and type 2 corticotropin-releasing hormone (CRH) receptor (CRHR2) and has anatomic and functional connections with the lateral hypothalamus (LH). We tested the effect of UCN in the LSi on feeding. Injection of 10 or 30 pmol UCN into LSi significantly decreased feeding in food-deprived rats for 24 h without producing conditioned taste aversion (CTA). Pretreatment with a CRH receptor antagonist, alpha-helical CRH (alpha-hCRH), blocked the inhibitory effect of UCN on deprivation-induced feeding at 1 and 2 h postinjection. Furthermore, UCN in the LSi significantly decreased feeding induced by LH-injected orexin A at 2 and 4 h postinjection, and addition of alpha-hCRH blocked the inhibitory effect of UCN on orexin A-induced feeding. In conclusion, UCN significantly inhibits feeding induced by deprivation and LH-injected orexin A without producing a CTA, an effect that is mediated by CRHR2. These data define the LSi as an important site for UCN-induced anorexia and indicate that LSi UCN may influence orexin A feeding signals in the LH.