Distinct lateral hypothalamic CaMKIIα neuronal populations regulate wakefulness and locomotor activity.

For nearly a century, evidence has accumulated indicating that the lateral hypothalamus (LH) contains neurons essential to sustain wakefulness. While lesion or inactivation of LH neurons produces a profound increase in sleep, stimulation of inhibitory LH neurons promotes wakefulness. To date, the primary wake-promoting cells that have been identified in the LH are the hypocretin/orexin (Hcrt) neurons, yet these neurons have little impact on total sleep or wake duration across the 24-h period. Recently, we and others have identified other LH populations that increase wakefulness. In the present study, we conducted microendoscopic calcium imaging in the LH concomitant with EEG and locomotor activity (LMA) recordings and found that a subset of LH neurons that express Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) are preferentially active during wakefulness. Chemogenetic activation of these neurons induced sustained wakefulness and greatly increased LMA even in the absence of Hcrt signaling. Few LH CaMKIIα-expressing neurons are hypocretinergic or histaminergic while a small but significant proportion are GABAergic. Ablation of LH inhibitory neurons followed by activation of the remaining LH CaMKIIα neurons induced similar levels of wakefulness but blunted the LMA increase. Ablated animals showed no significant changes in sleep architecture but both spontaneous LMA and high theta (8 to 10 Hz) power during wakefulness were reduced. Together, these findings indicate the existence of two subpopulations of LH CaMKIIα neurons: an inhibitory population that promotes locomotion without affecting sleep architecture and an excitatory population that promotes prolonged wakefulness even in the absence of Hcrt signaling.

Deficiency of orexin signaling during sleep is involved in abnormal REM sleep architecture in narcolepsy.

Narcolepsy is a sleep disorder caused by deficiency of orexin signaling. However, the neural mechanisms by which deficient orexin signaling causes the abnormal rapid eye movement (REM) sleep characteristics of narcolepsy, such as cataplexy and frequent transitions to REM states, are not fully understood. Here, we determined the activity dynamics of orexin neurons during sleep that suppress the abnormal REM sleep architecture of narcolepsy. Orexin neurons were highly active during wakefulness, showed intermittent synchronous activity during non-REM (NREM) sleep, were quiescent prior to the transition from NREM to REM sleep, and a small subpopulation of these cells was active during REM sleep. Orexin neurons that lacked orexin peptides were less active during REM sleep and were mostly silent during cataplexy. Optogenetic inhibition of orexin neurons established that the activity dynamics of these cells during NREM sleep regulate NREM-REM sleep transitions. Inhibition of orexin neurons during REM sleep increased subsequent REM sleep in "orexin intact" mice and subsequent cataplexy in mice lacking orexin peptides, indicating that the activity of a subpopulation of orexin neurons during the preceding REM sleep suppresses subsequent REM sleep and cataplexy. Thus, these results identify how deficient orexin signaling during sleep results in the abnormal REM sleep architecture characteristic of narcolepsy.

Biological sex influences sleep phenotype in mice experiencing spontaneous opioid withdrawal.

Aversive symptoms, including insomnia experienced during opioid withdrawal, are a major drive to relapse; however, withdrawal-associated sleep symptomatology has been little explored in preclinical models. We describe here a model of opioid withdrawal in mice that resembles the sleep phenotype characteristic of withdrawal in humans. Male and female C57BL/6 mice were instrumented with telemeters to record electroencephalogram, electromyogram, activity and subcutaneous temperature. All mice received two treatments separated by a 16-day washout period: (1) saline (volume: 10 ml kg-1 ); or (2) ascending doses of morphine (5, 10, 20, 40 and 80 mg kg-1 ; volume: 10 ml kg-1 ) for 5 days at Zeitgeber time 1 and Zeitgeber time 13. Recordings for the first 71 hr after treatment discontinuation (withdrawal days 1-3) and for 24 hr on withdrawal days 5 and 7 were scored for sleep/wake state, and sleep architecture and electroencephalogram spectral data were analysed. Morphine was acutely wake- and activity-promoting, and non-rapid eye movement and rapid eye movement sleep were increased during the dark phase on withdrawal day 2 in both sexes. While non-rapid eye movement delta power (0.5-4.0 Hz), a measure of sleep intensity, was reduced during the light phase on withdrawal day 1 and the dark phase on withdrawal day 2 in both sexes, female mice also exhibited changes in the duration and the number of bouts of sleep/wake states. These observations of fragmented sleep on withdrawal days 1-3 suggest poorer sleep consolidation and a more pronounced withdrawal-associated sleep phenotype in female than in male mice. These data may indicate a greater sensitivity to morphine, a more distinct aversive sleep phenotype and/or a faster escalation to dependence in female mice.

Peripheral vs. core body temperature as hypocretin/orexin neurons degenerate: Exercise mitigates increased heat loss.

Hypocretins/Orexins (Hcrt/Ox) are hypothalamic neuropeptides implicated in diverse functions, including body temperature regulation through modulation of sympathetic vasoconstrictor tone. In the current study, we measured subcutaneous (Tsc) and core (Tb) body temperature as well as activity in a conditional transgenic mouse strain that allows the inducible ablation of Hcrt/Ox-containing neurons by removal of doxycycline (DOX) from their diet (orexin-DTA mice). Measurements were made during a baseline, when mice were being maintained on food containing DOX, and over 42 days while the mice were fed normal chow which resulted in Hcrt/Ox neuron degeneration. The home cages of the orexin-DTA mice were equipped with running wheels that were either locked or unlocked. In the presence of a locked running wheel, Tsc progressively decreased on days 28 and 42 in the DOX(-) condition, primarily during the dark phase (the major active period for rodents). This nocturnal reduction in Tsc was mitigated when mice had access to unlocked running wheels. In contrast to Tsc, Tb was largely maintained until day 42 in the DOX(-) condition even when the running wheel was locked. Acute changes in both Tsc and Tb were observed preceding, during, and following cataplexy. Our results suggest that ablation of Hcrt/Ox-containing neurons results in elevated heat loss, likely through reduced sympathetic vasoconstrictor tone, and that exercise may have some therapeutic benefit to patients with narcolepsy, a disorder caused by Hcrt/Ox deficiency. Acute changes in body temperature may facilitate prediction of cataplexy onset and lead to interventions to mitigate its occurrence.

Activation of the nociceptin/orphanin-FQ receptor promotes NREM sleep and EEG slow wave activity.

Sleep/wake control involves several neurotransmitter and neuromodulatory systems yet the coordination of the behavioral and physiological processes underlying sleep is incompletely understood. Previous studies have suggested that activation of the Nociceptin/orphanin FQ (N/OFQ) receptor (NOPR) reduces locomotor activity and produces a sedation-like effect in rodents. In the present study, we systematically evaluated the efficacy of two NOPR agonists, Ro64-6198 and SR16835, on sleep/wake in rats, mice, and Cynomolgus macaques. We found a profound, dose-related increase in non-Rapid Eye Movement (NREM) sleep and electroencephalogram (EEG) slow wave activity (SWA) and suppression of Rapid Eye Movement sleep (REM) sleep in all three species. At the highest dose tested in rats, the increase in NREM sleep and EEG SWA was accompanied by a prolonged inhibition of REM sleep, hypothermia, and reduced locomotor activity. However, even at the highest dose tested, rats were immediately arousable upon sensory stimulation, suggesting sleep rather than an anesthetic state. NOPR agonism also resulted in increased expression of c-Fos in the anterodorsal preoptic and parastrial nuclei, two GABAergic nuclei that are highly interconnected with brain regions involved in physiological regulation. These results suggest that the N/OFQ-NOPR system may have a previously unrecognized role in sleep/wake control and potential promise as a therapeutic target for the treatment of insomnia.

Evaluation of the efficacy of the hypocretin/orexin receptor agonists TAK-925 and ARN-776 in narcoleptic orexin/tTA; TetO-DTA mice.

The sleep disorder narcolepsy, a hypocretin deficiency disorder thought to be due to degeneration of hypothalamic hypocretin/orexin neurons, is currently treated symptomatically. We evaluated the efficacy of two small molecule hypocretin/orexin receptor-2 (HCRTR2) agonists in narcoleptic male orexin/tTA; TetO-DTA mice. TAK-925 (1-10 mg/kg, s.c.) and ARN-776 (1-10 mg/kg, i.p.) were injected 15 min before dark onset in a repeated measures design. EEG, EMG, subcutaneous temperature (Tsc ) and activity were recorded by telemetry; recordings for the first 6 h of the dark period were scored for sleep/wake and cataplexy. At all doses tested, TAK-925 and ARN-776 caused continuous wakefulness and eliminated sleep for the first hour. Both TAK-925 and ARN-776 caused dose-related delays in NREM sleep onset. All doses of TAK-925 and all but the lowest dose of ARN-776 eliminated cataplexy during the first hour after treatment; the anti-cataplectic effect of TAK-925 persisted into the second hour for the highest dose. TAK-925 and ARN-776 also reduced the cumulative amount of cataplexy during the 6 h post-dosing period. The acute increase in wakefulness produced by both HCRTR2 agonists was characterised by increased spectral power in the gamma EEG band. Although neither compound provoked a NREM sleep rebound, both compounds affected NREM EEG during the second hour post-dosing. TAK-925 and ARN-776 also increased gross motor activity, running wheel activity, and Tsc , suggesting that the wake-promoting and sleep-suppressing activities of these compounds could be a consequence of hyperactivity. Nonetheless, the anti-cataplectic activity of TAK-925 and ARN-776 is encouraging for the development of HCRTR2 agonists.

The development of sleep/wake disruption and cataplexy as hypocretin/orexin neurons degenerate in male vs. female Orexin/tTA; TetO-DTA Mice.

Narcolepsy Type 1 (NT1), a sleep disorder with similar prevalence in both sexes, is thought to be due to loss of the hypocretin/orexin (Hcrt) neurons. Several transgenic strains have been created to model this disorder and are increasingly being used for preclinical drug development and basic science studies, yet most studies have solely used male mice. We compared the development of narcoleptic symptomatology in male vs. female orexin-tTA; TetO-DTA mice, a model in which Hcrt neuron degeneration can be initiated by removal of doxycycline (DOX) from the diet. EEG, EMG, subcutaneous temperature, gross motor activity, and video recordings were conducted for 24-h at baseline and 1, 2, 4, and 6 weeks after DOX removal. Female DTA mice exhibited cataplexy, the pathognomonic symptom of NT1, by Week 1 in the DOX(-) condition but cataplexy was not consistently present in males until Week 2. By Week 2, both sexes showed an impaired ability to sustain long wake bouts during the active period, the murine equivalent of excessive daytime sleepiness in NT1. Subcutaneous temperature appeared to be regulated at lower levels in both sexes as the Hcrt neurons degenerated. During degeneration, both sexes also exhibited the "Delta State", characterized by sudden cessation of activity, high delta activity in the EEG, maintenance of muscle tone and posture, and the absence of phasic EMG activity. Since the phenotypes of the two sexes were indistinguishable by Week 6, we conclude that both sexes can be safely combined in future studies to reduce cost and animal use.

Hypocretin/Orexin Receptor Pharmacology and Sleep Phases.

The hypocretins/orexins are two excitatory neuropeptides, alternately called HCRT1 or orexin-A and HCRT2 or orexin-B, that are the endogenous ligands for two G-protein-coupled receptors, HCRTR1/OX1R and HCRTR2/OX2R. Shortly after the discovery of this system, degeneration of hypocretin/orexin-producing neurons was implicated in the etiology of the sleep disorder narcolepsy. The involvement of this system in a disorder characterized by the loss of control over arousal state boundaries also suggested its role as a critical component of endogenous sleep-wake regulatory circuitry. The broad projections of the hypocretin/orexin-producing neurons, along with differential expression of the two receptors in the projection fields of these neurons, suggest distinct roles for these receptors. While HCRTR1/OX1R is associated with regulation of motivation, reward, and autonomic functions, HCRTR2/OX2R is strongly linked to sleep-wake control. The association of hypocretin/orexin with these physiological processes has led to intense interest in the therapeutic potential of compounds targeting these receptors. Agonists and antagonists for the hypocretin/orexin receptors have shown potential for the treatment of disorders of excessive daytime somnolence and nocturnal hyperarousal, respectively, with the first antagonists approved by the US Food and Drug Administration (FDA) in 2014 and 2019 for the treatment of insomnia. These and related compounds have also been useful tools to advance hypocretin/orexin neurobiology.

Cerebrospinal fluid monoamine levels in central disorders of hypersomnolence.

STUDY OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Animal models of narcolepsy and the hypocretin/orexin system: Past, present, and future.

Animal models have advanced not only our understanding of the etiology and phenotype of the sleep disorder narcolepsy but have also informed sleep/wake regulation more generally. The identification of an inheritable narcolepsy phenotype in dogs in the 1970s allowed the establishment of a breeding colony at Stanford University, resulting in studies that provided the first insights into the genetics and neurotransmitter systems that underlie cataplexy and rapid-eye movement sleep atonia. Although the discovery of the hypocretin/orexin neuropeptides in 1998 initially seemed unrelated to sleep/wake control, the description of the phenotype of the prepro-orexin knockout (KO) mouse as strongly resembling cataplexy, the pathognomonic symptom of narcolepsy, along with identification of a mutation in hypocretin receptor-2 gene as the source of canine narcolepsy, unequivocally established the relationship between this system and narcolepsy. The subsequent discovery of hypocretin neuron degeneration in human narcolepsy demystified a disorder whose etiology had been unknown since its initial description 120 years earlier. These breakthroughs prompted the development of numerous other animal models that have allowed manipulation of the hypocretin/orexin system, thereby advancing our understanding of sleep/wake circuitry. While animal models have greatly informed understanding of this fascinating disorder and the role of the hypocretin/orexin system in sleep/wake control, the question of why these neurons degenerate in human narcolepsy is only beginning to be understood. The development of new immune-mediated narcolepsy models are likely to further inform the etiology of this sleep disorder and animal models will undoubtedly play a critical role in the development of novel narcolepsy therapeutics.

Acute cognitive effects of the hypocretin receptor antagonist almorexant relative to zolpidem and placebo: a randomized clinical trial.

STUDY OBJECTIVES: Hypnotic medications can adversely affect behavior during unanticipated awakenings during the night. Animals treated with the hypocretin (Hcrt) receptor antagonist almorexant (ALM) have less acute cognitive impairment compared to the GABAA receptor modulator zolpidem (ZOL). This study aimed to determine whether ALM produces less acute cognitive impairment than ZOL in human subjects. METHODS: Healthy, young adult, unmedicated male and female subjects participated in a controlled trial of a single dose of ALM 100 mg (N = 48), ALM 200 mg (N = 53), ZOL 10 mg (N = 49), and placebo (PBO, N = 52). RESULTS: ZOL and both doses of ALM produced similar levels of subjective sleepiness and impaired the ability of subjects to remain awake in a dark, low-stimulus setting relative to PBO. For most cognitive measures, performance under ZOL was significantly worse than ALM or PBO. For tasks involving verbal memory or visual-motor coordination, ZOL impaired performance, whereas the two doses of ALM were no different than PBO. For tasks involving higher-order executive function, ZOL produced impairment in processing speed and inhibitory control, whereas the two doses of ALM were no different than PBO. Performance decrements for ALM were less than ZOL but greater than PBO for some reaction time measures. CONCLUSIONS: The data provide support for the hypothesis that Hcrt receptor antagonists produce less functional impairment than a benzodiazepine receptor agonist (BzRA). These observations are particularly relevant to patients treated with sedative-hypnotics who are at elevated risk for falls and other untoward events during the intended hours for sleep.

Dual orexin and MCH neuron-ablated mice display severe sleep attacks and cataplexy.

Orexin/hypocretin-producing and melanin-concentrating hormone-producing (MCH) neurons are co-extensive in the hypothalamus and project throughout the brain to regulate sleep/wakefulness. Ablation of orexin neurons decreases wakefulness and results in a narcolepsy-like phenotype, whereas ablation of MCH neurons increases wakefulness. Since it is unclear how orexin and MCH neurons interact to regulate sleep/wakefulness, we generated transgenic mice in which both orexin and MCH neurons could be ablated. Double-ablated mice exhibited increased wakefulness and decreased both rapid eye movement (REM) and non-REM (NREM) sleep. Double-ablated mice showed severe cataplexy compared with orexin neuron-ablated mice, suggesting that MCH neurons normally suppress cataplexy. Double-ablated mice also showed frequent sleep attacks with elevated spectral power in the delta and theta range, a unique state that we call 'delta-theta sleep'. Together, these results indicate a functional interaction between orexin and MCH neurons in vivo that suggests the synergistic involvement of these neuronal populations in the sleep/wakefulness cycle.

Shallow metabolic depression and human spaceflight: a feasible first step.

Synthetic torpor is an induced state of deep metabolic depression (MD) in an organism that does not naturally employ regulated and reversible MD. If applied to spaceflight crewmembers, this metabolic state may theoretically mitigate numerous biological and logistical challenges of human spaceflight. These benefits have been the focus of numerous recent articles where, invariably, they are discussed in the context of hypothetical deep MD states in which the metabolism of crewmembers is profoundly depressed relative to basal rates. However, inducing these deep MD states in humans, particularly humans aboard spacecraft, is currently impossible. Here, we discuss shallow MD as a feasible first step toward synthetic torpor during spaceflight and summarize perspectives following a recent NASA-hosted workshop. We discuss methods to safely induce shallow MD (e.g., sleep and slow wave enhancement via acoustic and photoperiod stimulation; moderate sedation via dexmedetomidine), which we define as an ~20% depression of metabolic rate relative to basal levels. We also discuss different modes of shallow MD application (e.g., habitual versus targeted, whereby shallow MD is induced routinely throughout a mission or only under certain circumstances, respectively) and different spaceflight scenarios that would benefit from its use. Finally, we propose a multistep development plan toward the application of synthetic torpor to human spaceflight, highlighting shallow MD's role. As space agencies develop missions to send humans further into space than ever before, shallow MD has the potential to confer health benefits for crewmembers, reduce demands on spacecraft capacities, and serve as a testbed for deeper MD technologies.

REM sleep-active MCH neurons are involved in forgetting hippocampus-dependent memories.

The neural mechanisms underlying memory regulation during sleep are not yet fully understood. We found that melanin concentrating hormone-producing neurons (MCH neurons) in the hypothalamus actively contribute to forgetting in rapid eye movement (REM) sleep. Hypothalamic MCH neurons densely innervated the dorsal hippocampus. Activation or inhibition of MCH neurons impaired or improved hippocampus-dependent memory, respectively. Activation of MCH nerve terminals in vitro reduced firing of hippocampal pyramidal neurons by increasing inhibitory inputs. Wake- and REM sleep-active MCH neurons were distinct populations that were randomly distributed in the hypothalamus. REM sleep state-dependent inhibition of MCH neurons impaired hippocampus-dependent memory without affecting sleep architecture or quality. REM sleep-active MCH neurons in the hypothalamus are thus involved in active forgetting in the hippocampus.

Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy.

Narcolepsy, characterized by excessive daytime sleepiness, is associated with dysfunction of the hypothalamic hypocretin/orexin (Hcrt) system, either due to extensive loss of Hcrt cells (Type 1, NT1) or hypothesized Hcrt signaling impairment (Type 2, NT2). Accordingly, efforts to recapitulate narcolepsy-like symptoms in mice have involved ablating these cells or interrupting Hcrt signaling. Here, we describe orexin/Arch mice, in which a modified archaerhodopsin-3 gene was inserted downstream of the prepro-orexin promoter, resulting in expression of the yellow light-sensitive Arch-3 proton pump specifically within Hcrt neurons. Histological examination along with ex vivo and in vivo electrophysiological recordings of male and female orexin/Arch mice demonstrated silencing of Hcrt neurons when these cells were photoilluminated. However, high expression of the Arch transgene affected cellular and physiological parameters independent of photoillumination. The excitability of Hcrt neurons was reduced, and both circadian and metabolic parameters were perturbed in a subset of orexin/Arch mice that exhibited high levels of Arch expression. Orexin/Arch mice also had increased REM sleep under baseline conditions but did not exhibit cataplexy, a sudden loss of muscle tone during wakefulness characteristic of NT1. These aberrations resembled some aspects of mouse models with Hcrt neuron ablation, yet the number of Hcrt neurons in orexin/Arch mice was not reduced. Thus, orexin/Arch mice may be useful to investigate Hcrt system dysfunction when these neurons are intact, as is thought to occur in narcolepsy without cataplexy (NT2). These results also demonstrate the utility of extended phenotypic screening of transgenic models when specific neural circuits have been manipulated.SIGNIFICANCE STATEMENT Optogenetics has become an invaluable tool for functional dissection of neural circuitry. While opsin expression is often achieved by viral injection, stably integrated transgenes offer some practical advantages. Here, we demonstrate successful transgenic expression of an inhibitory opsin in hypocretin/orexin neurons, which are thought to promote or maintain wakefulness. Both brief and prolonged illumination resulted in inhibition of these neurons and induced sleep. However, even in the absence of illumination, these cells exhibited altered electrical characteristics, particularly when transgene expression was high. These aberrant properties affected metabolism and sleep, resulting in a phenotype reminiscent of the narcolepsy Type 2, a sleep disorder for which no good animal model currently exists.

Electrophysiological characterization of sleep/wake, activity and the response to caffeine in adult cynomolgus macaques.

Most preclinical sleep studies are conducted in nocturnal rodents that have fragmented sleep in comparison to humans who are primarily diurnal, typically with a consolidated sleep period. Consequently, we sought to define basal sleep characteristics, sleep/wake architecture and electroencephalographic (EEG) activity in a diurnal non-human primate (NHP) to evaluate the utility of this species for pharmacological manipulation of the sleep/wake cycle. Adult, 9-11 y.o. male cynomolgus macaques (n = 6) were implanted with telemetry transmitters to record EEG and electromyogram (EMG) activity and Acticals to assess locomotor activity under baseline conditions and following injections either with vehicle or the caffeine (CAF; 10 mg/kg, i.m.) prior to the 12 h dark phase. EEG/EMG recordings (12-36 h in duration) were analyzed for sleep/wake states and EEG spectral composition. Macaques exhibited a sleep state distribution and architecture similar to previous NHP and human sleep studies. Acute administration of CAF prior to light offset enhanced wakefulness nearly 4-fold during the dark phase with consequent reductions in both NREM and REM sleep, decreased slow wave activity during wakefulness, and increased higher EEG frequency activity during NREM sleep. Despite the large increase in wakefulness and profound reduction in sleep during the dark phase, no sleep rebound was observed during the 24 h light and dark phases following caffeine administration. Cynomolgus macaques show sleep characteristics, EEG spectral structure, and respond to CAF in a similar manner to humans. Consequently, monitoring EEG/EMG by telemetry in this species may be useful both for basic sleep/wake studies and for pre-clinical assessments of drug-induced effects on sleep/wake.

Trace amine-associated receptor 1 agonism promotes wakefulness without impairment of cognition in Cynomolgus macaques.

Trace amine-associated receptor 1 (TAAR1) is a G-protein coupled receptor with affinity for the trace amines. TAAR1 agonists have pro-cognitive, antidepressant-, and antipsychotic-like properties in both rodents and non-human primates (NHPs). TAAR1 agonism also increases wakefulness and suppresses rapid-eye movement (REM) sleep in mice and rats and reduces cataplexy in two mouse models of narcolepsy. We investigated the effects of TAAR1 agonism in Cynomolgus macaques, a diurnal species that exhibits consolidated night-time sleep, and evaluated the effects of TAAR1 agonists on cognition using a working memory (WM) paradigm in this species. Adult male Cynomolgus macaques (n = 6) were surgically implanted to record the electroencephalogram (EEG), electromyogram, and locomotor activity (LMA) and the efficacy of the TAAR1 partial agonist RO5263397 (0.1,1,10 mg/kg, p.o.) on sleep/wake, EEG spectra, and LMA was determined. In a second experiment, the acute effects of RO5263397 (0.1,1,10 mg/kg, p.o.) were assessed on a delayed-match-to-sample test of WM in adult male macaques (n = 7). RO5263397 (10 mg/kg) administered at lights off, when sleep pressure was high, promoted wakefulness and reduced both REM and non-REM sleep without inducing hyperlocomotion. RO5263397 (10 mg/kg) also increased delta/theta activity during all vigilance states. RO5263397 had no effect on WM at either short (2 sec) or long (10 sec) delay intervals. The wake-enhancing and REM-suppressing effects of R05263397 shown here in a diurnal primate are consistent with previous results in nocturnal rodents. These effects and the associated alterations in EEG spectra occurred without inducing hyperlocomotion or affecting WM, encouraging further study of TAAR1 agonists as potential narcolepsy therapeutics.

Excitation of Cortical nNOS/NK1R Neurons by Hypocretin 1 is Independent of Sleep Homeostasis.

We have proposed that cortical nNOS/NK1R interneurons have a role in sleep homeostasis. The hypocretins (orexins) are wake-promoting neuropeptides and hypocretin/orexin (Hcrt) neurons project to the cortex. Hcrt peptides affect deep layer cortical neurons, and Hcrt receptor 1 (Hcrtr1; Ox1r) mRNA is expressed in cortical nNOS/NK1R cells. Therefore, we investigated whether Hcrt neuron stimulation affects cingulate cortex nNOS/NK1R neurons. Bath application of HCRT1/orexin-A evoked an inward current and membrane depolarization in most nNOS/NK1R cells which persisted in tetrodotoxin; optogenetic stimulation of Hcrt terminals expressing channelrhodopsin-2 confirmed these results, and pharmacological studies determined that HCRTR1 mediated these responses. Single-cell RT-PCR found Hcrtr1 mRNA in 31% of nNOS/NK1R cells without any Hcrtr2 mRNA expression; immunohistochemical studies of Hcrtr1-EGFP mice confirmed that a minority of nNOS/NK1R cells express HCRTR1. When Hcrt neurons degenerated in orexin-tTA;TetO DTA mice, the increased EEG delta power during NREM sleep produced in response to 4 h sleep deprivation and c-FOS expression in cortical nNOS/NK1R cells during recovery sleep were indistinguishable from that of controls. We conclude that Hcrt excitatory input to these deep layer cells is mediated through HCRTR1 but is unlikely to be involved in the putative role of cortical nNOS/NK1R neurons in sleep homeostasis.