Stability and Volatility of Human Rest-Activity Rhythms: Insights from Very Long Actograms (VLAs).

Importance: Wrist-worn activity monitors provide biomarkers of health by non-obtrusively measuring the timing and amount of rest and physical activity (rest-activity rhythms, RARs). The morphology and robustness of RARs vary by age, gender, and sociodemographic factors, and are perturbed in various chronic illnesses. However, these are cross-sectionally derived associations from recordings lasting 4-10 days, providing little insights into how RARs vary with time. Objective: To describe how RAR parameters can vary or evolve with time (~months). Design Setting and Participants: 48 very long actograms ("VLAs", ≥90 days in duration) were identified from subjects enrolled in the STAGES (Stanford Technology, Analytics and Genomics in Sleep) study, a prospective cross-sectional, multi-site assessment of individuals > 13 years of age that required diagnostic polysomnography to address a sleep complaint. A single 3-year long VLA (author GD) is also described. Exposures/Intervention: None planned. Main Outcomes and Measures: For each VLA, we assessed the following parameters in 14-day windows: circadian/ultradian spectrum, pseudo-F statistic ("F"), cosinor amplitude, intradaily variability, interdaily stability, acrophase and estimates of "sleep" and non-wearing. Results: Included STAGES subjects (n = 48, 30 female) had a median age of 51, BMI of 29.4kg/m2, Epworth Sleepiness Scale score (ESS) of 10/24 and a median recording duration of 120 days. We observed marked within-subject undulations in all six RAR parameters, with many subjects displaying ultradian rhythms of activity that waxed and waned in intensity. When appraised at the group level (nomothetic), averaged RAR parameters remained remarkably stable over a ~4 month recording period. Cohort-level deficits in average RAR robustness associated with unemployment or high BMI (>29.4) also remained stable over time. Conclusions and Relevance: Through an exemplary set of months-long wrist actigraphy recordings, this study quantitatively depicts the longitudinal stability and dynamic range of human rest-activity rhythms. We propose that continuous and long-term actigraphy may have broad potential as a holistic, transdiagnostic and ecologically valid monitoring biomarker of changes in chronobiological health. Prospective recordings from willing subjects will be necessary to precisely define contexts of use.

Acoustic stimulation time-locked to the beginning of sleep apnea events reduces oxygen desaturations: a pilot-study.

STUDY OBJECTIVES: We aimed to determine whether bone-conducted acoustic stimulation could prematurely terminate sleep apnea events, thereby decreasing amplitude and duration of subsequent oxygen desaturation. As oxygen desaturation has been linked to cardiovascular consequences, we postulate this could be a viable therapy in some cases. METHODS: Eight patients with severe Obstructive Sleep Apnea (2 women, 45 [20-68] y.o. Apnea-Hypopnea Index: 77.7 ± 22.3/h) underwent polysomnography at the Lausanne University Sleep Center. Short acoustic stimulations were administered by bone conduction every second event of sleep apnea. Sounds were remotely administered using a Dreem® headband worn by patients while undergoing nocturnal polysomnography. Amplitude (%) and duration(s) of oxygen desaturations following terminated apneas were compared to that of non-stimulated previous and subsequent events. RESULTS: 549 stimulations (68.6 ± 38 sounds per patient) in N1 (16.2%), N2 (69.9%), N3 (4.2%), and REM(9.6%) were conducted. Compared to the previous and subsequent non-stimulated apnea, stimulations reduced event duration by 21.4% (-3.4 ± 7.2 s, p < 0.0001) while oxygen desaturation amplitude and duration were reduced by 30.4% (mean absolute difference ± SD: -1.9 ± 2.8%, p < 0.0001), and 39.6% (-5.7 ± 9.2 s, p < 0.0001) respectively. For these variables, each patient showed a significant improvement following acoustic stimulation. Sound-associated discomfort was rated 1.14 ± 1.53 on an 8 points scale (8 = worst) and only 6.8% of emitted sounds were perceived by the patients, suggesting a well-tolerated intervention. CONCLUSIONS: Bone-conducted sound stimuli decreased apnea events duration as well as duration and amplitude of associated oxygen desaturations. Stimulations were well tolerated and rarely perceived by patients. This therapeutic approach deserves further investigation, with monitoring of effects on sleep quality, daytime function/sleepiness and cardiovascular parameters.

Risk of narcolepsy associated with inactivated adjuvanted (AS03) A/H1N1 (2009) pandemic influenza vaccine in Quebec.

CONTEXT: An association between an adjuvanted (AS03) A/H1N1 pandemic vaccine and narcolepsy has been reported in Europe. OBJECTIVE: To assess narcolepsy risk following administration of a similar vaccine in Quebec. DESIGN: Retrospective population-based study. SETTING: Neurologists and lung specialists in the province were invited to report narcolepsy cases to a single reference centre. POPULATION: Patients were interviewed by two sleep experts and standard diagnostic tests were performed. Immunization status was verified in the provincial pandemic influenza vaccination registry. MAIN OUTCOME MEASURES: Confirmed narcolepsy with or without cataplexy with onset of excessive daytime sleepiness between January 1st, 2009, and December 31st, 2010. Relative risks (RRs) were calculated using a Poisson model in a cohort analysis, by a self-controlled case series (SCCS) and a case-control method. RESULTS: A total of 24 cases were included and overall incidence rate was 1.5 per million person-years. A cluster of 7 cases was observed among vaccinated persons in the winter 2009-2010. In the primary cohort analysis, 16-week post-vaccination RR was 4.32 (95% CI: 1.50-11.12). RR was 2.07 (0.70-6.17) in the SCCS, and 1.48 (0.37-7.03) using the case-control method. Estimates were lower when observation was restricted to the period of pandemic influenza circulation, and tended to be higher in persons <20 years old and for cataplexy cases. CONCLUSIONS: Results are compatible with an excess risk of approximately one case per million vaccine doses, mainly in persons less than 20 years of age. However, a confounding effect of the influenza infection cannot be ruled out.

Transcriptional regulation of the hypocretin/orexin gene by NR6A1.

The hypocretin (also known as orexin) neuropeptide system coordinates the regulation of various physiological processes. A reduction in Nr6a1 expression was observed in hypocretin neuron-ablated transgenic mice. To show that prepro-hypocretin transcription is functionally modulated by NR6A1, we performed chromatin immunoprecipitation (ChIP) analysis, double-immunostaining, a luciferase reporter assay, and an in utero electroporation study. ChIP analysis showed that endogenous NR6A1 binds to a putative NR6A1-binding site. Double-immunostaining indicated almost all hypocretin neurons were positive for NR6A1 immunoreactivity. NR6A1 overexpression in SH-SY5Y cells modulated hypocretin promoter activity, an effect that was countered by lacking a putative NR6A1-binding site. Electroporation with Nr6a1 in the foetal hypothalamus promoted hypocretin transcription as compared to GFP-electroporation. These experiments confirmed that NR6A1 works as a regulator for hypocretin transcription.

Characterization of two melanin-concentrating hormone genes in zebrafish reveals evolutionary and physiological links with the mammalian MCH system.

Melanin-concentrating hormone (MCH) regulates feeding and complex behaviors in mammals and pigmentation in fish. The relationship between fish and mammalian MCH systems is not well understood. Here, we identify and characterize two MCH genes in zebrafish, Pmch1 and Pmch2. Whereas Pmch1 and its corresponding MCH1 peptide resemble MCH found in other fish, the zebrafish Pmch2 gene and MCH2 peptide share genomic structure, synteny, and high peptide sequence homology with mammalian MCH. Zebrafish Pmch genes are expressed in closely associated but non-overlapping neurons within the hypothalamus, and MCH2 neurons send numerous projections to multiple MCH receptor-rich targets with presumed roles in sensory perception, learning and memory, arousal, and homeostatic regulation. Preliminary functional analysis showed that whereas changes in zebrafish Pmch1 expression correlate with pigmentation changes, the number of MCH2-expressing neurons increases in response to chronic food deprivation. These findings demonstrate that zebrafish MCH2 is the putative structural and functional ortholog of mammalian MCH and help elucidate the nature of MCH evolution among vertebrates.

Narcolepsy is strongly associated with the T-cell receptor alpha locus.

Narcolepsy with cataplexy, characterized by sleepiness and rapid onset into REM sleep, affects 1 in 2,000 individuals. Narcolepsy was first shown to be tightly associated with HLA-DR2 (ref. 3) and later sublocalized to DQB1*0602 (ref. 4). Following studies in dogs and mice, a 95% loss of hypocretin-producing cells in postmortem hypothalami from narcoleptic individuals was reported. Using genome-wide association (GWA) in Caucasians with replication in three ethnic groups, we found association between narcolepsy and polymorphisms in the TRA@ (T-cell receptor alpha) locus, with highest significance at rs1154155 (average allelic odds ratio 1.69, genotypic odds ratios 1.94 and 2.55, P < 10(-21), 1,830 cases, 2,164 controls). This is the first documented genetic involvement of the TRA@ locus, encoding the major receptor for HLA-peptide presentation, in any disease. It is still unclear how specific HLA alleles confer susceptibility to over 100 HLA-associated disorders; thus, narcolepsy will provide new insights on how HLA-TCR interactions contribute to organ-specific autoimmune targeting and may serve as a model for over 100 other HLA-associated disorders.

IGFBP3 colocalizes with and regulates hypocretin (orexin).

BACKGROUND: The sleep disorder narcolepsy is caused by a vast reduction in neurons producing the hypocretin (orexin) neuropeptides. Based on the tight association with HLA, narcolepsy is believed to result from an autoimmune attack, but the cause of hypocretin cell loss is still unknown. We performed gene expression profiling in the hypothalamus to identify novel genes dysregulated in narcolepsy, as these may be the target of autoimmune attack or modulate hypocretin gene expression. METHODOLOGY/PRINCIPAL FINDINGS: We used microarrays to compare the transcriptome in the posterior hypothalamus of (1) narcoleptic versus control postmortem human brains and (2) transgenic mice lacking hypocretin neurons versus wild type mice. Hypocretin was the most downregulated gene in human narcolepsy brains. Among many additional candidates, only one, insulin-like growth factor binding protein 3 (IGFBP3), was downregulated in both human and mouse models and co-expressed in hypocretin neurons. Functional analysis indicated decreased hypocretin messenger RNA and peptide content, and increased sleep in transgenic mice overexpressing human IGFBP3, an effect possibly mediated through decreased hypocretin promotor activity in the presence of excessive IGFBP3. Although we found no IGFBP3 autoantibodies nor a genetic association with IGFBP3 polymorphisms in human narcolepsy, we found that an IGFBP3 polymorphism known to increase serum IGFBP3 levels was associated with lower CSF hypocretin-1 in normal individuals. CONCLUSIONS/SIGNIFICANCE: Comparison of the transcriptome in narcolepsy and narcolepsy model mouse brains revealed a novel dysregulated gene which colocalized in hypocretin cells. Functional analysis indicated that the identified IGFBP3 is a new regulator of hypocretin cell physiology that may be involved not only in the pathophysiology of narcolepsy, but also in the regulation of sleep in normal individuals, most notably during adolescence. Further studies are required to address the hypothesis that excessive IGFBP3 expression may initiate hypocretin cell death and cause narcolepsy.

Anomalous hypothalamic responses to humor in cataplexy.

BACKGROUND: Cataplexy is observed in a subset of patients with narcolepsy and affects approximately 1 in 2,000 persons. Cataplexy is most often triggered by strong emotions such as laughter, which can result in transient, yet debilitating, muscle atonia. The objective of this study was to examine the neural systems underlying humor processing in individuals with cataplexy. METHODOLOGY/PRINCIPAL FINDINGS: While undergoing functional Magnetic Resonance Imaging (fMRI), we showed ten narcolepsy-cataplexy patients and ten healthy controls humorous cartoons. In addition, we examined the brain activity of one subject while in a full-blown cataplectic attack. Behavioral results showed that participants with cataplexy rated significantly fewer humorous cartoons as funny compared to controls. Concurrent fMRI showed that patients, when compared to controls and in the absence of overt cataplexy symptoms, showed pronounced activity in the emotional network including the ventral striatum and hypothalamus while viewing humorous versus non-humorous cartoons. Increased activity was also observed in the right inferior frontal gyri--a core component of the inhibitory circuitry. In comparison, the one subject who experienced a cataplectic attack showed dramatic reductions in hypothalamic activity. CONCLUSIONS: These findings suggest an overdrive of the emotional circuitry and possible compensatory suppression by cortical inhibitory regions in cataplexy. Moreover, during cataplectic attacks, the hypothalamus is characterized by a marked decrease in activity similar to that observed during sleep. One possible explanation for these findings is an initial overdrive and compensatory shutdown of the hypothalamus resulting in full cataplectic symptoms.

The diurnal rhythm of hypocretin in young and old F344 rats.

STUDY OBJECTIVES: Hypocretins (HCRT-1 and HCRT-2), also known as orexins, are neuropeptides localized in neurons surrounding the perifornical region of the posterior hypothalamus. These neurons project to major arousal centers in the brain and are implicated in regulating wakefulness. In young rats and monkeys, levels of HCRT-1 are highest at the end of the wake-active period and lowest toward the end of the sleep period. However, the effects of age on the diurnal rhythm of HCRT-1 are not known. DESIGN: To provide such data, cerebrospinal fluid (CSF) was collected from the cisterna magna of young (2-month-old, n = 9), middle-aged (12 months, n = 10), and old (24 months, n = 10) F344 rats at 4-hour intervals, (beginning at zeitgeber [ZT]0, lights on). CSF was collected once from each rat every 4 days at 1 ZT point. After collecting the CSF at all of the time points, the rats were kept awake by gentle handling for 8 hours (ZT 0-ZT8), and the CSF was collected again at the end of the sleep-deprivation procedure. HCRT-1 levels in the CSF were determined by radioimmunoassay SETTINGS: Basic neuroscience research lab. MEASUREMENTS AND RESULTS: Old rats had significantly less HCRT-1 in the CSF versus young and middle-aged rats (P < .002) during the lights-on and lights-off periods and over the 24-hour period. In old rats, significantly low levels of HCRT-1 were evident at the end of the lights-off period (predominantly wake-active period). The old rats continued to have less HCRT-1 even after 8 hours of prolonged waking. Northern blot analysis did not show a difference in pre-proHCRT mRNA between age groups. CONCLUSIONS: In old rats there is a 10% decline in CSF HCRT-1 over the 24-hour period. Functionally, if there is less HCRT-1, which our findings indicated, and there is also a decline in HCRT receptor mRNA, as has been previously found, then the overall consequence would be diminished action of HCRT at target sites. This would diminish the waking drive, which in the elderly could contribute to the increased tendency to fall asleep during the normal wake period.

Increased hypocretin-1 levels in cerebrospinal fluid after REM sleep deprivation.

Rat cisternal (CSF) hypocretin-1 in cerebrospinal fluid was measured after 6 or 96 h of REM sleep deprivation and following 24 h of REM sleep rebound. REM deprivation was found to increase CSF hypocretin-1 collected at zeitgeber time (ZT) 8 but not ZT0. Decreased CSF hypocretin levels were also observed at ZT8 after 24 h of REM sleep rebound. These results suggest that REM sleep deprivation activates and REM sleep rebound inhibits the hypocretin system. Increased hypocretin tone during REM deprivation may be important in mediating some of the effects of REM sleep deprivation such as antidepressant effects, hyperphagia and increased sympathetic activity.

Sleep disturbances and hypocretin deficiency in Niemann-Pick disease type C.

DESIGN AND PATIENTS: Subjects with Niemann-Pick disease, type C have been reported to display narcolepsylike symptoms, including cataplexy. In this study, 5 patients with juvenile Niemann-Pick disease were evaluted for sleep abnormalities using nocturnal polysomnography, clinical evaluation, and the Multiple Sleep Latency Test. HLA typing and cerebrospinal fluid hypocretin levels were also evaluated in 4 patients. Niemann-Pick disease diagnosis was confirmed in all cases biochemically and by the presence of foam cells in the bone marrow. RESULTS: Deterioration of intellectual function; the presence of pyramidal, dystonic and cerebellar features; and splenomegaly were observed in all cases. Cataplexy was reported in 1 patient. Nocturnal polysomnography revealed disrupted sleep in all patients. Total sleep time, sleep efficiency, rapid eye movement sleep, and delta sleep amounts were decreased when compared to age-matched controls. Altered sleep patterns included sudden increases in muscle tone during delta sleep, electroencephalographic sigma activity connected with rapid eye movements and muscle atonia, atypical K-complexes and spindle activity, and the presence of alpha-delta sleep. All Niemann-Pick disease cases exhibited fragmentary myoclonus. Shortened mean sleep latencies were observed in 3 patients during the Multiple Sleep Latency Test, but sleep-onset rapid eye movement periods were observed only in the case with cataplexy. This patient was HLA DQB1*0602 positive, while the other subjects were HLA negative. Cerebrospinal fluid hypocretin-1 levels were reduced in 2 patients (1 with cataplexy) while in the 2 other patients, the levels were at the lower range of the normal values. Hypocretin levels in the Niemann-Pick disease group (204.8 +/- 39.3 pg/mL) were significantly reduced when compared to controls (265.8 +/- 48.8 pg/mL). CONCLUSIONS: The findings suggest that lysozomal storage abnormalities in Niemann-Pick disease patients may impact the hypothalamus and, more specifically, hypocretin-containing cells. These changes might be partially responsible for sleep abnormalities and cataplexy in patients with Niemann-Pick disease.

Voxel-based morphometry in hypocretin-deficient narcolepsy.

STUDY OBJECTIVES: Recent studies suggest that narcolepsy is caused by degeneration of hypocretin (orexin) producing neurons. To find evidence for this hypothesis, we aimed to detect structural changes in the hypothalamus and/or hypocretin projection areas of patients with narcolepsy. DESIGN: We used voxel-based morphometry (VBM), an unbiased MRI morphometric method with a high sensitivity for subtle changes in gray and white matter volumes. SETTING: Image acquisition was carried out in the department of Radiology at Leiden University Medical Center; image post-processing was performed in the Wellcome Department of Cognitive Neurology, London. PARTICIPANTS: Fifteen narcoleptic patients were studied, all having cataplexy and typical findings on Multiple Sleep Latency Testing. All patients were HLA-DQB1*0602 positive and hypocretin-1 deficient. The control group consisted of 15 age and sex matched healthy subjects. MEASUREMENTS AND RESULTS: We found no differences in global gray or white matter volumes between patients and controls. Furthermore, regional gray or white matter volumes in the hypothalamus and hypocretin projection areas did not differ between patients and controls. CONCLUSIONS: VBM failed to show structural changes in the brains of patients with narcolepsy. This suggests that narcolepsy either is associated with microscopic changes undetectable by VBM or that functional abnormalities of hypocretin neurons are not associated with structural correlates.

Relationship between CSF hypocretin levels and hypocretin neuronal loss.

The sleep disorder narcolepsy may now be considered a neurodegenerative disease, as there is a massive reduction in the number of neurons containing the neuropeptide, hypocretin (HCRT). Most narcoleptic patients have low to negligible levels of HCRT in the cerebrospinal fluid (CSF), and such measurements serve as an important diagnostic tool. However, the relationship between HCRT neurons and HCRT levels in CSF in human narcoleptics is not known and cannot be directly assessed. To identify this relationship in the present study, the neurotoxin, hypocretin-2-saporin (HCRT2-SAP), was administered to the lateral hypothalamus (LH) to lesion HCRT neurons. CSF was extracted at circadian times (ZT) 0 (time of lights-on) or ZT8 at various intervals (2, 4, 6, 12, 21, 36, 60 days) after neurotoxin administration. Compared to animals given saline in the LH, rats with an average loss of 73% of HCRT neurons had a 50% decline in CSF HCRT levels on day 60. The decline in HCRT levels was evident by day 6 and there was no recovery or further decrease. The decline in HCRT was correlated with increased REM sleep. Lesioned rats that were kept awake for 6 h were not able to release HCRT to match the output of saline rats. As most human narcoleptics have more than 80% reduction of CSF HCRT, the results from this study lead us to conclude that in these patients, virtually all of the HCRT neurons might be lost. In those narcoleptics where CSF levels are within the normal range, it is possible that not all of the HCRT neurons are lost and that the surviving HCRT neurons might be increasing output of CSF HCRT.

Transient total sleep loss in cerebral Whipple's disease: a longitudinal study.

A case with transient, almost complete sleep loss caused by cerebral manifestation of Whipple's disease (WD) is presented. Cerebral WD is rare and in most cases occurs after gastrointestinal infection. In our case, a progressive and finally almost complete sleep loss was the initial and predominant symptom. Polysomnographic studies in several consecutive nights and over 24 h showed a total abolition of the sleep-wake cycle with nocturnal sleep duration of less than 15 min. Endocrine tests revealed hypothalamic dysfunction with flattening of circadian rhythmicity of cortisol, TSH, growth hormone and melatonin. Cerebrospinal fluid (CSF) hypocretin was reduced. [18F]Deoxyglucose positron emission tomography (FDG-PET) revealed hypermetabolic areas in cortical and subcortical areas including the brainstem, which might explain sleep pathology and vertical gaze palsy. In the course of treatment with antibiotics and additional carbamazepine for 1 year, insomnia slowly and gradually improved. Endocrine investigations at 1-year follow-up showed persistent flattening of circadian rhythmicity. The FDG-PET indicated normalized metabolism in distinct regions of the brain stem which paralleled restoration of sleep length. The extent of sleep disruption in this case of organic insomnia was similar to cases of familial fatal insomnia, but was at least partially reversible with treatment.

Sleeping with the hypothalamus: emerging therapeutic targets for sleep disorders.

Delineating the basic mechanisms that regulate sleep will likely result in the development of better treatments for sleep disorders. The hypothalamus is now recognized as a key center for sleep regulation, with hypothalamic neurotransmitter systems providing the framework for therapeutic advances. An increased awareness of the close interaction between sleep and homeostatic systems is also emerging. Progress has occurred in the understanding of narcolepsy--molecular techniques have identified the lateral hypothalamic hypocretin (orexin) neuropeptide system as key to the disorder. Other sleep disorders are now being tackled in the same way and are likely to yield to efforts combining basic and clinical research. Here we highlight the role of the hypothalamus in sleep physiology and discuss neurotransmitter systems, such as adenosine, dopamine, GABA, histamine and hypocretin, that may have therapeutic applications for sleep disorders.

The role of hypocretins (orexins) in sleep regulation and narcolepsy.

The hypocretins (orexins) are two novel neuropeptides (Hcrt-1 and Hcrt-2), derived from the same precursor gene, that are synthesized by neurons located exclusively in the lateral, posterior, and perifornical hypothalamus. Hypocretin-containing neurons have widespread projections throughout the CNS with particularly dense excitatory projections to monoaminergic centers such as the noradrenergic locus coeruleus, histaminergic tuberomammillary nucleus, serotoninergic raphe nucleus, and dopaminergic ventral tegmental area. The hypocretins were originally believed to be primarily important in the regulation of appetite; however, a major function emerging from research on these neuropeptides is the regulation of sleep and wakefulness. Deficiency in hypocretin neurotransmission results in the sleep disorder narcolepsy in mice, dogs, and humans. The hypocretins are also uniquely positioned to link sleep, appetite, and neuroendocrine control. The aim of this review is to describe and discuss the current knowledge regarding the hypocretin neurotransmitter system in narcolepsy and normal sleep.