Publications

Sensory information is transmitted from peripheral nerves, through the spinal cord, andup to the brain. Sensory information may be modulated by projections from the brain tothe spinal cord, but the neural substrates for top-down sensory control are incompletelyunderstood. We identified a novel population of inhibitory neurons in the mousebrainstem, distinguished by their expression of prodynorphin, which we named LJA5.Here, we identify a similar group of Pdyn+ neurons in the human brainstem, and we definethe efferent and afferent projection patterns of LJA5 neurons in mouse. Using specificgenetic tools, we selectively traced the projections of the Pdyn-expressing LJA5 neuronsthrough the brain and spinal cord. Terminal fields were densest in the lateral and ventro-lateral periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), caudal pressor area,and lamina I of the spinal trigeminal nucleus and all levels of the spinal cord. We thenlabeled cell types in the PAG, LPB, medulla, and spinal cord to better define the specifictargets of LJA5 boutons. LJA5 neurons send the only known inhibitory descending pro-jection specifically to lamina I of the spinal cord, which transmits afferent pain, tempera-ture, and itch information up to the brain. Using retrograde tracing, we found LJA5neurons receive inputs from sensory and stress areas such as somatosensory/insular cor-tex, preoptic area, paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus,PAG, and LPB. This pattern of inputs and outputs suggest LJA5 neurons are uniquelypositioned to be activated by sensation and stress, and in turn, inhibit pain and itch.

Study Objectives: We determine if young people with narcolepsy type 1 (NT1), narcolepsy type 2 (NT2), and idiopathic hypersomnia (IH) have distinct nocturnal sleep stability phenotypes compared to subjectively sleepy controls. Methods: Participants were 5- to 21-year old and drug-naïve or drug free: NT1 (n = 46), NT2 (n = 12), IH (n = 18), and subjectively sleepy controls (n = 48). We compared the following sleep stability measures from polysomnogram recording between each hypersomnolence disorder to subjectively sleepy controls: number of wake and sleep stage bouts, Kaplan–Meier survival curves for wake and sleep stages, and median bout durations. Results: Compared to the subjectively sleepy control group, NT1 participants had more bouts of wake and all sleep stages (p ≤ .005) except stage N3. NT1 participants had worse survival of nocturnal wake, stage N2, and rapid eye movement (REM) bouts (p < .005). In the first 8 hours of sleep, NT1 participants had longer stage N1 bouts but shorter REM (all ps < .004). IH participants had a similar number of bouts but better survival of stage N2 bouts (p = .001), and shorter stage N3 bouts in the first 8 hours of sleep (p = .003). In contrast, NT2 participants showed better stage N1 bout survival (p = .006) and longer stage N1 bouts (p = .02). Conclusions: NT1, NT2, and IH have unique sleep physiology compared to subjectively sleepy controls, with only NT1 demonstrating clear nocturnal wake and sleep instability. Overall, sleep stability measures may aid in diagnoses and management of these central nervous system disorders of hypersomnolence.

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.

The wake-active orexin system plays a central role in the dynamic regulation of glucose homeostasis. Here we show orexin receptor type 1 and 2 are predominantly expressed in dorsal raphe nucleus-dorsal and -ventral, respectively. Serotonergic neurons in ventral median raphe nucleus and raphe pallidus selectively express orexin receptor type 1. Inactivation of orexin receptor type 1 in serotonin transporter-expressing cells of mice reduced insulin sensitivity in diet-induced obesity, mainly by decreasing glucose utilization in brown adipose tissue and skeletal muscle. Selective inactivation of orexin receptor type 2 improved glucose tolerance and insulin sensitivity in obese mice, mainly through a decrease in hepatic gluconeogenesis. Optogenetic activation of orexin neurons in lateral hypothalamus or orexinergic fibers innervating raphe pallidus impaired or improved glucose tolerance, respectively. Collectively, the present study assigns orexin signaling in serotonergic neurons critical, yet differential orexin receptor type 1- and 2-dependent functions in the regulation of systemic glucose homeostasis.

Origin and functions of intermittent transitions among sleep stages, including brief awakenings and arousals, constitute a challenge to the current homeostatic framework for sleep regulation, focusing on factors modulating sleep over large time scales. Here we propose that the complex micro-architecture characterizing sleep on scales of seconds and minutes results from intrinsic non-equilibrium critical dynamics. We investigate θ- and δ-wave dynamics in control rats and in rats where the sleep-promoting ventrolateral preoptic nucleus (VLPO) is lesioned (male Sprague-Dawley rats). We demonstrate that bursts in θ and δ cortical rhythms exhibit complex temporal organization, with long-range correlations and robust duality of power-law (θ-bursts, active phase) and exponential-like (δ-bursts, quiescent phase) duration distributions, features typical of non-equilibrium systems self-organizing at criticality. We show that such non-equilibrium behavior relates to anti-correlated coupling between θ- and δ-bursts, persists across a range of time scales, and is independent of the dominant physiologic state; indications of a basic principle in sleep regulation. Further, we find that VLPO lesions lead to a modulation of cortical dynamics resulting in altered dynamical parameters of θ- and δ-bursts and significant reduction in θ–δ coupling. Our empirical findings and model simulations demonstrate that θ–δ coupling is essential for the emerging non-equilibrium critical dynamics observed across the sleep–wake cycle, and indicate that VLPO neurons may have dual role for both sleep and arousal/brief wake activation. The uncovered critical behavior in sleep- and wake-related cortical rhythms indicates a mechanism essential for the micro-architecture of spontaneous sleep-stage and arousal transitions within a novel, non-homeostatic paradigm of sleep regulation.

Orexin receptor antagonists are clinically useful for treating insomnia, but thorough blockade of orexin signaling could cause narcolepsy-like symptoms. Specifically, while sleepiness is a desirable effect, an orexin antagonist could also produce cataplexy, sudden episodes of muscle weakness often triggered by strong, positive emotions. In this study, we examined the effects of dual orexin receptor antagonists (DORAs), lemborexant (E2006) and almorexant, on sleep-wake behavior and cataplexy during the dark period in wild-type (WT) mice and prepro-orexin knockout (OXKO) mice. In WT mice, lemborexant at 10 and 30 mg/kg quickly induced NREM sleep in a dose-dependent fashion. In contrast, lemborexant did not alter sleep-wake behavior in OXKO mice. Under the baseline condition, cataplexy was rare in lemborexant-treated WT mice, but when mice were given chocolate as a rewarding stimulus, lemborexant dose-dependently increased cataplexy. Almorexant produced similar results. Collectively, these results demonstrate that DORAs potently increase NREM and REM sleep in mice via blockade of orexin signaling, and higher doses can cause cataplexy when co-administered with a likely rewarding stimulus.

The sleep disorder narcolepsy is associated with symptoms related to either boundary state control that include excessive daytime sleepiness and sleep fragmentation, or rapid eye movement (REM) sleep features including cataplexy, sleep paralysis, hallucinations, and sleep-onset REM sleep events (SOREMs). Although the loss of Hypocretin/Orexin (Hcrt/ Ox) peptides or their receptors have been associated with the disease, here we propose a circuit perspective of the pathophysiological mechanisms of these narcolepsy symptoms that encompasses brain regions, neuronal circuits, cell types, and transmitters beyond the Hcrt/Ox system. We further discuss future experimental strategies to investigate brainwide mechanisms of narcolepsy that will be essential for a better understanding and treatment of the disease.

Researchers have hypothesized that narcolepsy type 1 (NT1) is caused by an autoimmune process since the 1990s, when narcolepsy was first found to be associated with the human leukocyte antigen (HLA) DQB1*06:02/DQA1*01:02 (DQ0602). In 2000, NT1 was discovered to be due to the selective loss of the hypocretin (HCRT, also known as orexin)-producing neurons in the hypothalamus [1–3]; and in 2010, Pandemrix, an H1N1 flu vaccine used in northern Europe, triggered numerous new cases of narcolepsy, further supporting an autoimmune trigger [4].

In the last year, several groups have established that people with NT1 have autoreactive T cells targeting the prepro-hypocretin peptides [5–8]. In addition, T cells expressing specific T-cell receptor alpha (TCRα) J24 gene segments cross-react with the C-terminal ends of HCRT-1 and HCRT-2 when presented by DQ0602 [6, 7, 9]; specifically, NT1 is associated with an F to L polymorphism in the CDR3 region of J24 [6]. Considered together, these findings suggest that hypocretin neurons are directly injured by these T cells, resulting in NT1, and that medications that inhibit migration of T cells into the brain could halt the destruction of the hypocretin neurons.

Given these new discoveries, we treated a young woman soon after NT1 onset with natalizumab (Tysabri), a drug that inhibits T cells and other leukocytes from crossing the blood-brain barrier, and then tracked her clinical symptoms and cerebrospinal fluid (CSF) hypocretin over time.

Study Objectives: Disrupted nighttime sleep (DNS) is a core narcolepsy symptom of unconsolidated sleep resulting from hypocretin neuron loss. In this study, we define a DNS objective measure and evaluate its diagnostic utility for pediatric narcolepsy type 1 (NT1). Methods: This was a retrospective, multisite, cross-sectional study of polysomnograms (PSGs) in 316 patients, ages 6–18 years (n = 150 NT1, n = 22 narcolepsy type 2, n = 27 idiopathic hypersomnia, and n = 117 subjectively sleepy subjects). We assessed sleep continuity PSG measures for (1) their associations with subjective and objective daytime sleepiness, daytime sleep onset REM periods (SOREMPs), self-reported disrupted nocturnal sleep and CSF hypocretin levels and (2) their predictive value for NT1 diagnosis. We then combined the best performing DNS measure with nocturnal SOREMP (nSOREMP) to assess the added value to the logistic regression model and the predictive accuracy for NT1 compared with nSOREMP alone. Results: The Wake/N1 Index (the number of transitions from any sleep stage to wake or NREM stage 1 normalized by total sleep time) was associated with objective daytime sleepiness, daytime SOREMPs, self-reported disrupted sleep, and CSF hypocretin levels (p’s < 0.003) and held highest area under the receiver operator characteristic curves (AUC) for NT1 diagnosis. When combined with nSOREMP, the DNS index had greater accuracy for diagnosing NT1 (AUC = 0.91 [0.02]) than nSOREMP alone (AUC = 0.84 [0.02], likelihood ratio [LR] test p < 0.0001). Conclusions: The Wake/N1 Index is an objective DNS measure that can quantify DNS severity in pediatric NT1. The Wake/N1 Index in combination with or without nSOREMP is a useful sleep biomarker that improves recognition of pediatric NT1 using only the nocturnal PSG.

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.