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Thomas Kilduff

SRI Author

Biomedical sciences publications

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.

Keywords: central disorders of hypersomnolence; cerebrospinal fluid; hypersomnia; hypocretin/orexin; monoamine; narcolepsy; sleepiness.

Biomedical sciences publications

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.

Biomedical sciences publications

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.

Biomedical sciences publications

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.

Biomedical sciences publications

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.

Biomedical sciences publications

Parallel Arousal Pathways in the Lateral Hypothalamus

Until recently, hypocretin (Hcrt) neurons were the only known wake-promoting neuronal population in the lateral hypothalamus (LH), but subpopulations of inhibitory neurons in this area and glutamatergic neurons in the nearby supramammillary nucleus (SuM) have recently been found that also promote wakefulness. We performed chemogenetic excitation of LH neurons in mice and observed increased wakefulness that lasted more than 4 h without unusual behavior or EEG anomalies. The increased wakefulness was similar in the presence or absence of the dual orexin receptor blocker almorexant (ALM). Analysis of hM3Dq transfection and c-FOS expression in LH inhibitory neurons and in the SuM failed to confirm that the increased wakefulness was due to these wake-promoting populations, although this possibility cannot be completely excluded. To evaluate the relationship to the Hcrt system, we repeated the study in Orexin-tTA mice in the presence or absence of dietary doxycycline (DOX), which enabled us to manipulate the percentage of Hcrt neurons that expressed hM3Dq. In DOX-fed mice, 18% of Hcrt neurons as well as many other LH neurons expressed hM3Dq; these mice showed a profound increase in wake after hM3Dq activation even in the presence of ALM. In mice switched to normal chow, 62% of Hcrt neurons expressed hM3Dq along with other LH cells; chemogenetic activation produced even more sustained arousal which could be reduced to previous levels by ALM treatment. Together, these results indicate an LH neuron population that promotes wakefulness through an Hcrt-independent pathway that can act synergistically with the Hcrt system to prolong arousal.