Orexin Neurons Research Articles

006 The effect of sodium oxybate on cataplexy in orexin knockout mice

Abstract Introduction Narcolepsy is a neurological disorder that is characterized by the loss of orexin neurons in the lateral hypothalamus. Cataplexy is a symptom of narcolepsy and is identified by a sudden loss of muscle tone during wakefulness. Cataplexy abruptly interrupts day-to-day activities, and makes activities like driving dangerous and potentially fatal. It is hypothesized that cataplexy occurs due to the intrusion of REM sleep muscle atonia during wakefulness. It has been demonstrated that a GABAergic mechanism is responsible for silencing the REM sleep atonia circuit and preventing REM sleep muscle atonia from occurring during wakefulness. Sodium oxybate (SXB), a low affinity agonist of GABAB receptors, is currently an approved treatment for narcolepsy; however, its mechanism of action in the brain remains unknown. Here, we investigate the hypothesis that SXB prevents cataplexy through a GABAB mediated mechanism by potentially suppressing the REM sleep atonia circuit. Methods We intraperitoneally (IP) injected orexin-/- mice with one of five treatments; either Lactated Ringers Solution, SXB at 50, 100, or 200 mg/kg, or Phaclophen at 10 mg/kg followed by SXB at 100 mg/kg. Cataplexy was assessed by video recordings. We gathered and analyzed data at three distinct chronological time points (i.e, at baseline, after three consecutive weeks of daily dosing with a treatment, and after one week of no treatment). These experiments were designed and conducted to determine whether; 1) SXB reduces cataplexy, 2) the cessation of SXB administration has an effect on cataplexy, 3) SXB is mediated through a GABAB mechanism. Results We first confirmed that mice used were indeed orexin-/- using immunohistochemical analysis to show that no orexin-a expressing neurons were located in the lateral hypothalamus. Then, we determined that: 1) SXB reduces cataplexy compared to time matched controls (unpaired t-test, p=0.0027, n=18); 2) When we stopped administering SXB IP injections, cataplexy levels increased towards control levels (paired t-test, p=0.0024, n=18); 3) it remains unclear if SXB’s effect is solely mediated through a GABAB mechanism. Conclusion Our findings demonstrate that SXB effectively reduces cataplexy in orexin-/- mice. Support (if any) This research was funded by Jazz Pharmaceuticals

Orexin facilitates the hypoxic ventilatory response in rats

Orexin is a hypothalamic neuropeptide involved in a variety of functions including arousal, body temperature regulation, and feeding. Orexin neurons are active in wakefulness (or the dark phase in rats) and mostly silent in sleep (light phase), implying the effects of orexin are greater in wakefulness and/or dark phase. Recent evidence implicates orexin in the control of cardiorespiratory function, including the respiratory response to CO2 and blood pressure regulation. Whether orexin influences normal breathing in a state-dependent manner is unresolved. In addition, although the role of orexin in the hypercapnic ventilatory response is well established, its effects on the acute hypoxic ventilatory response (HVR) has not been investigated. We hypothesized that orexin receptor blockade would lead to hypoventilation in wakefulness but not sleep and would reduce the HVR. To test these hypotheses, we conducted two separate experiments, both of which were performed in the light phase. First, we used whole-body plethysmography to measure the ventilation of adult rats in room air. Rats were implanted with EEG and EMG electrodes to determine vigilance state, which was confirmed by applying Fast Fourier Transform on the EEG signals. After a 1hr baseline period, we injected i.p. either suvorexant (an OxR1 and OxR2 antagonist, 20 mg/kg i.p.) or vehicle (100% DMSO, i.p.) and allowed the rats to freely behave for 4 hours in the chamber while breathing room air. Separate groups of rats, also instrumented with EEG and EMG electrodes, were exposed to graded hypoxia (15%, 13%, 11%, and 9% O2) following administration of either 20 mg/kg i.p. suvorexant or vehicle. We found that, irrespective of state of vigilance, OxR1 and OxR2 blockade had no effect on baseline breathing, but significantly reduced the HVR (treatment × O2 level: p=0.03). The effect on the HVR was especially evident at 13% and 11% O2 when the response was reduced by ~30%. We show that orexin facilitates the HVR in the light phase, even though the orexin neurons are silent. Whether orexin has a larger effect on the HVR during the dark phase, when orexin neurons are active, has yet to be determined.

Orexin in Dorsal Raphe Nucleus enhances Physical Activity and Energy Expenditure

Background Lateral hypothalamic (LH) orexin neurons modulate spontaneous physical activity (SPA), and energy expenditure (EE). Orexin deficiency results in weight gain, whereas heightened LH orexin sensitivity results in obesity resistance. Orexins act on multiple brain sites, including the serotonergic dorsal raphe nucleus (DRN), which receives excitatory projections from the orexin neurons. Our earlier studies show presence of both orexin-1 (OX1R) and orexin-2 receptors (OX2R) in DRN, and higher expression of OX1R and OX2R in DRN of obesity resistant (OR) rats. We hypothesized that orexin in DRN enhances SPA and EE. Secondarily, we hypothesized that DRN GABA (gamma-aminobutyric acid) signaling mediates the effect of orexin on SPA and EE. Methods We manipulated orexin tone in DRN either by direct injections of orexin A into DRN or by chemogenetic activation of LH orexin neurons in aged (~15 mo) orexin-cre mice. Mice were implanted with a guide cannula targeting DRN, and simultaneously prepared with the stimulatory (hM3Dq floxed) designer receptors exclusively activated by designer drugs (DREADD) virus in LH (N = 16). All mice recovered for 3 weeks to allow transfection of the DREADD virus, and randomly assigned to saline or clozapine N-oxide (CNO, DREADD activator) treatment. Mice were transferred to Promethion indirect calorimetry cages (Sable Systems International) to continuously monitor SPA and EE. Body weight and food intake were measured daily. After acclimation, mice received either IP vehicle or CNO (1 mg/kg) in a repeated measure design. Fifteen minutes prior to IP CNO or saline, animals were infused with either the GABA agonist muscimol (32.5 pmol/0.2 μl), GABA antagonist bicuculline (32.5 pmol/0.2 μl) or vehicle into DRN. In a separate experiment, either orexin A (250 pmol/0.2 μl) or saline was injected into DRN without IP CNO injection, to study the direct effect of DRN orexin on SPA and EE for 24h post-injection. Results We found that DREADD activation of LH orexin neurons increases SPA and EE. Manipulation of GABA receptors (by muscimol or bicuculline) in DRN did not affect orexin neuron activation induced enhancement of SPA. On the other hand, intra-DRN OXA enhanced SPA and EE in these mice up to 4h post-injection, without affecting food intake. Conclusions These results support the idea that OXA in DRN facilitates negative energy balance by increasing physical activity-induced energy expenditure. In addition, these data suggest that the DRN is a prominent site modulating OXA-stimulated SPA and EE, and that modulation of DRN OXA is a potential strategy to mitigate age-induced reductions in SPA and EE.

Pitolisant in an Adolescent with Prader-Willi Syndrome.

This case report evaluates the potential benefit of pitolisant in a 15-year-old female with Prader-Willi syndrome, obsessive-compulsive disorder, autism spectrum disorder, and mild intellectual disability. Due to its action on the H3 receptor, it enhances central activity of histaminergic neurons resulting in increased alertness, irrespective of the loss of orexin neurons seen in narcolepsy. Additionally, it is thought to modulate various other neurotransmitter systems including acetylcholine, norepinephrine, and dopamine. Pitolisant has the potential to improve many symptoms in patients with Prader-Willi syndrome and it appears to be well tolerated with minimal side effects observed. Therefore, the use of pitolisant should be considered in patients with Prader-Willi syndrome who fail a psychostimulant trial.

Orexin-A inhibits fictive air breathing responses to respiratory stimuli in the bullfrog tadpole (Lithobates catesbeianus).

In pre-metamorphic tadpoles, the neural network generating lung ventilation is present but actively inhibited; the mechanisms leading to the onset of air breathing are not well understood. Orexin (ORX) is a hypothalamic neuropeptide that regulates several homeostatic functions, including breathing. While ORX has limited effects on breathing at rest, it potentiates reflexive responses to respiratory stimuli mainly via ORX receptor 1 (OX1R). Here, we tested the hypothesis that OX1Rs facilitate the expression of the motor command associated with air breathing in pre-metamorphic bullfrog tadpoles (Lithobates catesbeianus). To do so, we used an isolated diencephalic brainstem preparation to determine the contributions of OX1Rs to respiratory motor output during baseline breathing, hypercapnia and hypoxia. A selective OX1R antagonist (SB-334867; 5-25 µmoll-1) or agonist (ORX-A; 200 nmol l-1 to 1 µmoll-1) was added to the superfusion media. Experiments were performed under basal conditions (media equilibrated with 98.2% O2 and 1.8% CO2), hypercapnia (5% CO2) or hypoxia (5-7% O2). Under resting conditions gill, but not lung, motor output was enhanced by the OX1R antagonist and ORX-A. Hypercapnia alone did not stimulate respiratory motor output, but its combination with SB-334867 increased lung burst frequency and amplitude, lung burst episodes, and the number of bursts per episode. Hypoxia alone increased lung burst frequency and its combination with SB-334867 enhanced this effect. Inactivation of OX1Rs during hypoxia also increased gill burst amplitude, but not frequency. In contrast with our initial hypothesis, we conclude that ORX neurons provide inhibitory modulation of the CO2 and O2 chemoreflexes in pre-metamorphic tadpoles.

Loss of the metabolism and sleep regulating neuronal populations expressing orexin and oxytocin in the hypothalamus in amyotrophic lateral sclerosis.

To determine the underlying cellular changes and clinical correlates associated with pathology of the hypothalamus in amyotrophic lateral sclerosis (ALS), as hypothalamic atrophy occurs in the preclinical phase of the disease. The hypothalamus was pathologically examined in nine patients with amyotrophic lateral sclerosis in comparison to eight healthy control subjects. The severity of regional atrophy (paraventricular nucleus: PVN, fornix and total hypothalamus) and peptidergic neuronal loss (oxytocin, vasopressin, cocaine- and amphetamine-regulating transcript: CART, and orexin) was correlated with changes in eating behaviour, sleep function, cognition, behaviour and disease progression. Tar DNA-binding protein 43 (TDP-43) inclusions were present in the hypothalamus of all patients with amyotrophic lateral sclerosis. When compared to controls, there was atrophy of the hypothalamus (average 21% atrophy, p=0.004), PVN (average 30% atrophy p=0.014) and a loss of paraventricular oxytocin-producing neurons (average 49% loss p=0.02) and lateral hypothalamic orexin-producing neurons (average 37% loss, significance p=0.02). Factor analysis identified strong relationships between abnormal eating behaviour, hypothalamic atrophy and loss of orexin-producing neurons. With increasing disease progression, abnormal sleep behaviour and cognition associated with atrophy of the fornix. Substantial loss of hypothalamic oxytocin-producing neurons occurs in ALS, with regional atrophy and the loss of orexin neurons relating to abnormal eating behaviour in ALS. Oxytocin- and orexin neurons display TDP43 inclusions. Our study points to significant pathology in the hypothalamus that may play a key role in metabolic and pathogenic changes in ALS.

TASK1 and TASK3 in orexin neuron of lateral hypothalamus contribute to respiratory chemoreflex by projecting to nucleus tractus solitarius.

TWIK-related acid-sensitive potassium channels (TASKs)-like current was recorded in orexin neurons in the lateral hypothalamus (LH), which are essential in respiratory chemoreflex. However, the specific mechanism responsible for the pH-sensitivity remains elusive. Thus, we hypothesized that TASKs contribute to respiratory chemoreflex. In the present study, we found that TASK1 and TASK3 were expressed in orexin neurons. Blocking TASKs or microinjecting acid artificial cerebrospinal fluid (ACSF) in the LH stimulated breathing. In contrast, alkaline ACSF inhibited breathing, which was attenuated by blocking TASK1. Damage of orexin neurons attenuated the stimulatory effect on respiration caused by microinjection of acid ACSF (at a pH of 6.5) or TASKs antagonists. The orexinA-positive fiber and orexin type 1 receptor (OX1R) neurons were located in the nucleus tractus solitarius (NTS). The exciting effect of acidosis in the LH on respiration was inhibited by blocking OX1R of the NTS. Taken together, we conclude that orexin neurons sense the extracellular pH change through TASKs and regulate respiration by projecting to the NTS.

Effect of Parecoxib on Hippocampus and Hypothalamic Orexin Neurons in Rats with Cerebral Infarction

Cerebral infarction has seriously threatened human life and health. Parecoxib is the first nonsteroidal analgesic for surgical analgesia. However, its effect on orexin neurons during cerebral infarction treatment is unclear. In this study, a rat model of cerebral infarction was established by suture method. The experiment was assigned into sham operation group, cerebral infarction model group (MCAO), high and low dose group of parecoxib. Western blotting and immunofluorescence staining was used to evaluate the activity of orexin neurons. The infarct size was evaluated by TTC staining. The apoptosis of neurons in hypothalamus and hippocampus was determined by AV-PI staining. TTC staining suggested that parecoxib treatment significantly reduced cerebral infarct size, increased orexin neuronal activity, and decreased neuronal apoptosis in hypothalamus and hippocampus, which were significantly different from sham-operated groups. This study demonstrates that parecoxib has a protective effect on cerebral infarction rats, which can inhibit the apoptosis of hypothalamic and hippocampal neurons through the orexin neuron pathway. It provides a theoretical basis for the protective effect of parecoxib, indicating that it might be a new target for the treatment of cerebral infarction.

Orexin/Hypocretin and MCH Neurons: Cognitive and Motor Roles Beyond Arousal.

The lateral hypothalamus (LH) is classically implicated in sleep-wake control. It is the main source of orexin/hypocretin and melanin-concentrating hormone (MCH) neuropeptides in the brain, which have been both implicated in arousal state switching. These neuropeptides are produced by non-overlapping LH neurons, which both project widely throughout the brain, where release of orexin and MCH activates specific postsynaptic G-protein-coupled receptors. Optogenetic manipulations of orexin and MCH neurons during sleep indicate that they promote awakening and REM sleep, respectively. However, recordings from orexin and MCH neurons in awake, moving animals suggest that they also act outside sleep/wake switching. Here, we review recent studies showing that both orexin and MCH neurons can rapidly (sub-second-timescale) change their firing when awake animals experience external stimuli, or during self-paced exploration of objects and places. However, the sensory-behavioral correlates of orexin and MCH neural activation can be quite different. Orexin neurons are generally more dynamic, with about 2/3rds of them activated before and during self-initiated running, and most activated by sensory stimulation across sensory modalities. MCH neurons are activated in a more select manner, for example upon self-paced investigation of novel objects and by certain other novel stimuli. We discuss optogenetic and chemogenetic manipulations of orexin and MCH neurons, which combined with pharmacological blockade of orexin and MCH receptors, imply that these rapid LH dynamics shape fundamental cognitive and motor processes due to orexin and MCH neuropeptide actions in the awake brain. Finally, we contemplate whether the awake control of psychomotor brain functions by orexin and MCH are distinct from their “arousal” effects.

Orexins (hypocretins): The intersection between homeostatic and hedonic feeding.

Orexins are hypothalamic neuropeptides originally discovered to play a role in the regulation of feeding behaviour. The broad connections of orexin neurons to mesocorticolimbic circuitry suggest they may play a role in mediating reward-related behaviour beyond homeostatic feeding. Here, we review the role of orexin in a variety of eating-related behaviour, with a focus on reward and motivation, and the neural circuits driving these effects. One emerging finding is the involvement of orexins in hedonic and appetitive behaviour towards palatable food, in addition to their role in homeostatic feeding. This review discusses the brain circuitry and possible mechanisms underlying the role of orexins in these behaviours. Overall, there is a marked bias in the literature towards studies involving male subjects. As such, future work needs to be done to involve female subjects. In summary, orexins play an important role in driving motivation for high salient rewards such as highly palatable food and may serve as the intersection between homeostatic and hedonic feeding.

The Impacts of Age and Sex in a Mouse Model of Childhood Narcolepsy.

Narcolepsy is a sleep disorder caused by selective death of the orexin neurons that often begins in childhood. Orexin neuron loss disinhibits REM sleep during the active period and produces cataplexy, episodes of paralysis during wakefulness. Cataplexy is often worse when narcolepsy develops in children compared to adults, but the reason for this difference remains unknown. We used orexin-tTA; TetO DTA mice to model narcolepsy at different ages. When doxycycline is removed from the diet, the orexin neurons of these mice express diphtheria toxin A and die within 2–3 weeks. We removed doxycycline at 4 weeks (young-onset) or 14 weeks (adult-onset) of age in male and female mice. We implanted electroencephalography (EEG) and electromyography (EMG) electrodes for sleep recordings two weeks later and then recorded EEG/EMG/video for 24 h at 3 and 13 weeks after removal of doxycycline. Age-matched controls had access to doxycycline diet for the entire experiment. Three weeks after doxycycline removal, both young-onset and adult-onset mice developed severe cataplexy and the sleep-wake fragmentation characteristic of narcolepsy. Cataplexy and maintenance of wake were no worse in young-onset compared to adult-onset mice, but female mice had more bouts of cataplexy than males. Orexin neuron loss was similarly rapid in both young- and adult-onset mice. As age of orexin neuron loss does not impact the severity of narcolepsy symptoms in mice, the worse symptoms in children with narcolepsy may be due to more rapid orexin neuron loss than in adults.

Cerebrospinal fluid monoamine levels in central disorders of hypersomnolence.

Whether the cause of daytime sleepiness in narcolepsy type 1 (NT1) is a direct consequence of the loss of orexin (ORX) neurons or whether low orexin reduces the efficacy of the monoaminergic systems to promote wakefulness is unclear. The neurobiology underlying sleepiness in other central hypersomnolence disorders, narcolepsy type 2 (NT2), and idiopathic hypersomnia (IH), is currently unknown. Eleven biogenic amines including the monoaminergic neurotransmitters and their metabolites and five trace amines were measured in the cerebrospinal fluid (CSF) of 94 drug-free subjects evaluated at the French National Reference Center for Narcolepsy: 39 NT1(orexin-deficient) patients, 31 patients with objective sleepiness non orexin-deficient (NT2 and IH), and 24 patients without objective sleepiness. Three trace amines were undetectable in the sample: tryptamine, octopamine, and 3-iodothyronamine. No significant differences were found among the three groups for quantified monoamines and their metabolites in crude and adjusted models; however, CSF 5-hydroxyindoleacetic acid (5-HIAA) levels tended to increase in NT1 compared to other patients after adjustment. Most of the biomarkers were not associated with ORX-A levels, clinical or neurophysiological parameters, but a few biomarkers (e.g. 3-methoxy-4-hydroxyphenylglycol and norepinephrine) correlated with daytime sleepiness and high rapid eye movement (REM) sleep propensity. We found no striking differences among CSF monoamines, their metabolites and trace amine levels, and few associations between them and key clinical or neurophysiological parameters in NT1, NT2/IH, and patients without objective sleepiness. Although mostly negative, these findings are a significant contribution to our understanding of the neurobiology of hypersomnolence in these disorders that remain mysterious and deserve further exploration.

Inhibition of Orexin/Hypocretin Neurons Ameliorates Elevated Physical Activity and Energy Expenditure in the A53T Mouse Model of Parkinson's Disease.

Aside from the classical motor symptoms, Parkinson’s disease also has various non-classical symptoms. Interestingly, orexin neurons, involved in the regulation of exploratory locomotion, spontaneous physical activity, and energy expenditure, are affected in Parkinson’s. In this study, we hypothesized that Parkinson’s-disease-associated pathology affects orexin neurons and therefore impairs functions they regulate. To test this, we used a transgenic animal model of Parkinson’s, the A53T mouse. We measured body composition, exploratory locomotion, spontaneous physical activity, and energy expenditure. Further, we assessed alpha-synuclein accumulation, inflammation, and astrogliosis. Finally, we hypothesized that chemogenetic inhibition of orexin neurons would ameliorate observed impairments in the A53T mice. We showed that aging in A53T mice was accompanied by reductions in fat mass and increases in exploratory locomotion, spontaneous physical activity, and energy expenditure. We detected the presence of alpha-synuclein accumulations in orexin neurons, increased astrogliosis, and microglial activation. Moreover, loss of inhibitory pre-synaptic terminals and a reduced number of orexin cells were observed in A53T mice. As hypothesized, this chemogenetic intervention mitigated the behavioral disturbances induced by Parkinson’s disease pathology. This study implicates the involvement of orexin in early Parkinson’s-disease-associated impairment of hypothalamic-regulated physiological functions and highlights the importance of orexin neurons in Parkinson’s disease symptomology.

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.

Interaction between Orexin Neurons and Monoaminergic Systems.

Orexins have received a lot of attention as potent endogenous arousal-promoting peptides, and orexin receptor antagonists have shown clinical efficacy for the treatment of insomnia. Orexin neurons are thought to act primarily on monoaminergic neurons to maintain arousal and vigilance. In this chapter, we discuss the functional interaction between monoaminergic systems, including noradrenaline, serotonin and histamine, and orexin neurons, as well as interactions between the acetylcholine system and the orexin neurons, focusing, in particular, on their function in the regulation of sleep-wakefulness states. Orexin also has close interactions with the dopaminergic system, and many studies have suggested roles of orexin signaling in the reward system and roles for orexins in drug addiction.

Subsecond Ensemble Dynamics of Orexin Neurons Link Sensation and Action.

Hypothalamic hypocretin/orexin neurons have been initially conceptualized as slow, modulatory controllers of behavior. Furthermore, their behavioral effects have been assumed to be a secondary consequence of their impact on arousal. However, cellular-resolution calcium imaging and optogenetic studies show that orexin neurons regulate self-generated and sensory-evoked movement on rapid, subsecond timescales. Orexin cell activity rapidly and transiently peaks before and during movements. Optogenetic prevention of this activation reduces the probability of locomotion initiation, and optogenetic mimicry of orexin cell activation rapidly causes locomotion. Neural ensemble calcium imaging experiments reveal that the same orexin cells whose activity underlies movement initiation display subsecond-latency responses to diverse sensory stimuli. These findings establish orexin neurons as rapid and strong sensorimotor controllers that are in many ways operationally similar to classic subcortical movement controllers, such as midbrain dopamine neurons. While a scientific definition of "arousal" is still lacking, the subsecond-scale sensorimotor control by orexin neurons could be viewed as reminiscent of a motor rather than an arousal system.

Elucidation of Neural Circuits Involved in the Regulation of Sleep/Wakefulness Using Optogenetics.

Although sleep is an absolutely essential physiological phenomenon for maintaining normal health in animals, little is known about its function to date. In this section, I introduce the application of optogenetics to freely behaving animals for the purpose of characterizing neural circuits involved in the regulation of sleep/wakefulness. Applying optogenetics to the specific neurons involved in sleep/wakefulness regulation enabled the precise control of the sleep/wakefulness states between wakefulness, non-rapid eye movement (NREM) sleep, and REM sleep states. For example, selective activation of orexin neurons using channelrhodopsin-2 and melanopsin induced a transition from sleep to wakefulness. In contrast, suppression of these neurons using halorhodopsin and archaerhodopsin induced a transition from wakefulness to NREM sleep and increased the time spent in NREM sleep. Selective activation of melanin-concentrating hormone (MCH) neurons induced a transition from NREM sleep to REM sleep and prolonged the time spent in REM sleep, which was accompanied by a decrease in NREM sleep time. Optogenetics was first introduced to orexin neurons in 2007 and has since rapidly spread throughout the field of neuroscience. In the last 13years or so, neural nuclei and the cell types that control sleep/wakefulness have been identified. The use of optogenetic studies has greatly contributed to the elucidation of the neural circuits involved in the regulation of sleep/wakefulness.

The Drosophila Microrna Bantam Regulates Excitability in Adult Mushroom Body Output Neurons to Promote Early Night Sleep

SummaryThe decision to sleep requires integration of information about the internal state of the animal and its immediate external environment. Sleep circuitry therefore evolved to have both dedicated and context-dependent modulatory neuronal elements [1-3]. Identifying these important subcircuits and understanding the molecular mechanisms regulating their roles is a major challenge for the sleep field. microRNAs, non-coding RNA transcripts which posttranscriptionally control gene expression [4], have been implicated in sleep regulation [5-10]. Our previous screen [11] identified 25 sleep-regulating microRNAs in Drosophila melanogaster , including the developmentally-important [12-15] microRNA bantam ( ban ). Here we show that ban acts in the adult brain to promote early nighttime sleep through a population of glutamatergic neurons that is intimately involved in applying contextual information to behaviors [16]- the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs). GCaMP calcium imaging revealed that bantam inhibits the neural activity of these cells during the early night, but not the day. Blocking synaptic transmission in these MBONs rescued the effect of ban knockdown. Together these results suggest ban promotes early night sleep via inhibition of the γ5β′2a/β′2mp/β′2mp_bilateral MBONs. RNAseq identifies Kelch and CCHamide-2 receptor as mediators of this role. These experiments establish a new role for bantam as an active regulator of sleep and neural activity in the adult fly brain. Notably, the gene for BRS3 (Bombesin receptor subtype-3), the human ortholog of CCHa2-R, hosts an MRE for the mammalian ortholog of ban , miR-450b-3p [17-19]. The regulation of orexin neurons by this receptor [20] implies this pathway is highly conserved.

Decreased Orexin Receptor 1 mRNA Expression in the Locus Coeruleus in Both Tau Transgenic rTg4510 and Tau Knockout Mice and Accompanying Ascending Arousal System Tau Invasion in rTg4510.

Sleep/wake disturbances (e.g., insomnia and sleep fragmentation) are common in neurodegenerative disorders, especially Alzheimer's disease (AD) and frontotemporal dementia (FTD). These symptoms are somewhat reminiscent of narcolepsy with cataplexy, caused by the loss of orexin-producing neurons. A bidirectional relationship between sleep disturbance and disease pathology suggests a detrimental cycle that accelerates disease progression and cognitive decline. The accumulation of brain tau fibrils is a core pathology of AD and FTD-tau and clinical evidence supports that tau may impair the orexin system in AD/FTD. This hypothesis was investigated using tau mutant mice. To characterize orexin receptor mRNA expression in sleep/wake regulatory brain centers and quantify noradrenergic locus coeruleus (LC) and orexinergic lateral hypothalamus (LH) neurons, in tau transgenic rTg4510 and tau-/- mice. We used i n situ hybridization and immunohistochemistry (IHC) in rTg4510 and tau-/- mice. rTg4510 and tau-/- mice exhibited a similar decrease in orexin receptor 1 (OX1R) mRNA expression in the LC compared with wildtype controls. IHC data indicated this was not due to decreased numbers of LC tyrosine hydroxylase-positive (TH) or orexin neurons and demonstrated that tau invades TH LC and orexinergic LH neurons in rTg4510 mice. In contrast, orexin receptor 2 (OX2R) mRNA levels were unaffected in either model. The LC is strongly implicated in the regulation of sleep/wakefulness and expresses high levels of OX1R. These findings raise interesting questions regarding the effects of altered tau on the orexin system, specifically LC OX1Rs, and emphasize a potential mechanism which may help explain sleep/wake disturbances in AD and FTD.

DLK1 Expressed in Mouse Orexin Neurons Modulates Anxio-Depressive Behavior but Not Energy Balance.

Lateral hypothalamic area (LHA) neurons expressing the neuropeptide orexin (OX) are implicated in obesity and anxio-depression. However, these neurons release OX as well as a host of other proteins that might contribute to normal physiology and disease states. We hypothesized that delta-like homolog 1 (DLK1), a protein reported to be co-expressed by all OX neurons, contributes to the regulation of energy balance and/or anxio-depression. Consistent with previous reports, we found that all rat OX neurons co-express DLK1. Yet, in mice and humans only a subset of OX neurons co-expressed DLK1. Since human OX-DLK1 distribution is more similar to mice than rats, mice are a comparable model to assess the human physiologic role of DLK1. We therefore used a viral lesion strategy to selectively delete DLK1 within the LHA of adult mice (DLK1Null) to reveal its role in body weight and behavior. Adult-onset DLK1 deletion had no impact on body weight or ingestive behavior. However, DLK1Null mice engaged in more locomotor activity than control mice and had decreased anxiety and depression measured via the elevated plus maze and forced swim tests. These data suggest that DLK1 expression via DLK1-expressing OX neurons primarily contributes to anxio-depression behaviors without impacting body weight.