Ageing-related modification of sleep and breathing in orexin-knockout narcoleptic mice.

Narcolepsy type-1 (NT1) is a lifelong sleep disease, characterised by impairment of the orexinergic system, with a typical onset during adolescence and young adulthood. Since the wake-sleep cycle physiologically changes with ageing, this study aims to compare sleep patterns between orexin-knockout (KO) and wild type (WT) control mice at different ages. Four groups of age-matched female KO and WT mice (16 weeks of age: 8 KO-YO and 9 WT-YO mice; 87 weeks of age: 13 KO-OLD and 12 WT-OLD mice) were implanted with electrodes for discriminating wakefulness, rapid-eye-movement sleep (REMS), and non-REMS (NREMS). Mice were recorded for 48 h in their home cages and for 7 more hours into a plethysmographic chamber to characterise their sleep-breathing pattern. Regardless of orexin deficiency, OLD mice spent less time awake and had fragmentation of this behavioural state showing more bouts of shorter length than YO mice. OLD mice also had more NREMS bouts and less frequent NREMS apneas than YO mice. Regardless of age, KO mice showed cataplexy-like episodes and shorter REMS latency than WT controls and had a faster breathing rate and an increased minute ventilation during REMS. KO mice also had more wakefulness, NREMS and REMS bouts, and a shorter mean length of wakefulness bouts than WT controls. Our experiment indicated that the lack of orexins as well as ageing importantly modulate the sleep and breathing phenotype in mice. The narcoleptic phenotype caused by orexin deficiency in female mice was substantially preserved with ageing.

How to study sleep apneas in mouse models of human pathology.

Sleep apnea, the most widespread sleep-related breathing disorder (SBD), consists of recurrent episodes of breathing cessation during sleep. This condition can be classified as either central (CSA) or obstructive (OSA) sleep apnea, with the latest being the most common and toxic. Due to the complexity of living organisms, animal models and, particularly, mice still represent an essential tool for the study of SBD. In the present review we first discuss the methodological pros and cons in the use of whole-body plethysmography to coupling respiratory and sleep measurements and to characterize CSA and OSA in mice; then, we draw an updated and objective picture of the methods used so far in the study of sleep apnea in mice. Most of the studies present in the literature used intermittent hypoxia to mimic OSA in mice and to investigate consequent pathological correlates. On the contrary, few studies using genetic manipulation or high-fat diets investigated the pathogenesis or potential treatments of sleep apnea. To date, mice lacking orexins, hemeoxygenase-2, monoamine oxidase A, Phox2b or Cdkl5 can be considered validated mouse models of sleep apnea. Moreover, genetically- or diet-induced obese mice, and mice recapitulating Down syndrome were proposed as OSA models. In conclusion, our review shows that despite the growing interest in the field and the need of new therapeutical approaches, technical complexity and inter-study variability strongly limit the availability of validated mouse of sleep apnea, which are essential in biomedical research.

Pilot Study of the Effects of Chronic Intracerebroventricular Infusion of Human Anti-IgLON5 Disease Antibodies in Mice.

BACKGROUND: Anti-IgLON5 disease is a rare late-onset neurological disease associated with autoantibodies against IgLON5, neuronal accumulation of phosphorylated Tau protein (p-Tau), and sleep, respiratory, and motor alterations. PURPOSE: We performed a pilot study of whether the neuropathological and clinical features of anti-IgLON5 disease may be recapitulated in mice with chronic intracerebroventricular infusion of human anti-IgLON5 disease IgG (Pt-IgG). METHODS: Humanized transgenic hTau mice expressing human Tau protein and wild-type (WT) control mice were infused intracerebroventricularly with Pt-IgG or with antibodies from a control subject for 14 days. The sleep, respiratory, and motor phenotype was evaluated at the end of the antibody infusion and at least 30 days thereafter, followed by immunohistochemical assessment of p-Tau deposition. RESULTS: In female hTau and WT mice infused with Pt-IgG, we found reproducible trends of diffuse neuronal cytoplasmic p-Tau deposits in the brainstem and hippocampus, increased ventilatory period during sleep, and decreased inter-lick interval during wakefulness. These findings were not replicated on male hTau mice. CONCLUSION: The results of our pilot study suggest, but do not prove, that chronic ICV infusion of mice with Pt-IgG may elicit neuropathological, respiratory, and motor alterations. These results should be considered as preliminary until replicated in larger studies taking account of potential sex differences in mice.

Early-life nicotine or cotinine exposure produces long-lasting sleep alterations and downregulation of hippocampal corticosteroid receptors in adult mice.

Early-life exposure to environmental toxins like tobacco can permanently re-program body structure and function. Here, we investigated the long-term effects on mouse adult sleep phenotype exerted by early-life exposure to nicotine or to its principal metabolite, cotinine. Moreover, we investigated whether these effects occurred together with a reprogramming of the activity of the hippocampus, a key structure to coordinate the hormonal stress response. Adult male mice born from dams subjected to nicotine (NIC), cotinine (COT) or vehicle (CTRL) treatment in drinking water were implanted with electrodes for sleep recordings. NIC and COT mice spent significantly more time awake than CTRL mice at the transition between the rest (light) and the activity (dark) period. NIC and COT mice showed hippocampal glucocorticoid receptor (GR) downregulation compared to CTRL mice, and NIC mice also showed hippocampal mineralocorticoid receptor downregulation. Hippocampal GR expression significantly and inversely correlated with the amount of wakefulness at the light-to-dark transition, while no changes in DNA methylation were found. We demonstrated that early-life exposure to nicotine (and cotinine) concomitantly entails long-lasting reprogramming of hippocampal activity and sleep phenotype suggesting that the adult sleep phenotype may be modulated by events that occurred during that critical period of life.

Obstructive sleep apneas naturally occur in mice during REM sleep and are highly prevalent in a mouse model of Down syndrome.

STUDY OBJECTIVES: The use of mouse models in sleep apnea study is limited by the belief that central (CSA) but not obstructive sleep apneas (OSA) occur in rodents. We aimed to develop a protocol to investigate the presence of OSAs in wild-type mice and, then, to apply it to a validated model of Down syndrome (Ts65Dn), a human pathology characterized by a high incidence of OSAs. METHODS: In a pilot study, nine C57BL/6J wild-type mice were implanted with electrodes for electroencephalography (EEG), neck electromyography (nEMG), and diaphragmatic activity (DIA), and then placed in a whole-body-plethysmographic (WBP) chamber for 8 h during the rest (light) phase to simultaneously record sleep and breathing activity. CSA and OSA were discriminated on the basis of WBP and DIA signals recorded simultaneously. The same protocol was then applied to 12 Ts65Dn mice and 14 euploid controls. RESULTS: OSAs represented about half of the apneic events recorded during rapid-eye-movement-sleep (REMS) in each experimental group, while the majority of CSAs were found during non-rapid eye movement sleep. Compared with euploid controls, Ts65Dn mice had a similar total occurrence rate of apneic events during sleep, but a significantly higher occurrence rate of OSAs during REMS, and a significantly lower occurrence rate of CSAs during NREMS. CONCLUSIONS: Mice physiologically exhibit both CSAs and OSAs. The latter appear almost exclusively during REMS, and are highly prevalent in Ts65Dn. Mice may, thus, represent a useful model to accelerate the understanding of the pathophysiology and genetics of sleep-disordered breathing and to help the development of new therapies.

Orexin/Hypocretin and Histamine Cross-Talk on Hypothalamic Neuron Counts in Mice.

The loss of hypothalamic neurons that produce wake-promoting orexin (hypocretin) neuropeptides is responsible for narcolepsy type 1 (NT1). While the number of histamine neurons is increased in patients with NT1, results on orexin-deficient mouse models of NT1 are inconsistent. On the other hand, the effect of histamine deficiency on orexin neuron number has never been tested on mammals, even though histamine has been reported to be essential for the development of a functional orexin system in zebrafish. The aim of this study was to test whether histamine neurons are increased in number in orexin-deficient mice and whether orexin neurons are decreased in number in histamine-deficient mice. The hypothalamic neurons expressing L-histidine decarboxylase (HDC), the histamine synthesis enzyme, and those expressing orexin A were counted in four orexin knock-out mice, four histamine-deficient HDC knock-out mice, and four wild-type C57BL/6J mice. The number of HDC-positive neurons was significantly higher in orexin knock-out than in wild-type mice (2,502 ± 77 vs. 1,800 ± 213, respectively, one-tailed t-test, P = 0.011). Conversely, the number of orexin neurons was not significantly lower in HDC knock-out than in wild-type mice (2,306 ± 56 vs. 2,320 ± 120, respectively, one-tailed t-test, P = 0.459). These data support the view that orexin peptide deficiency is sufficient to increase histamine neuron number, supporting the involvement of the histamine waking system in the pathophysiology of NT1. Conversely, these data do not support a significant role of histamine in orexin neuron development in mammals.

Autonomic mechanisms of blood pressure alterations during sleep in orexin/hypocretin-deficient narcoleptic mice.

STUDY OBJECTIVES: Increase in arterial pressure (AP) during sleep and smaller differences in AP between sleep and wakefulness have been reported in orexin (hypocretin)-deficient mouse models of narcolepsy type 1 (NT1) and confirmed in NT1 patients. We tested whether these alterations are mediated by parasympathetic or sympathetic control of the heart and/or resistance vessels in an orexin-deficient mouse model of NT1. METHODS: Thirteen orexin knock-out (ORX-KO) mice were compared with 12 congenic wild-type (WT) mice. The electroencephalogram, electromyogram, and AP of the mice were recorded in the light (rest) period during intraperitoneal infusion of atropine methyl nitrate, atenolol, or prazosin to block muscarinic cholinergic, ß 1-adrenergic, or α 1-adrenergic receptors, respectively, while saline was infused as control. RESULTS: AP significantly depended on a three-way interaction among the mouse group (ORX-KO vs WT), the wake-sleep state, and the drug or vehicle infused. During the control vehicle infusion, ORX-KO had significantly higher AP values during REM sleep, smaller decreases in AP from wakefulness to either non-rapid-eye-movement (non-REM) sleep or REM sleep, and greater increases in AP from non-REM sleep to REM sleep compared to WT. These differences remained significant with atropine methyl nitrate, whereas they were abolished by prazosin and, except for the smaller AP decrease from wakefulness to REM sleep in ORX-KO, also by atenolol. CONCLUSIONS: Sleep-related alterations of AP due to orexin deficiency significantly depend on alterations in cardiovascular sympathetic control in a mouse model of NT1.

Tibialis anterior electromyographic bursts during sleep in histamine-deficient mice.

Antihistamine medications have been suggested to elicit clinical features of restless legs syndrome. The available data are limited, particularly concerning periodic leg movements during sleep, which are common in restless legs syndrome and involve bursts of tibialis anterior electromyogram. Here, we tested whether the occurrence of tibialis anterior electromyogram bursts during non-rapid eye movement sleep is altered in histidine decarboxylase knockout mice with congenital histamine deficiency compared with that in wild-type control mice. We implanted six histidine decarboxylase knockout and nine wild-type mice to record neck muscle electromyogram, bilateral tibialis anterior electromyogram, and electroencephalogram during the rest (light) period. The histidine decarboxylase knockout and wild-type mice did not differ significantly in terms of sleep architecture. In both histidine decarboxylase knockout and wild-type mice, the distribution of intervals between tibialis anterior electromyogram bursts had a single peak for intervals < 10 s. The total occurrence rate of tibialis anterior electromyogram bursts during non-rapid eye movement sleep and the occurrence rate of the tibialis anterior electromyogram bursts separated by intervals < 10 s were significantly lower in histidine decarboxylase knockout than in wild-type mice. These data do not support the hypothesis that preventing brain histamine signalling may promote restless legs syndrome. Rather, the data suggest that limb movements during sleep, including those separated by short intervals, are a manifestation of subcortical arousal requiring the integrity of brain histamine signalling.

Effect of ambient temperature on sleep breathing phenotype in mice: the role of orexins.

The loss of orexinergic neurons, which release orexins, results in narcolepsy. Orexins participate in the regulation of many physiological functions, and their role as wake-promoting molecules has been widely described. Less is known about the involvement of orexins in body temperature and respiratory regulation. The aim of this study was to investigate if orexin peptides modulate respiratory regulation as a function of ambient temperature (Ta) during different sleep stages. Respiratory phenotype of male orexin knockout (KO-ORX, N=9) and wild-type (WT, N=8) mice was studied at thermoneutrality (Ta=30°C) or during mild cold exposure (Ta=20°C) inside a whole-body plethysmography chamber. The states of wakefulness (W), non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were scored non-invasively, using a previously validated technique. In both WT and KO-ORX mice, Ta strongly and significantly affected ventilatory period and minute ventilation values during NREMS and REMS; moreover, the occurrence rate of sleep apneas in NREMS was significantly reduced at Ta=20°C compared with Ta=30°C. Overall, there were no differences in respiratory regulation during sleep between WT and KO-ORX mice, except for sigh occurrence rate, which was significantly increased at Ta=20°C compared with Ta=30°C in WT mice, but not in KO-ORX mice. These results do not support a main role for orexin peptides in the temperature-dependent modulation of respiratory regulation during sleep. However, we showed that the occurrence rate of sleep apneas critically depends on Ta, without any significant effect of orexin peptides.