Local network regulation of orexin neurons in the lateral hypothalamus

Obesity and inadequate sleep are among the most common causes of health problems in modern society. Thus, the discovery that orexin (hypocretin) neurons play a pivotal role in sleep/wake regulation, energy balance, and consummatory behaviors has sparked immense interest in understanding the regulatory mechanisms of these neurons. The local network consisting of neurons and astrocytes within the lateral hypothalamus and perifornical area (LH/PFA), where orexin neurons reside, shapes the output of orexin neurons and the LH/PFA. Orexin neurons not only send projections to remote brain areas but also contribute to the local network where they release multiple neurotransmitters to modulate its activity. These neurotransmitters have opposing actions, whose balance is determined by the amount released and postsynaptic receptor desensitization. Modulation and negative feedback regulation of excitatory glutamatergic inputs as well as release of astrocyte-derived factors, such as lactate and ATP, can also affect the excitability of orexin neurons. Furthermore, distinct populations of LH/PFA neurons express neurotransmitters with known electrophysiological actions on orexin neurons, such as melanin-concentrating hormone, corticotropin-releasing factor, thyrotropin-releasing hormone, neurotensin, and GABA. These LH/PFA-specific mechanisms may be important for fine tuning the firing activity of orexin neurons to maintain optimal levels of prolonged output to sustain wakefulness and stimulate consummatory behaviors. Building on these exciting findings should shed further light onto the cellular mechanisms of energy balance and sleep-wake regulation.     

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression,sleep and behaviour

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF‐α) is an important neuroinflammation mediator. Here, we examined the effects of TNF‐α treatment on hypocretin expression in vivo and behaviour in mice. TNF‐α decreased hypocretin 1 and hypocretin 2 expression in a dose‐dependent manner in cultured hypothalamic neurons. TNF‐α decreased mRNA stability of prepro‐hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF‐α demonstrated decreased expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF‐α, prepro‐hypocretin mRNA decay was increased in hypothalamus. TNF‐α neutralizing antibody restored the expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF‐α challenged mice, supporting hypocretin system can be impaired by increased TNF‐α through decreasing hypocretin expression. Repeated TNF‐α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF‐α neutralizing antibody blocked the effects of TNF‐α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF‐α. The data support that TNF‐α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.     

CSF hypocretin-1/orexin-A concentrations in patients with subarachnoid hemorrhage (SAH)

The aim of this study was to examine the role of the hypothalamic hypocretin/orexin system in complications of delayed ischemic neuronal deficit (DIND) resulting from symptomatic vasospasm in patients with aneurysmal subarachnoid hemorrhage (SAH). CSF hypocretin-1/orexin-A levels were measured in 15 SAH patients. DIND complications occurred in seven patients with symptomatic vasospasm. Hypocretin-1/orexin-A levels were low in SAH patients during the 10 days following the SAH event. CSF hypocretin-1/orexin-A levels were lower in patients with DIND complications than in those who did not develop DIND. A significant transient decline in CSF hypocretin-1/orexin-A levels was also observed at the onset of DIND in all patients with symptomatic vasospasm. The reduced hypocretin/orexin production observed in SAH patients may reflect reduced brain function due to the decrease in cerebral blood flow. These results, taken together with recent experimental findings in rats that indicate hypocretin receptor 1 (orexin 1 receptor) mRNA and protein are elevated following middle cerebral artery occlusion, suggest that a reduction in hypocretin/orexin production in SAH and DIND patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia.     

Lesions of orexin neurons block conditioned place preference for sexual behavior in male rats

The hypothalamic neuropeptide orexin (hypocretin) mediates reward related to drugs of abuse and food intake. However, a role for orexin in sexual reward has yet to be investigated. Orexin neurons are activated by sexual behavior, but endogenous orexin does not appear to be essential for sexual performance and motivation in male rats. Therefore, the goal of the current study was to test the hypothesis that orexin is critically involved in processing of sexual reward in male rats. First, it was demonstrated following exposure to conditioned contextual cues associated with sexual behavior in a conditioned place preference paradigm that cFos expression is induced in orexin neurons, indicating activation of orexin neurons by cues predicting sexual reward. Next, orexin-cell specific lesions were utilized to determine the functional role of orexin in sexual reward processing. Hypothalami of adult male rats were infused with orexin-B-conjugated saporin, resulting in greater than 80% loss of orexin neurons in the perifornical-dorsomedial and lateral hypothalamus. Orexin lesions did not affect expression of sexual behavior, but prevented formation of conditioned place preference for a sexual behavior paired chamber. In contrast, intact sham-treated males or males with partial lesions developed a conditioned place preference for mating. Orexin lesioned males maintained the ability to form a conditioned place aversion to lithium chloride-induced visceral illness, indicating that orexin lesions did not disrupt associative contextual memory. Overall, these findings suggest that orexin is not essential for sexual performance or motivation, but is critical for reward processing and conditioned cue-induced seeking of sexual behavior.     

Extinction and Renewal of Conditioned Sexual Responses

Introduction

Extinction involves an inhibitory form of new learning that is highly dependent on the context for expression. This is supported by phenomena such as renewal and spontaneous recovery, which may help explain the persistence of appetitive behavior, and related problems such as addictions. Research on these phenomena in the sexual domain is lacking, where it may help to explain the persistence of learned sexual responses.

Method

Men (n = 40) and women (n = 62) participated in a differential conditioning paradigm, with genital vibrotactile stimulation as US and neutral pictures as conditional stimuli (CSs). Dependent variables were genital and subjective sexual arousal, affect, US expectancy, and approach and avoid tendencies towards the CSs. Extinction and renewal of conditioned sexual responses were studied by context manipulation (AAA vs. ABA condition).

Results

No renewal effect of genital conditioned responding could be detected, but an obvious recovery of US expectancy following a context change after extinction (ABA) was demonstrated. Additionally, women demonstrated recovery of subjective affect and subjective sexual arousal. Participants in the ABA demonstrated more approach biases towards stimuli.

Conclusions

The findings support the context dependency of extinction and renewal of conditioned sexual responses in humans. This knowledge may have implications for the treatment of disturbances in sexual appetitive responses such as hypo- and hypersexuality.     

An arousing discovery on catalepsy: orexin regulates vestibular motor functions

A study by Zhang et?al. in this issue of Neuron reveals a novel mechanism of control of vestibular motor functions by the orexin (hypocretin) system in the perifornical/LH area through the lateral vestibular nucleus in the brainstem. This knowledge provides new insights into the understanding of brain circuitry that controls motor functions and diseases/conditions related to impairments in this circuitry.     

Orexin synthesis and response in the gut

Orexin (hypocretin) appears to play a role in the regulation of energy balances. Previous reports have indicated that orexin-containing neurons are found only in the lateral hypothalamic (LH) area. We show that a subset of neurons in the gut which also express leptin receptors display orexin-like immunoreactivity and express functional orexin receptors. Orexin excites secretomotor neurons in the guinea pig submucosal plexus and increases motility. Moreover, fasting upregulates the phosphorylated form of cAMP response element-binding protein (pCREB) in orexin-immunoreactive neurons, indicating a functional response to food status in these cells. Together, these data suggest that orexin in the gut may play an even more intimate role in regulating energy homeostasis than it does in the CNS.     

Review. Context-induced relapse to drug seeking: a review

In humans, exposure to environmental contexts previously associated with drug intake often provokes relapse to drug use, but the mechanisms mediating this relapse are unknown. Based on early studies by Bouton & Bolles on context-induced 'renewal' of learned behaviours, we developed a procedure to study context-induced relapse to drug seeking. In this procedure, rats are first trained to self-administer drug in one context. Next, drug-reinforced lever responding is extinguished in a different (non-drug) context. Subsequently, context-induced reinstatement of drug seeking is assessed by re-exposing rats to the drug-associated context. Using variations of this procedure, we and others reported reliable context-induced reinstatement in rats with a history of heroin, cocaine, heroin-cocaine combination, alcohol and nicotine self-administration. Here, we first discuss potential psychological mechanisms of context-induced reinstatement, including excitatory and inhibitory Pavlovian conditioning, and occasion setting. We then summarize results from pharmacological and neuroanatomical studies on the role of several neurotransmitter systems (dopamine, glutamate, serotonin and opioids) and brain areas (ventral tegmental area, accumbens shell, dorsal striatum, basolateral amygdala, prefrontal cortex, dorsal hippocampus and lateral hypothalamus) in context-induced reinstatement. We conclude by discussing the clinical implications of rat studies on context-induced reinstatement of drug seeking.     

The Hypocretin/Orexin System: Implications for Drug Reward and Relapse

Hypocretins (also known as orexins) are hypothalamic neuropeptides involved in the regulation of sleep/wake states and feeding behavior. Recent studies have also demonstrated an important role for the hypocretin/orexin system in the addictive properties of drugs of abuse, consistent with the reciprocal innervations between hypocretin neurons and brain areas involved in reward processing. This system participates in the primary reinforcing effects of opioids, nicotine, and alcohol. Hypocretins are also involved in the neurobiological mechanisms underlying relapse to drug-seeking behavior induced by drug-related environmental stimuli and stress, as mainly described in the case of psychostimulants. Based on these preclinical studies, the use of selective ligands targeting hypocretin receptors could represent a new therapeutical strategy for the treatment of substance abuse disorders. In this review, we discuss and update the current knowledge about the participation of the hypocretin system in drug addiction and the possible neurobiological mechanisms involved in these processes regulated by hypocretin transmission.     

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo. Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product. Conversely, processed mRNA can be added to the embryo to increase levels of a gene product. The vehicle for adding either mRNA or morpholino to an embryo is microinjection. Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour. This video shows all the steps involved in microinjection. Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish. Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume. Finally, the needle is plunged into the embryo''s yolk and the morpholino or mRNA is expelled.     

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.     

Long-lasting up-regulation of orexin receptor type 2 protein levels in the rat nucleus accumbens after chronic cocaine administration

Hypothalamic orexin (hypocretin) neurons project to the key structures of the limbic system and orexin receptors, both orexin receptor type 1 (OXR1) and type 2 (OXR2), are expressed in most limbic regions. Emerging evidence suggests that orexin is among important neurotransmitters that regulate addictive properties of drugs of abuse. In this study, we examined the effect of psychostimulant cocaine on orexin receptor protein abundance in the rat limbic system in vivo. Intermittent administration of cocaine (20 mg/kg, i.p., once daily for 5 days) caused a typical behavioral sensitization response to a challenge cocaine injection at a 14-day withdrawal period. Repeated cocaine administration at the same withdrawal time also increased OXR2 protein levels in the nucleus accumbens while repeated cocaine had no effect on OXR1 and orexin neuropeptide (both orexin-A and orexin-B) levels in this region. In contrast to the nucleus accumbens, OXR2 levels in the frontal cortex, the ventral tegmental area, the hippocampus, and the dorsal striatum (caudate putamen) were not altered by cocaine. Remarkably, the up-regulated OXR2 levels in the nucleus accumbens showed a long-lasting nature as it persisted up to 60 days after the discontinuation of repeated cocaine treatments. In contrast to chronic cocaine administration, an acute cocaine injection was insufficient to modify levels of any orexin receptor and peptide. Our data identify the up-regulation of OXR2 in the nucleus accumbens as an enduring molecular event that is correlated well with behavioral plasticity in response to chronic psychostimulant administration. This OXR2 up-regulation may reflect a key adaptation of limbic orexinergic transmission to chronic drug exposure and may thus be critical for the expression of motor plasticity.     

Orexin A/Hypocretin Modulates Leptin Receptor-Mediated Signaling by Allosteric Modulations Mediated by the Ghrelin GHS-R1A Receptor in Hypothalamic Neurons

The hypothalamus is a key integrator of nutrient-seeking signals in the form of hormones and metabolites originated in both the central nervous system and the periphery. The main autocrine and paracrine target of orexinergic-related hormones such as leptin, orexin/hypocretin, and ghrelin are neuropeptide Y neurons located in the arcuate nucleus of the hypothalamus. The aim of this study was to investigate the expression and the molecular and functional relationships between leptin, orexin/hypocretin and ghrelin receptors. Biophysical studies in a heterologous system showed physical interactions between them, with potential formation of heterotrimeric complexes. Functional assays showed robust allosteric interactions particularly different when the three receptors are expressed together. Further biochemical and pharmacological assays provided evidence of heterotrimer functional expression in primary cultures of hypothalamic neurons. These findings constitute evidence of close relationships in the action of the three hormones already starting at the receptor level in hypothalamic cells.     

Neurogenesis and neurite outgrowth in the spinal cord of chicken embryos and in primary cultures of spinal neurons following knockdown of Class III beta tubulin with antisense morpholinos

Microtubules are the primary cytoskeletal constituent of extending neurites. We used antisense morpholinos to knock down expression of neuron-specific Class III beta tubulin in the right half of the neural tube of chicken embryos in ovo. There was a significant (p < 0.01) reduction in the number of Class III beta tubulin immunostained interneurons 24 h following electroporation of the morpholinos when compared with the contralateral side of the neural tube. However, neural crest-derived sensory neurons labeled with the fluorescently tagged morpholinos developed distinct processes. Moreover, there was no significant difference in the number of interneurons labeled on either side of the neural tube with a second marker of developing neurons, anti-microtubule associated protein (MAP) 1b. Neural tubes were also excised and dissociated following antisense or control morpholino electroporation. The resulting neurons were cultured for 48 h and immunostained with anti-Class III beta tubulin and anti-MAP 1b. Neurons that had taken up the antisense morpholino had significantly shorter neurites (p < 0.01) than neurons from the same neural tubes that did not; they also had significantly shorter neurites (p < 0.05) than labeled neurons from neural tubes electroporated with a control morpholino. Thus, normal expression of Class III beta tubulin may not be necessary for neurogenesis in the early avian spinal cord in situ, but is required for neurite outgrowth in vitro.     

Gonadal steroids modulated hypocretin/orexin type-1 receptor expression in a brain region, sex and daytime specific manner

Orexins A and B (hypocretins A and B) are regulatory peptides that control a variety of neuroendocrine and autonomic functions including feeding and sleep-wakefulness. Previously, we described a clear relationship between the hormonal milieu of the estrous cycle and the mRNA expression of the components of the orexinergic system, in the hypothalamus, pituitary and ovary. Here, we investigate whether steroid hormones are involved in the modulation of the hypocretin/orexin type-1 receptor expression at the protein level, and its time of the day dependence, in hypothalamus and pituitary of castrated male and female rats and castrated receiving hormone replacement.Orchidectomy decreased the hypocretin/orexin type-1 receptor expression in anterior hypothalamus, but not in mediobasal hypothalamus or cortex; in pituitary this treatment resulted in an increase. Testosterone and dihydrotestosterone were able to restore receptor expression and gonadotropins.In females, pituitary and ovarian hormones increased during proestrous afternoon. Hypocretin/orexin type-1 receptor expression was higher at 19:00 of proestrus in hypothalamus and pituitary. Ovariectomized treated with estradiol or oil and sacrificed at 11:00 h showed the receptor expression similar to 11:00 h of proestrus in hypothalamus and pituitary. At 19:00 h, low expression persisted in these areas in oil-treated ovariectomized rats; in contrast, estradiol replacement increased the expression to high levels of normal cycling rats at 19:00 h.Sexual steroids modulate the orexinergic system and the anatomical regions, hormones and times of the day all have to be considered when the roles of orexins, and probably other peptides, are under consideration.     

Acid sensing ion channel 1 in lateral hypothalamus contributes to breathing control

Acid-sensing ion channels (ASICs) are present in neurons and may contribute to chemoreception. Among six subunits of ASICs, ASIC1 is mainly expressed in the central nervous system. Recently, multiple sites in the brain including the lateral hypothalamus (LH) have been found to be sensitive to extracellular acidification. Since LH contains orexin neurons and innervates the medulla respiratory center, we hypothesize that ASIC1 is expressed on the orexin neuron and contributes to acid-induced increase in respiratory drive. To test this hypothesis, we used double immunofluorescence to determine whether ASIC1 is expressed on orexin neurons in the LH, and assessed integrated phrenic nerve discharge (iPND) in intact rats in response to acidification of the LH. We found that ASIC1 was co-localized with orexinA in the LH. Microinjection of acidified artificial cerebrospinal fluid increased the amplitude of iPND by 70% (pH 7.4 v.s. pH 6.5∶1.05±0.12 v.s. 1.70±0.10, n = 6, P<0.001) and increased the respiratory drive (peak amplitude of iPND/inspiratory time, PA/Ti) by 40% (1.10±0.23 v.s. 1.50±0.38, P<0.05). This stimulatory effect was abolished by blocking ASIC1 with a nonselective inhibitor (amiloride 10 mM), a selective inhibitor (PcTX1, 10 nM) or by damaging orexin neurons in the LH. Current results support our hypothesis that the orexin neuron in the LH can exert an excitation on respiration via ASIC1 during local acidosis. Since central acidification is involved in breathing dysfunction in a variety of pulmonary diseases, understanding its underlying mechanism may improve patient management.     

Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The cold and drought-induced expression of these genes was ABA-dependent, but divergent in various ABA signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue-specific expression as well as in temporal induction in response to low temperature. To elucidate the function of AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition. Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in antisense plants. These plants displayed increased sensitivity to ABA both during development of frost tolerance and during seed germination, but not in their drought responses. Furthermore, the expression of cold-and ABA-induced genes was enhanced in transgenic antisense plants. Our results suggest that AtPP2CA is a negative regulator of ABA responses during cold acclimation.     

Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology

For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.