Orexin Neurons Research Articles

Role of orexin in exercise-induced leptin sensitivity in the mediobasal hypothalamus of mice

Orexin is known as an important neuropeptide in the regulation of energy metabolism. However, the role of orexin in exercise-induced leptin sensitivity in the hypothalamus has been unclear. In this study, we determined the effect of transient treadmill exercise on leptin sensitivity in the mediobasal hypothalamus (MBH) of mice and examined the role of orexin in post-exercise leptin sensitivity. Treadmill running for 45 min increased the orexin neuron activity in mice. Intraperitoneal injection of a submaximal dose of leptin after exercise stimulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MBH of mice post-exercise compared with that in non-exercised mice, although intracerebroventricular (icv) injection of leptin did not enhance STAT3 phosphorylation, even after exercise. Icv injection of an orexin receptor antagonist, SB334867 reduced STAT3 phosphorylation, which was enhanced by icv injection of orexin but not by direct injection of orexin into MBH. Exercise increased the phosphorylation of extracellular signal-regulated kinases (ERKs) in the MBH of mice, while ERK phosphorylation was reduced by SB334867. Leptin injection after exercise increased the leptin level in MBH, whereas icv injection of SB334867 suppressed the increase in the leptin level in MBH of mice. These results indicate that the activation of orexin neurons by exercise may contribute to the enhancement of leptin sensitivity in MBH. This effect may be mediated by increased transportation of circulating leptin into MBH, with the involvement of ERK phosphorylation.

Identification of nutrient metabolites capable of altering the epigenetic status at specific loci

Nutrients can influence cellular functions associated with physiological and pathological processes. Major energy sources in our body, such as glucose and amino acids, are decomposed, resynthesized via numerous synthetic pathways, and finally, metabolized into energy molecules (e.g., ATP) and metabolites involved in various molecular events such as gene expression. Our previous study demonstrates that a sugar metabolite, N‐acetyl mannosamine, when used as a supplement in the culture medium, can induce the differentiation of pluripotent stem cells into orexin neurons and activate the epigenetic status at the orexin‐coding gene locus. Thus, our observations suggest that some metabolites can change the fate of a cell by participating in epigenetic alterations at specific loci. However, there is insufficient information about the role of specific nutrients and their metabolites in altering the epigenetic status. In the present study, we aimed to screen for metabolites that function as epigenetic modulators. We supplemented the culture medium with 129 metabolites individually during the induction of neural differentiation from human iPS cells (hiPSCs). We investigated the epigenetic status at 40 loci of the neural peptide‐coding genes by ChIP‐qPCR, using a high‐throughput platform. Trimethylation of lysine 4 of histone H3 (H3K4me3) and histone O‐GlcNAcylation of serine 40 of H2A (H2AS40Gc) were used as active epigenetic markers. Metabolites 4 and 12 considerably altered the levels of H3K4me3 and H2AS40Gc, respectively, at the loci of most genes, except at those of genes encoding pan‐neural markers. Among them, metabolites produced via the kynurenine pathway (one of the tryptophan metabolic pathways) increased H3K4me3 and H2AS40Gc levels at multiple neural peptide‐encoding gene loci such as CART (cocaine‐and amphetamine‐regulated transcript) and OXT (oxytocin). The mRNA expression levels of these genes were also found to have increased following supplementation of the culture medium with metabolites of the kynurenine pathway. In other words, supplementation with these metabolites could effectively induce the differentiation of hiPSCs into CART‐ and OXT‐producing neurons. Collectively, we have identified metabolites that can alter the epigenetic status at specific loci. Our findings provide a better understanding of the use of nutrients or bioactive food components in modifying epigenetic mechanisms.Support or Funding InformationThis work was supported by Lotte Shigemitsu Prize.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoventilation and autonomic dysfunction in infant rats following orexin receptor blockade

Orexin (hypocretin) is a neuropeptide expressed by neurons in the lateral and perifornical hypothalamus that project widely to respiratory and autonomic regions of the brainstem. The activity of orexin neurons depends on vigilance state; they are most active in wakefulness, less active in quiet sleep, and silent during active sleep. Although there are well‐described facilitatory effects of orexin in adult animals on the control of breathing and autonomic response to stress, its role in infancy has not been studied. This is an important issue because there is accumulating pathological evidence of orexinergic dysfunction in some cases of the Sudden Infant Death Syndrome (SIDS), a leading cause of death in infancy that is highly associated with abnormal respiratory and autonomic control during periods of sleep. We hypothesized that in infant (~2 week old) rat pups, orexin receptor blockade would: 1) lead to respiratory dysfunction, more so in wakefulness and quiet sleep than in active sleep, and 2) compromise the thermogenic response to mild environmental cooling. To test these hypotheses we used whole‐body plethysmography to monitor breathing and metabolic O2 consumption in rat pups treated with suvorexant, a selective orexin 1 and 2 receptor antagonist. Vigilance state was determined using high‐definition video to monitor and confirm standard behavioral criteria associated with quiet sleep, active sleep, and arousal in infant rat pups. Experiment 1: Pups cycled through wakefulness, quiet and active sleep for 1 hr at thermoneutral ambient temperature (TA=31°C), at which point suvorexant (1mg/kg in 50% DMSO; n=3) or vehicle alone (n=2) was injected via an intra‐abdominal cannula, and pups cycled between wakefulness and sleep for another 1 hr. The two groups were compared with respect to respiratory frequency (f), tidal volume (VT), ventilation (VE) and metabolic O2 consumption (VO2). Experiment 2: Pups were kept at thermoneutral TA for 1 hr, then exposed to a ~2°C drop in TA over the following 15 min, and then returned to baseline TA. Pups were then injected with either suvorexant (1mg/kg; n=8) or vehicle alone (n=8), and after another 1 hr, the TA challenge was repeated. In each animal, the change in metabolic O2 consumption in response to cooling was measured before and after drug or vehicle injection. In wakefulness and quiet sleep, suvorexant reduced respiratory frequency by 48 ± 4 breaths/min (~30%; p=0.01), and VE by 770 ± 254 ml/min/kg (~40%; p<0.001), with no effect on VO2. These effects were not consistently observed during active sleep, and were absent in pups given vehicle alone. Suvorexant nearly abolished the metabolic response to cooling (p=0.01). These data suggest that orexin dysfunction can severely compromise breathing and autonomic function in infancy during wakefulness and quiet sleep.Support or Funding InformationFunding for this project provided by NHLBI grant 1R01HL136710‐01A1 (PI Cummings)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Consequences of Microglial Depletion on the Hypercapnic Ventilatory Response of Female Rats

Panic disorder (PD) is a neurological disorder associated with an abnormally high hypercapnic ventilatory response. In humans and rodents, early life stress is a significant risk factor for PD. Based on recent data indicating that CO2/H+ sensing by microglia is an important mechanism in the translation of hypercapnic stimulation into physiological responses, we tested the hypothesis that depletion of microglia by pharmacological treatment can decrease the hypercapnic ventilatory response. We also proposed that this effect is greater in stressed rats than controls. The prevalence of PD being ~2 times greater in women than men, experiments were performed on female rats. Following birth, rats were either raised under control conditions or subjected to neonatal maternal separation (NMS; 3h/day, postnatal days 3 to 12). At 8 weeks, females were fed a palatable treat containing either vehicle (DMSO) or PLX3397 (40mg/kg). Treatment was performed daily for 10 consecutive days. 1 day after completion of the treatment, rats were placed in a plethysmography chamber to measure ventilatory activity at rest (room air) followed by hypercapnic exposure (FiCO2 = 0.05; 10 min). Iba‐1 immunohistochemistry was used to quantify microglial density; measurements performed in hypothalamic structures containing orexin neurons showed that PLX3397 treatment educed microglial density by 31%; this effect did not differ between the structures quantified. Furthermore, our preliminary results suggest that oestrus cycle influences the impact of microglial depletion on the CO2 response. During the estrus phase, PLX3397 attenuated the hypercapnic ventilatory response of controls but augmented the response of NMS females. These data suggest that microglia contribute to the abnormal effects that ovarian hormones exert on the neural networks regulating CO2/H+ chemoreflex respiratory regulation in NMS rats. These data provide the basis for the exploration of new treatment for respiratory manifestations of PD.Support or Funding InformationSupported by the Canadian Institutes of Health Research (RK & FB).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of orexinergic and histaminergic pathway involved in therapeutic effect of histamine H4 receptor antagonist against cisplatin-induced anorexia in mice.

We previously reported that hypothalamic tumor necrosis factor-alpha (TNF-α) mRNA expression via histamine H4 receptors contributes to the development of cisplatin-induced anorexia; however, its precise mechanisms remain unclear. It has been reported that chemotherapeutic agents induce the suppression of orexin neuron activity, and the administration of orexin inhibits chemotherapeutic agent-induced gastric discomfort. Other studies demonstrated that the central administration of TNF-α impairs the orexinergic system, and that orexin excites the histaminergic system. We investigated the involvement of orexinergic and histaminergic systems in the therapeutic effect of an H4 receptor antagonist against cisplatin-induced anorexia. Cisplatin decreased the expression of prepro-orexin mRNA, which encodes precursors of orexin, in the hypothalamus of mice. The period of expression decreased in parallel with the onset of anorexia, and treatment with an H4 receptor antagonist (JNJ7777120, 10mg/kg) inhibited the decrease in expression. The effect of the H4 receptor antagonist on cisplatin-induced anorexia in mice was antagonized by an orexin OX2 receptor antagonist (JNJ10397049, 5mg/kg) rather than an orexin OX1 receptor antagonist (SB408124, 30mg/kg). Although an OX2 receptor agonist (YNT-185, 20mg/kg) or a histamine H3 receptor inverse agonist (ciproxifan, 1mg/kg) inhibited the cisplatin-induced anorexia, the inhibitory effect of the OX2 receptor agonist was antagonized by an H3 receptor silent antagonist (VUF5681, 5mg/kg). The combination of JNJ7777120 (10mg/kg) and ciproxifan (0.5mg/kg) completely resolved the cisplatin-induced anorexia. These results suggest that activation of the orexinergic and histaminergic pathway is involved in the therapeutic effect of an H4 receptor antagonist against cisplatin-induced anorexia.

New nociceptive circuits to the hypothalamic perifornical area from the spinal cord and spinal trigeminal nucleus via the parabrachial nucleus

Neurons of the parabrachial nucleus (PB) receive nociceptive input from the dorsal horn (DH) of the spinal cord and caudal part of the spinal trigeminal nucleus (Vc). Previously, we demonstrated that glutamatergic lateral PB neurons innervate orexin (ORX) neurons in the perifornical area (PeF) of the hypothalamus. However, the neural circuit via which ORX neurons receive nociceptive input from the DH and brainstem remains to be determined. In the present study, we aimed to clarify the potential nociceptive circuit from DH/Vc to PeF via lateral PB. We first examined the neuronal activity of fluorogold (FG)-labeled, PeF-projecting lateral PB neurons in Wistar rats following either saline or formalin injection to the forepaw or lips. We clearly detected more abundant c-Fos-positive, FG-labeled neurons in the PB nucleus. To investigate the relay from the DH/Vc to the PeF via the lateral PB, we injected FG into the PeF and biotinylated dextranamine (BDA) into the contralateral DH or ipsilateral Vc. We observed the most prominent overlap between BDA-labeled axon terminals and FG-labeled neurons in the dorsal lateral and central lateral subnuclei. Furthermore, we found that FG-labeled neurons formed close contact sites with BDA-labeled axons with synaptophysin immunoreactivity. Using electron microscopy, we confirmed that these contact sites were truly synapses. Taken together, our results indicate that the DH/Vc transmits nociceptive information to the PeF via the lateral PB, suggesting the involvement of ORX neurons in the pain pathway.

Lateral septum inhibition reduces motivation for cocaine: Reversal by diazepam.

The lateral septum (LS) is a brain region implicated in motivation, addiction, anxiety, and affect. We recently found that LS is necessary for cocaine-seeking behaviors including conditioned place preference and reinstatement of extinguished drug seeking, which involve LS input to limbic regions including ventral tegmental area (VTA) and orexin neurons in hypothalamus. Here, we microinjected baclofen-muscimol (B-M) in LS prior to testing in a behavioral economics (BE) paradigm. We found that intra-LS B-M decreased motivation (increased demand elasticity; α) for cocaine, but did not change consumption at low effort (Q0 ). We also compared the effects of LS inhibition with the effects of treatment with the benzodiazepine diazepam, which has been shown to facilitate reward pathways and disinhibit VTA dopamine neurons. Pretreatment with diazepam blocked the effects of LS inhibition and restored cocaine demand to that following vehicle treatment. These changes in cocaine demand after LS inhibition or diazepam were not due to effects on anxiety, as both manipulations produced similar effects on anxiety measures but opposing effects on drug taking. Collectively, these studies point to LS as a critical region driving motivation for cocaine, likely through its interactions with the mesolimbic dopamine system.

Serotonergic neurons in the dorsal raphe nucleus mediate the arousal-promoting effect of orexin during isoflurane anesthesia in male rats

Previous studies have demonstrated that the activation of orexinergic neurons facilitates the recovery of animals from general anesthesia. Moreover, serotonergic neurons that receive projections from orexin neurons have also been shown to participate in sleep-wakefulness regulation. In the present study, we aimed to explore whether orexinergic neurons facilitate emergence from isoflurane anesthesia in rats by activating serotonergic neurons. Orexin A (30 or 100 pmol), orexin B (30 or 100 pmol), and their respective antagonists SB-334867 and TCS-OX2–29 (5 or 20 μg) were microinjected into the dorsal raphe nucleus (DRN) of rats, and their effects on induction and emergence times were analyzed. Electroencephalogram (EEG) changes were also recorded and analyzed to illuminate the effect of orexin injection into the DRN on cortical excitability under isoflurane anesthesia. Activation of serotonergic neurons was detected via immunohistochemical analysis of c-Fos expression following orexin administration. Our results indicated that injection of neither orexins nor orexin antagonists into the rat DRN exerted an impact on induction time, whereas orexin-A injection (100 pmol) enhanced arousal when compared with the saline group. In contrast, administration of orexin receptor type 1 antagonist SB-334867 (20 μg) prolonged emergence time from isoflurane anesthesia. Microinjection of orexin-A induced an arousal pattern on EEG, and decreased the burst suppression ratio under isoflurane anesthesia. Isoflurane anesthesia inhibited the activity of serotonergic neurons, as shown by decrease in the number of c-Fos-immunoreactive serotonergic neurons when compared with the sham group. This inhibitory effect was partially reversed by administration of orexin-A. Taken together, our findings suggest that orexinergic signals facilitate emergence from isoflurane anesthesia, at least partially, by reversing the effects of isoflurane on serotonergic neurons of the DRN.

Lateral parabrachial neurons innervate orexin neurons projecting to brainstem arousal areas in the rat

Orexin (ORX) neurons in the hypothalamus send their axons to arousal-promoting areas. We have previously shown that glutamatergic neurons in the lateral parabrachial nucleus (LPB) innervate ORX neurons. In this study, we examined potential pathways from the LPB to ORX neurons projecting to arousal-promoting areas in the brainstem by a combination of tract-tracing techniques in male Wistar rats. We injected the anterograde tracer biotinylated dextranamine (BDA) into the LPB and the retrograde tracer cholera toxin B subunit (CTb) into the ventral tegmental area, dorsal raphe nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental area, or locus coeruleus (LC). We then analyzed the BDA-labeled fibers and ORX-immunoreactive neurons in the hypothalamus. We found that double-labeled ORX and CTb neurons were the most abundant after CTb was injected into the LC. We also observed prominently overlapping distribution of BDA-labeled fibers, arising from neurons located in the lateral-most part of the dorsomedial nucleus and adjacent dorsal perifornical area. In these areas, we confirmed by confocal microscopy that BDA-labeled synaptophysin-immunoreactive axon terminals were in contiguity with cell bodies and dendrites of CTb-labeled ORX-immunoreactive neurons. These results suggest that the LPB innervates arousal-promoting areas via ORX neurons and is likely to promote arousal responses to stimuli.

Involvement of hypothalamic perifornical astrocytes in the emergence from isoflurane anaesthesia

The mechanism of general anaesthesia remains unclear1. Most studies on the mechanism of general anaesthesia are focused on neurones including orexin neurones,2,3 whereas the role of astrocytes, the most abundant cells inside the brain, is unclear. This study is aimed to elucidate the effects of hypothalamic periforical astrocytes, where orexin neurones are concentrated, on the emergence of anaesthesia. We especially focused on the role of astrocyte-derived lactate in this process. Recovery of righting reflex and EEG were recorded to determine emergence time after isoflurane anaesthesia after inhibiting the transportation of astrocytic lactate with α-cyano-4-hydroxy-cinnamate (4-CIN). The spontaneous firing rate was recorded to verify the effect of astrocytes on orexin neurones. Emergence time was significantly prolonged after injection of 4-CIN into the perifornical orexin neurones, in comparison with the control group (12.86±1.258 vs 8.255±1.05 min, **P<0.01). Furthermore, the 4-CIN group showed higher EEG burst suppression rate. The suppression pattern after administration of 4-CIN was prolonged compared with that in the control group. In brain slices, 4-CIN significantly decreased the spontaneous firing frequency of orexin neurones (15.17±7.542 vs 58.8±14.45 Hz, *P<0.05). Similarly, spontaneous firing rate was significantly decreased with the inhibition of astrocytes by pharmacological inhibitor and chemical genetic methods. This study demonstrates that astrocytes in the hypothalamic perifornical area contribute to the emergence from isoflurane anaesthesia and astrocytic lactate plays a key factor in mediation of this effect.

Chemogenetic activation of orexin/hypocretin neurons ameliorates aging-induced changes in behavior and energy expenditure.

Aging affects numerous physiological processes, as well as behavior. A large number of these processes are regulated, at least partially, by hypothalamic orexin neurons, and orexin tone may decrease with normal aging. In this study, we hypothesized that designer receptors exclusively activated by designer drugs (DREADD) stimulation of orexin neuronal activity will ameliorate the effect of aging on behavioral and metabolic alterations in young and middle-aged mice. DREADD targeting was achieved by stereotaxic injection of AAV vectors (AAV2-hSyn-DIO-hM3D(Gq)-mCherry) into the lateral hypothalamus of 5- and 12-mo old orexin-cre female mice and was confirmed by immunohistochemistry (IHC) analysis of orexin A and mCherry expression. After recovery, animals were subjected to a behavioral test battery consisting of the elevated plus maze (EPM), open field (OFT), and novel object recognition tests (NORT) to assess effects of aging on anxiety-like behavior, general locomotion, and working memory. A comprehensive laboratory animal monitoring system (CLAMS) was used to measure spontaneous physical activity (SPA) and energy expenditure (EE). The results indicate that activation of orexin neurons mitigates aging-induced reductions in anxiety-like behavior in middle-aged mice (P < 0.005) and increases locomotion in both young and middle-aged mice (P < 0.05). Activation of orexin neurons increases SPA (P < 0.01) and EE (P < 0.005) in middle-aged mice, restoring the levels to that observed in young animals. Results from this study identify orexin neurons as potential therapeutic targets for age-related impairments in cognitive and anxiety-related behavior, and energy balance.

Sleep well. Untangling the role of melatonin MT1 and MT2 receptors in sleep.

The pharmacological potential of targeting selectively melatonin MT1 or MT2 receptors has not yet been exploited in medicine. Research using selective MT1/MT2 receptor ligands and MT1/MT2 receptor knockout mice has indicated that the activation of MT2 receptors selectively increases non-rapid eye movement (NREM) sleep whereas MT1 receptors seem mostly implicated in the regulation of REM sleep. Moreover, MT1 knockout mice show an increase in NREM sleep, while MT2 knockout a decrease, suggesting an opposite role of these two receptors. A recent paper in mice by Sharma et al (J Pineal Res, 2018, e12498) found that MT1 but not MT2 receptors are expressed on orexin neurons in the perifornical lateral hypothalamus (PFH). Moreover, after injecting melatonin or luzindole into the mouse PFH, the authors suggest that melatonin promotes NREM sleep because activates PFH MT1 receptors, which in turn inhibit orexin neurons that are important in promoting arousal and maintaining wakefulness. In this commentary, we have critically commented on some of these findings on the bases of previous literature. In addition, we highlighted the fact that no conclusions could be drawn on the melatonin receptor subtype mediating the effects of melatonin on sleep because the authors used the non-selective MT1/MT2 receptors antagonist luzindole. More solid research should further characterize the pharmacological function of these two melatonin receptors in sleep.

Orexin- and MCH-Neurons Double-Ablated Mice Display Severe Sleep Attacks and Cataplexy

Orexin-producing neurons (orexin neurons) and melanin-concentrating hormone-producing neurons (MCH neurons) are exclusively distributed in the lateral hypothalamus and project throughout the whole brain to regulate sleep/wakefulness. Ablation of orexin neurons in mice decreases wakefulness in the dark period and they display a narcolepsy-like phenotype, whereas ablation of MCH neurons increases wakefulness. It is unclear how interaction between orexin and MCH neurons regulates sleep/wakefulness. Here we generated transgenic mice in which both orexin and MCH neurons are ablated. These double-ablated mice show increased wakefulness and decreased rapid eye movement (REM) and non-REM sleep. Total time and duration of cataplexy were significantly increased in double-ablated mice compared with orexin neurons-ablated mice. These results suggest that MCH neurons have a suppressive role in cataplexy. Interestingly, these mice showed frequent sleep attacks with high delta and theta wave power via electroencephalograms during wakefulness, which is not classified under conventional criteria of sleep/wakefulness or cataplexy.

Functional identification of neurons regulate sleep and wakefulness

Sleep/wakefulness state change is regulated by neurons, however, its regulatory mechanism is still unclear. Here we found that GABAergic neurons in the ventral tegmental area (VTA) have an important role in the regulation of sleep/wakefulness. Adeno-associate virus (AAV) vectors were injected into VTA of glutamic acid decarboxylase (GAD)-Cre mice in which GABAergic neurons are exclusively express Cre. To manipulate GABAergic neurons in the VTA, channelrhodopsin2 (ChR2), anion-channelrhodopsin2 (ACR2) or hM3Dq was expressed by AAV. Chemogenetical activation of these neurons significantly increased time in NREM sleep. To reveal neural mechanism, slice patch clamp was performed. AAV was injected to VTA to express ChR2 in the GABAergic neurons in the VTA. Orexin neurons expressing fluorescent protein were identified and recorded. Then, GABAergic nerve terminals from VTA were stimulated by blue light. Blue light significantly inhibited activity of orexin neurons. On the other hand, optogenetical inhibition of these neurons using ACR2 immediately induced wakefulness. To evaluate physiological importance of this response, these neurons were inhibited during recovery sleep after sleep deprivation for 4 hr. Inhibition of GABAergic neurons induced wakefulness even in the very sleepy condition. These results suggest that activity of GABAergic neurons in the VTA is critical to change sleep/wakefulness state especially in the regulation of NREM sleep and wakefulness.

Orexin and fear; potential role of OX1R antagonist for treating anxiety disorders and PTSD

Fear is an important physiological function for survival. It appears when animals or humans are confronted with an environmental threat. The amygdala has been shown to play a highly important role in emergence of a fear-related behavior such as freezing that only occurs when the environment contains some elements suggestive of danger. Previous studies showed that hypothalamic orexin neurons are activated by fearful stimuli to evoke a 'defense reaction' with an increase in arousal level and sympathetic outflow to deal with the imminent danger. However, how this system contributes to the emergence of fear-related behavior is still unclear. We took cued and contextual fear conditioning experiment using orexin 1 receptor knockout (OX1R-/-) and orexin 2 receptor knockout (OX2R-/-) and orexin double receptor knockout (OX1R-/-; OX2R -/-) and prepro-orexin knockout (preproOX-/-) mice. We found that OX1R-/- and OX1R-/-; OX2R -/- and preproOX-/- showed abnormality in both cued and contextual fear conditioning test suggesting the orexin system might be important for fear memory formation and/or consolidation and/or retrieval mainly via OX1R signaling. Orexin neurons in the hypothalamus send excitatory innervations to noradrenergic neurons in the locus coeruleus (NALC) which abundantly express OX1R and send projections to the lateral amygdala (LA), which is known as the critical region for fear memory formation. Opto/chemogenetic inhibition of this di-synaptic orexin → NALC → LA pathway or pharmacological blockade of OX1R reduces cue-induced fear expression. Inversely, excitatory manipulation of this pathway after fear conditioning induces fear-related behavior. Although, fear memory helps animals respond precisely to a context or cue previously paired with an aversive stimulus, fear-related behavior is sometimes evoked even in a distinct context containing some similar elements, which is known as fear generalization. Our data suggests that the orexin → NALC → LA pathway might explain a part of the mechanism of fear generalization. We also discuss about the potential effectiveness of OX1R antagonists for treating excessive fear response or overgeneralization seen in anxiety disorder and post-traumatic stress disorder (PTSD).

Sleep deprivation-induced pre- and postsynaptic modulation of orexin neurons

Sleep/wake states are controlled by sleep- and wake-promoting systems, and transitions between states are thought to be regulated by their reciprocal inhibition and homeostatic sleep need. Orexin neurons are known to promote wake maintenance and stabilize the sleep/wake switch. Thus, we asked whether orexin neurons are modulated by homeostatic sleep need. Rats were sleep deprived or left undisturbed to rest for 6 h, then acute brain slices were generated for patch clamp recordings. We found that sleep deprivation increased firing and reduced spike frequency adaptation in response to excitatory drive in orexin neurons. These changes were specific to D-type orexin neurons which, unlike H-type orexin neurons, lack A-type current. In D-type orexin neurons, sleep deprivation decreased afterhyperpolarizing potential, which was associated with increased gain, measured as the slope of the input-output relationship. These effects were mimicked by inhibition of SK channels. Furthermore, sleep deprivation resulted in presynaptic inhibition of excitatory inputs to both D-type and H-type orexin neurons, which preferentially affected sparse synaptic inputs while sparing high frequency synaptic activities. Taken together, our results indicate that sleep deprivation modulates the gain control and synaptic gating in orexin neurons. These pre-and postsynaptic changes would tune orexin neurons to strong wake-promoting excitatory signals, while dampening weak synaptic inputs to allow transition to sleep in the absence of such strong signals. These mechanisms are consistent with a role of orexin neurons not only as a key state stabilizer, but also as a homeostatic wake integrator in the sleep/wake switch.This article is part of the Special Issue entitled ‘Hypothalamic Control of Homeostasis’.

Involvement of orexin-2 receptor in the ventral tegmental area in stress- and drug priming-induced reinstatement of conditioned place preference in rats

Orexin neurons originate from the lateral hypothalamus and send their projections broadly throughout the central nervous system including to the ventral tegmental area (VTA). In the current study, the reinstatement model has been used to examine the effects of intra-VTA administration of TCS-OX2-29, an orexin-2 receptor (OX2R) antagonist, on drug priming- and forced swim stress (FSS)-induced reinstatement of morphine-induced conditioned place preference (CPP). A total of 73 adult male Wistar rats weighing 200–280 g were bilaterally implanted with cannulas into the VTA. For induction of CPP, subcutaneous (sc) injection of morphine (5 mg/kg) was done daily during a 3-day conditioning phase. After a 24-h “off” period following achievement of extinction criterion, the rats were tested for drug priming-induced reinstatement with a priming dose of morphine (1 mg/kg;sc) or after application of FSS on the reinstatement day. The animals then received different doses of the OX2R antagonist, TCS-OX2-29 (0.3,3,1 and 10 nM) bilaterally in the VTA (0.3 μl/side) and were tested for FSS- and morphine priming-induced reinstatement of CPP. Our findings indicate that intra-VTA administration of OX2R antagonist suppresses FSS and morphine priming-induced reinstatement in a dose-dependent manner. The role of the orexinergic system in morphine-induced reinstatement through activation of OX2R in the VTA provides evidence that the OX2R antagonist could be a useful therapeutic target for prevention of reinstatement of morphine-induced CPP.

Orexin Depolarizes Central Amygdala Neurons via Orexin Receptor 1, Phospholipase C and Sodium-Calcium Exchanger and Modulates Conditioned Fear.

Orexins (OX), also known as hypocretins, are excitatory neuropeptides with well-described roles in regulation of wakefulness, arousal, energy homeostasis, and anxiety. An additional and recently recognized role of OX is modulation of fear responses. We studied the OX neurons of the perifornical hypothalamus (PeF) which send projections to the amygdala, a region critical in fear learning and fear expression. Within the amygdala, the highest density of OX-positive fibers was detected in the central nucleus (CeA). The specific mechanisms underlying OX neurotransmission within the CeA were explored utilizing rat brain slice electrophysiology, pharmacology, and chemogenetic stimulation. We show that OX induces postsynaptic depolarization of medial CeA neurons that is mediated by OX receptor 1 (OXR1) but not OX receptor 2 (OXR2). We further characterized the mechanism of CeA depolarization by OX as phospholipase C (PLC)- and sodium-calcium exchanger (NCX)- dependent. Selective chemogenetic stimulation of OX PeF fibers recapitulated OXR1 dependent depolarization of CeA neurons. We also observed that OXR1 activity modified presynaptic release of glutamate within the CeA. Finally, either systemic or intra-CeA perfusion of OXR1 antagonist reduced the expression of conditioned fear. Together, these data suggest the PeF-CeA orexinergic pathway can modulate conditioned fear through a signal transduction mechanism involving PLC and NCX activity and that selective OXR1 antagonism may be a putative treatment for fear-related disorders.

The neurobiological basis of narcolepsy.

Narcolepsy is the most common neurological cause of chronic sleepiness. The discovery about 20 years ago that narcolepsy is caused by selective loss of the neurons producing orexins (also known as hypocretins) sparked great advances in the field. Here, we review the current understanding of how orexin neurons regulate sleep-wake behaviour and the consequences of the loss of orexin neurons. We also summarize the developing evidence that narcolepsy is an autoimmune disorder that may be caused by a T cell-mediated attack on the orexin neurons and explain how these new perspectives can inform better therapeutic approaches.

Systemic Administration of Orexin a Loaded Liposomes Potentiates Nucleus Accumbens Shell Dopamine Release by Sucrose Feeding.

Orexin neurons originate in the lateral and dorsomedial hypothalamus and perifornical area and produce two different neuropeptides: orexin A (OxA) and orexin B (OxB), which activate OxR1 and OxR2 receptors. In the lateral hypothalamus (LH) orexin neurons are involved in behavior motivated by natural rewards such as palatable food (sugar, high-fat food) and it has been demonstrated similarly that the orexin signaling in the ventral tegmental area (VTA) is implicated in the intake of high-fat food. The VTA is an important area involved in reward processing. Given the involvement of nucleus accumbens (NAc) shell dopamine (DA) in motivation for food, we intended to investigate the effect of OxA on the basal and feeding-activated DA transmission in the NAc shell. OxA is a large peptide and does not cross the blood–brain barrier and for this reason was loaded on two kinds of liposomes: anti-transferrin-monoclonal antibodies (OX26-mAb) and lactoferrin-modified stealth liposomes. The effect of IV administration of both OxA liposomes on NAc shell DA was studied by microdialysis in freely moving rats. OxA, administered using both kinds of liposomes, produced a delayed and transitory increase in dialysate DA in the NAc shell, strongly and lastingly potentiated the increase in dialysate DA elicited by sucrose pellet consumption and increased the number of eaten pellets. These effects of OxA on DA transmission and feeding were prevented by the OxR1 antagonist SB 334867. Hence, OxA acting on VTA OxR1 can facilitate sucrose-stimulated NAc shell DA transmission directly by increasing the basal activity of VTA DA neurons that send their projections to the NAc shell.