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Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

No therapy is currently available for CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. Herein, we used a secretable TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement due to secretion and transduction of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle-treated Cdkl5 KO mice or Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector, indicating that a gene therapy based on a secretable recombinant TATk-CDKL5 protein is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5.

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.

CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene, a member of a highly conserved family of serine-threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF-β action, is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF-β1 treatment normalized defective neuronal survival and maturation in Cdkl5 knockout (KO) neurons. Moreover, we demonstrate that Cdkl5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF-β1 prevents increased NMDA-induced cell death in hippocampal neurons from Cdkl5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of Cdkl5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.

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, β 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.

Neuropeptides orexin A and B (OX-A/B, also called hypocretin 1 and 2) are released selectively by a population of neurons which projects widely into the entire central nervous system but are localized in a restricted area of the tuberal region of the hypothalamus, caudal to the paraventricular nucleus. The OX system prominently targets brain structures involved in the regulation of wake-sleep state switching, and also orchestrates multiple physiological functions. The degeneration and dysregulation of the OX system promotes narcoleptic phenotypes both in humans and animals. Hence, this review begins with the already proven involvement of OX in narcolepsy, but it mainly discusses the new pre-clinical and clinical insights of the role of OX in three major neurological disorders characterized by sleep impairment which have been recently associated with OX dysfunction, such as Alzheimer's disease, stroke and Prader Willi syndrome, and have been emerged over the past 10 years to be strongly associated with the OX dysfunction and should be more considered in the future. In the light of the impairment of the OX system in these neurological disorders, it is conceivable to speculate that the integrity of the OX system is necessary for a healthy functioning body.

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 < 10s. 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 < 10s 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.

Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader–Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11–q13, is characterized by hypothalamic insufficiency. Here, we investigate the role of the paternally expressed Snord116 gene within the context of sleep and metabolic abnormalities of PWS, and we report a significant role of this imprinted gene in the function and organization of the 2 main neuromodulatory systems of the lateral hypothalamus (LH) — namely, the orexin (OX) and melanin concentrating hormone (MCH) — systems. We observed that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion of Snord116 (PWScr(m+/p–)) are compromised. This abnormal state–dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutant mice. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutant mice reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake, and temperature control.

Torpor is a peculiar mammalian behaviour, characterized by the active reduction of metabolic rate, followed by a drop in body temperature. To enter torpor, the activation of all thermogenic organs that could potentially defend body temperature must be prevented. Most of these organs, such as the brown adipose tissue, are controlled by the key thermoregulatory region of the Raphe Pallidus (RPa). Currently, it is not known which brain areas mediate the entrance into torpor. To identify these areas, the expression of the early gene c-Fos at torpor onset was assessed in different brain regions in mice injected with a retrograde tracer (Cholera Toxin subunit b, CTb) into the RPa region. The results show a network of hypothalamic neurons that are specifically activated at torpor onset and a direct torpor-specific projection from the Dorsomedial Hypothalamus to the RPa that could putatively mediate the suppression of thermogenesis during torpor.

Under conditions of scarce food availability and cool ambient temperature, the mouse (Mus Musculus) enters into torpor, a state of transient metabolic suppression mediated in part by the autonomic nervous system. Hypothalamic orexins are involved in the coordination of behaviors and autonomic function. We tested whether orexins are necessary for the coordinated changes in physiological variables, which underlie torpor and represent its physiological signature. We performed simultaneous measurements of brain temperature, electroencephalographic, and electromyographic activity allowing objective assessment of wake-sleep behavior, and cardiovascular, respiratory, and metabolic variables in orexin knockout mice (ORX-KO) and wild-type mice (WT) during torpor bouts elicited by caloric restriction and mild cold stress. We found that torpor bouts in WT are characterized by an exquisitely coordinated physiological signature. The characteristics of torpor bouts in terms of duration and rate of change of brain temperature and electromyographic activity at torpor entrance and exit did not differ significantly between ORX-KO and WT, and neither did the cardiovascular, respiratory, and metabolic characteristics of torpor. ORX-KO and WT also had similar wake-sleep state changes associated with torpor bouts, with the exception of a significantly higher rapid-eye movement sleep time in ORX-KO at torpor entrance. Our results demonstrate that orexins are not necessary either for the normal physiological adaptations occurring during torpor in mice or for their coordination, suggesting that mechanisms different from orexin peptide signaling may be involved in the regulation and the coordination of these physiological responses.

The loss of orexinergic neurons, releasing 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 whether orexin peptides modulate respiratory regulation as a function of ambient temperature (T°a) 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 (T°a=30°C) or during mild cold exposure (T°a=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. Both in WT and KO-ORX mice T°a 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 T°a=20°C compared to T°a=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 T°a=20°C with respect to T°a =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 T°a, without any significant effect of orexin peptides.

Autosomal dominant leukodystrophy (ADLD) is a rare adult-onset demyelinating disease caused by overexpression of lamin B1, a nuclear lamina filament. Early autonomic dysfunction involving the cardiovascular system before progressive somatic motor dysfunction is a striking feature of most cases of ADLD. In the Plp-FLAG-LMNB1 transgenic mouse model, lamin B1 overexpression in oligodendrocytes elicits somatic motor dysfunction and neuropathology akin to ADLD. Here, we investigate whether Plp-FLAG-LMNB1 mice also develop autonomic cardiovascular dysfunction before or after somatic motor dysfunction. We find that Plp-FLAG-LMNB1 mice have preserved cardiovascular responses to changes in wake-sleep state and ambient temperature and normal indexes of autonomic modulation at 37–42 weeks of age despite a progressive somatic motor dysfunction, which includes impairments of walking ability (the ability to walk on a narrow path was impaired in 80% of mice at 34–38 weeks of age) and subtle breathing derangements. Only late in the development of the disease phenotype did Plp-FLAG-LMNB1 mice develop a structural deficit of sympathetic noradrenergic fibers, with a 38% decrease in fiber profiles in the kidneys at 44–47 weeks of age. We demonstrate that while the Plp-FLAG-LMNB1 mouse model recapitulates the age-dependent motor dysfunction of ADLD, it does not show signs of early autonomic cardiovascular dysfunction, raising the possibility that oligodendrocyte dysfunction may not be sufficient to cause the full spectrum of clinical features present in ADLD.

KEY POINTS While values of arterial pressure during sleep are predictive of cardiovascular risk, the autonomic mechanisms underlying the cardiovascular effects of sleep remain poorly understood. Here, we assess the autonomic mechanisms of the cardiovascular effects of sleep in C57Bl/6J mice, taking advantage of a novel technique for continuous intraperitoneal infusion of autonomic blockers. Our results indicate that non-REM sleep decreases arterial pressure by decreasing sympathetic vasoconstriction, decreases heart rate by balancing parasympathetic activation and sympathetic withdrawal, and increases cardiac baroreflex sensitivity mainly by increasing fluctuations in parasympathetic activity. Our results also indicate that REM sleep increases arterial pressure by increasing sympathetic activity to the heart and blood vessels, and increases heart rate, at least in part, by increasing cardiac sympathetic activity. These results provide a framework for generating and testing hypotheses on cardiovascular derangements during sleep in mouse models and human patients. ABSTRACT The values of arterial pressure (AP) during sleep predict cardiovascular risk. Sleep exerts similar effects on cardiovascular control in human subjects and mice. We aimed to determine the underlying autonomic mechanisms in 12 C57Bl/6J mice with a novel technique of intraperitoneal infusion of autonomic blockers, while monitoring the electroencephalogram, electromyogram, AP and heart period (HP, i.e. 1/heart rate). In different sessions, we administered atropine methyl nitrate, atenolol and prazosin to block muscarinic cholinergic, β1 -adrenergic and α1 -adrenergic receptors, respectively, and compared each drug infusion with a matched vehicle infusion. The decrease in AP from wakefulness to non-rapid-eye-movement sleep (N) was abolished by prazosin but was not significantly affected by atropine and atenolol, which, however, blunted the accompanying increase in HP to a similar extent. On passing from N to rapid-eye-movement sleep (R), the increase in AP was significantly blunted by prazosin and atenolol, whereas the accompanying decrease in HP was blunted by atropine and abolished by atenolol. Cardiac baroreflex sensitivity (cBRS, sequence technique) was dramatically decreased by atropine and slightly increased by prazosin. These data indicate that in C57Bl/6J mice, N decreases mean AP by decreasing sympathetic vasoconstriction, increases HP by balancing parasympathetic activation and sympathetic withdrawal, and increases cBRS mainly by increasing fluctuations in parasympathetic activity. R increases mean AP by increasing sympathetic vasoconstriction and cardiac sympathetic activity, which also explains, at least in part, the concomitant decrease in HP. These data represent the first comprehensive assessment of the autonomic mechanisms of cardiovascular control during sleep in mice.

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.

Narcolepsy is a rare disease that entails excessive daytime sleepiness, often associated with sudden episodes of muscle weakness known as cataplexy. Narcolepsy with cataplexy (NC) is due to the loss of hypothalamic neurons that release the neuropeptides orexin A and B. Orexin neuron projections prominently target brain structures involved in wake-sleep state switching and the central autonomic network. This review provides an updated summary of the links between NC and autonomic cardiovascular dysfunction from a translational perspective. The available evidence suggests that, compared with control subjects, the heart rate in patients and animal models with NC is variable during wakefulness and normal to high during sleep. Responses of the heart rate to internal stimuli (arousal from sleep, leg movements during sleep, defense response) are blunted. These alterations result from orexin deficiency and, at least during wakefulness before sleep, involve decreased parasympathetic modulation of the heart rate. On the other hand, NC in patients and animal models is associated with a blunted fall in arterial blood pressure from wakefulness to sleep, and particularly to the REM state, coupled to a variable decrease in arterial blood pressure during wakefulness. The former effect is caused, at least in part, by deranged control of the heart, whereas the latter may be due to decreased vasoconstrictor sympathetic activity. Systematic studies are warranted to help clarify whether and how the links between NC and autonomic dysfunction impact on the cardiovascular risk of patients with narcolepsy.

The Raphe Pallidus (RPa) is a region of the brainstem that was shown to modulate the sympathetic outflow to many tissues and organs involved in thermoregulation and energy expenditure. In rodents, the pharmacological activation of RPa neurons was shown to increase the activity of the brown adipose tissue, heart rate, and expired CO2, whereas their inhibition was shown to induce cutaneous vasodilation and a state of hypothermia that, when prolonged, leads to a state resembling torpor referred to as synthetic torpor. If translatable to humans, this synthetic torpor-inducing procedure would be advantageous in many clinical settings. A first step to explore such translatability, has been to verify whether the neurons within the RPa play the same role described for rodents in a larger mammal such as the pig. In the present study, we show that the physiological responses inducible by the pharmacological stimulation of RPa neurons are very similar to those observed in rodents. Injection of the GABAA agonist GABAzine in the RPa induced an increase in heart rate (from 99 to 174bpm), systolic (from 87 to 170mmHg) and diastolic (from 51 to 98mmHg) arterial pressure, and end-tidal CO2 (from 49 to 62 mmHg). All these changes were reversed by the injection in the same area of the GABAA agonist muscimol. These results support the possibility for RPa neurons to be a key target in the research for a safe and effective procedure for the induction of synthetic torpor in humans.

Sleep apneas can be categorized as post-sigh (prevailing in non-rapid eye movement sleep) or spontaneous (prevailing in rapid eye movement sleep) according to whether or not they are preceded by an augmented breath (sigh). Notably, the occurrence of these apnea subtypes changes differently in hypoxic/hypercapnic environments and in some genetic diseases, highlighting the importance of an objective discrimination. We aim to: (a) systematically review the literature comparing the criteria used in categorizing mouse sleep apneas; and (b) provide data-driven criteria for this categorization, with the final goal of reducing experimental variability in future studies. Twenty-two wild-type mice, instrumented with electroencephalographic/electromyographic electrodes, were placed inside a whole-body plethysmographic chamber to quantify sleep apneas and sighs. Wake-sleep states were scored on 4-s epochs based on electroencephalographic/electromyographic signals. Literature revision showed that highly different criteria were used for post-sigh apnea definition, the intervals for apnea occurrence after sigh ranging from 1 breath up to 20 s. In our data, the apnea occurrence rate during non-rapid eye movement sleep was significantly higher than that calculated before the sigh only in the 1st and 2nd 4-s epochs following a sigh. These data suggest that, in mice, apneas should be categorized as post-sigh only if they start within 8 s from a sigh; the choice of shorter or longer time windows might underestimate or slightly overestimate their occurrence rate, respectively.

Torpor is a peculiar mammalian behaviour, characterized by the active reduction of metabolic rate, followed by a drop in body temperature. To enter torpor, the activation of all thermogenic organs that could potentially defend body temperature must be prevented. Most of these organs, such as the brown adipose tissue, are controlled by the key thermoregulatory region of the Raphe Pallidus (RPa). Currently, it is not known which brain areas mediate the entrance into torpor. To identify these areas, the expression of the early gene c-Fos at torpor onset was assessed in different brain regions in mice injected with a retrograde tracer (Cholera Toxin subunit b, CTb) into the RPa region. The results show a network of hypothalamic neurons that are specifically activated at torpor onset and a direct torpor-specific projection from the Dorsomedial Hypothalamus to the RPa that could putatively mediate the suppression of thermogenesis during torpor.

While values of arterial pressure during sleep are predictive of cardiovascular risk, the autonomic mechanisms underlying the cardiovascular effects of sleep remain poorly understood. Here, we assess the autonomic mechanisms of the cardiovascular effects of sleep in C57Bl/6J mice, taking advantage of a novel technique for continuous intraperitoneal infusion of autonomic blockers. Our results indicate that non-REM sleep decreases arterial pressure by decreasing sympathetic vasoconstriction, decreases heart rate by balancing parasympathetic activation and sympathetic withdrawal, and increases cardiac baroreflex sensitivity mainly by increasing fluctuations in parasympathetic activity. Our results also indicate that REM sleep increases arterial pressure by increasing sympathetic activity to the heart and blood vessels, and increases heart rate, at least in part, by increasing cardiac sympathetic activity. These results provide a framework for generating and testing hypotheses on cardiovascular derangements during sleep in mouse models and human patients.The values of arterial pressure (AP) during sleep predict cardiovascular risk. Sleep exerts similar effects on cardiovascular control in human subjects and mice. We aimed to determine the underlying autonomic mechanisms in 12 C57Bl/6J mice with a novel technique of intraperitoneal infusion of autonomic blockers, while monitoring the electroencephalogram, electromyogram, AP and heart period (HP, i.e. 1/heart rate). In different sessions, we administered atropine methyl nitrate, atenolol and prazosin to block muscarinic cholinergic, β

AIM Gather 'proof-of-concept' evidence of the adverse developmental potential of cotinine (a seemingly benign biomarker of recent nicotine/tobacco smoke exposure). METHODS Pregnant C57 mice drank nicotine- or cotinine-laced water for 6 wks from conception (NPRE = 2% saccharin + 100 μg nicotine/mL; CPRE = 2% saccharin + 10 μg cotinine/mL) or 3 wks after birth (CPOST = 2% saccharin + 30 μg cotinine/mL). Controls drank 2% saccharin (CTRL). At 17 ± 1 weeks (male pups; CTRL n = 6; CPOST n = 6; CPRE n = 8; NPRE n = 9), we assessed (i) cardiovascular control during sleep; (ii) arterial reactivity ex vivo; and (iii) expression of genes involved in arterial constriction/dilation. RESULTS Blood cotinine levels recapitulated those of passive smoker mothers' infants. Pups exposed to cotinine exhibited (i) mild bradycardia - hypotension at rest (p < 0.001); (ii) attenuated (CPRE , p < 0.0001) or reverse (CPOST ; p < 0.0001) BP stress reactivity; (iii) adrenergic hypocontractility (p < 0.0003), low protein kinase C (p < 0.001) and elevated adrenergic receptor mRNA (p < 0.05; all drug-treated arteries); and (iv) endothelial dysfunction (NPRE only). CONCLUSION Cotinine has subtle, enduring developmental consequences. Some cardiovascular effects of nicotine can plausibly arise via conversion into cotinine. Low-level exposure to this metabolite may pose unrecognised perinatal risks. Adults must avoid inadvertently exposing a foetus or infant to cotinine as well as nicotine.

Summary Narcolepsy with cataplexy (NC) is a lifelong disorder caused by loss of hypothalamic hypocretin/orexin (HCRT) neurones, often starting in childhood. NC patients show altered control of heart rate (HR) and a normotensive non-dipper blood pressure (BP) profile, but the natural history and prognostic significance of these alterations remain unclear. Similar alterations have been observed in HCRT-ataxin-3 transgenic (TG) NC mice lacking HCRT neurones, but studies have been limited to young adult individuals

The validation of rodent models for restless legs syndrome (Willis-Ekbom disease) and periodic limb movements during sleep requires knowledge of physiological limb motor activity during sleep in rodents. This study aimed to determine the physiological time structure of tibialis anterior activity during sleep in mice and rats, and compare it with that of healthy humans. Wild-type mice (n = 9) and rats (n = 8) were instrumented with electrodes for recording the electroencephalogram and electromyogram of neck muscles and both tibialis anterior muscles. Healthy human subjects (31 ± 1 years, n = 21) underwent overnight polysomnography. An algorithm for automatic scoring of tibialis anterior electromyogram events of mice and rats during non-rapid eye movement sleep was developed and validated. Visual scoring assisted by this algorithm had inter-rater sensitivity of 92-95% and false-positive rates of 13-19% in mice and rats. The distribution of the time intervals between consecutive tibialis anterior electromyogram events during non-rapid eye movement sleep had a single peak extending up to 10 s in mice, rats and human subjects. The tibialis anterior electromyogram events separated by intervals

BackgroundCotinine - a nicotine by-product and biomarker of passive perinatal tobacco smoke exposure - is historically considered to lack significant health effects. We challenged this notion and sought “proof-of-concept” evidence of the adverse developmental potential of exposure to this substance at real-life levels.MethodsPregnant C57 mice drank nicotine or cotinine-laced water for 6wks from conception (NPRE = 2% saccharin+100μg nicotine/ml; CPRE = 2% saccharin + 10μg cotinine/ml) or for 3wks after birth (CPOST = 2% saccharin + 30μg cotinine/ml). Controls drank 2% saccharin (CTRL). At 17±1weeks male pups (CTRL n=6; CPOST n=6; CPRE n=8; NPRE n=9) were instrumented for EEG and blood pressure (BP) telemetry. We evaluated (i) cardiovascular control during sleep (at rest / during stress); (ii) arterial reactivity ex vivo; (iii) expression of genes involved in arterial constriction/dilation.ResultsBlood cotinine levels (ELISA) recapitulated passive smoker mothers-infants. Pups exposed only to cotinine exhibited (i) mild bradycardia - hypotension at rest (pPRE, pPOST; pPRE pups also exhibited endothelium-mediated dysfunction.ConclusionsCotinine has subtle, enduring developmental consequences. Some cardiovascular effects of nicotine can plausibly arise via conversion to cotinine. Low-level exposure to this metabolite may pose unrecognized perinatal risks. Adults must avoid inadvertently exposing a fetus or infant to cotinine as well as nicotine.

A major limitation in the study of sleep breathing disorders in mouse models of pathology is the need to combine whole-body plethysmography (WBP) to measure respiration with electroencephalography/electromyography (EEG/EMG) to discriminate wake-sleep states. However, murine wake-sleep states may be discriminated from breathing and body movements registered by the WBP signal alone. Our goal was to compare the EEG/EMG-based and the WBP-based scoring of wake-sleep states of mice, and provide formal guidelines for the latter. EEG, EMG, blood pressure and WBP signals were simultaneously recorded from 20 mice. Wake-sleep states were scored based either on EEG/EMG or on WBP signals and sleep-dependent respiratory and cardiovascular estimates were calculated. We found that the overall agreement between the 2 methods was 90%, with a high Cohen’s Kappa index (0.82). The inter-rater agreement between 2 experts and between 1 expert and 1 naïve sleep investigators gave similar results. Sleep-dependent respiratory and cardiovascular estimates did not depend on the scoring method. We show that non-invasive discrimination of the wake-sleep states of mice based on visual inspection of the WBP signal is accurate, reliable and reproducible. This work may set the stage for non-invasive high-throughput experiments evaluating sleep and breathing patterns on mouse models of pathophysiology.

Study Objectives rapid-eye-movement (REM) sleep without atonia (RSWA) is a marker of REM sleep behavior disorder (RBD) and is common in narcolepsy. Available techniques for electromyogram (EMG) analysis are species-specific, limiting translational research on RSWA. We developed an automated technique based on distributions of normalized EMG values (DNE) to overcome this limitation. With DNE, we tested whether the control of neck and tibialis anterior (TA) muscles during sleep in wild-type rats and mice validly models the control of submentalis (chin) and TA muscles in healthy humans. We then applied DNE to REM sleep recordings of patients with idiopathic RBD and of mouse models of narcolepsy, testing for a common DNE signature of RSWA. Methods retrospective analysis of sleep recordings from 20 idiopathic RBD patients, 34 control subjects, 8 wild-type rats, 21 orexin-neuron deficient mice, 8 orexin knock-out mice, and 22 wild-type mice. Results neck EMG of rats and mice and human chin EMG progressively decreased from wakefulness to non-REM sleep and REM sleep, whereas the effects of sleep on TA EMG differed between rats, mice, and humans. DNE discriminated idiopathic RBD patients from controls based on higher median values of normalized chin EMG during REM sleep. The same parameter, computed on neck muscle EMG, discriminated narcoleptic mice from wild-type mice. Conclusions these results support the application of DNE in translational research on RSWA. Increased median values of normalized EMG of chin (humans) and neck (rats and mice) muscles may be a signature of RSWA in different species and pathological conditions.

Histamine and orexins are wake promoters released by hypothalamic neurons. The activity of histamine neurons is increased by orexin neurons. Recently, it has been shown that orexin deficiency entails high-amplitude theta wave bursts during rapid eye movement sleep and cataplexy in narcoleptic mice. The primary aim of this study was to assess whether histamine system is involved in high-amplitude theta wave burst generation during rapid eye movement sleep. The secondary aim was to assess the effects of combined histamine and orexin deficiency on high-amplitude theta wave bursts during rapid eye movement sleep in mice. Twelve histidine-decarboxylase knockout mice with congenital histamine deficiency, seven double mutant mice with combined deficiency of orexin neurons and histamine, and 11 wild-type control mice were studied with electrodes for sleep recordings and a telemetric blood pressure transducer. High-amplitude theta wave bursts during rapid eye movement sleep were detected in each of the histidine-decarboxylase knockout and double mutant mice, whereas only one burst was found in a wild-type control mouse. High-amplitude theta wave bursts occurred significantly more often and were significantly longer in double mutant than in histidine-decarboxylase knockout mice. In conclusion, it was demonstrated that, similarly to orexin, the chronic impairment of histamine entailed high-amplitude theta wave bursts during rapid eye movement sleep. The current data also suggested a synergistic role of orexin and histamine signalling on high-amplitude theta wave bursts during rapid eye movement sleep in mice.

The central neural mechanisms underlying differences in cardiovascular variability between wakefulness, non-rapid-eye-movement sleep (NREMS), and rapid-eye-movement sleep (REMS) remain poorly understood. These mechanisms may involve hypocretin (HCRT)/orexin signaling. HCRT signaling is linked to wake-sleep states, involved in central autonomic control, and impaired in narcoleptic patients. Thus, we investigated whether HCRT signaling plays a role in controlling cardiovascular variability during spontaneous behavior in HCRT-deficient mice. HCRT-ataxin3 transgenic mice lacking HCRT neurons (TG), knockout mice lacking HCRT peptides (KO), and wild-type controls (WT) were instrumented with electrodes for sleep recordings and a telemetric blood pressure transducer. Fluctuations of systolic blood pressure (SBP) and heart period (HP) during undisturbed wake-sleep behavior were analyzed with the sequence technique, cross-correlation functions, and coherent averaging of SBP surges. During NREMS, all mice had lower SBP variability, greater baroreflex contribution to HP control at low frequencies, and greater amplitude of the central autonomic and baroreflex changes in HP associated with SBP surges than during wakefulness. During REMS, all mice had higher SBP variability and depressed central autonomic and baroreflex HP controls relative to NREMS. HP variability during REMS was higher than during NREMS in WT only. TG and KO also had lower amplitude of the cardiac baroreflex response to SBP surges during REMS than WT. These results indicate that chronic lack of HCRT signaling may cause subtle alterations in the control of HP during spontaneous behavior. Conversely, the integrity of HCRT signaling is not necessary for the occurrence of physiological sleep-dependent changes in SBP variability.

SUMMARY Neurons that release hypocretin (HCRT; orexin) peptides control wake‐ sleep states and autonomic functions, and are lost in patients with narcolepsy with cataplexy. Bursts of high-amplitude electroencephalographic (EEG) activity have been reported during behavioural arrests and rapid eye movement sleep (REMS) episodes at sleep onset in HCRTdeficient narcoleptic mice. Quantitative information on these EEG phenomena is lacking. We aimed to quantify EEG frequency, occurrence rate, daily rhythm and cardiovascular correlates of high-amplitude EEG bursts during REMS and cataplexy. Twenty HCRT-deficient mice and 15 congenic wild-type controls were instrumented with electrodes for sleep recordings and a telemetric blood pressure transducer. Short (1‐2 s) high-amplitude bursts of pointed theta waves (7 Hz) occurred during either REMS or cataplexy in 80% of HCRT-deficient mice without any significant accompanying modification in systolic blood pressure or heart period. Theta bursts were significantly more likely to occur during the dark period and in the last third of REMS episodes. Similar EEG events were detected in a significantly lower fraction (27%) of wild-type mice and with a significantly lower occurrence rate (0.8 versus 5 per hour of REMS). These data demonstrate that occurrence of high-amplitude theta bursts is facilitated during REMS and cataplexy in narcoleptic mice. Analysis of EEG frequency and daily and intra-episode patterns of event occurrence do not support interpretation of theta bursts as temporally displaced pre-REMS spindles. Facilitation of high-amplitude theta bursts may thus represent a novel neurophysiological abnormality associated with chronic HCRT deficiency.

A recently discovered neurodevelopmental disorder caused by the mutation of the cyclin-dependent kinase-like 5 gene (CDKL5) entails complex autistic-like behaviours similar to Rett syndrome, but its impact upon physiological functions remains largely unexplored. Sleep-disordered breathing is common and potentially life-threatening in patients with Rett syndrome; however, evidence is limited in children with CDKL5 disorder, and is lacking altogether in adults. The aim of this study was to test whether the breathing pattern during sleep differs between adult Cdkl5 knockout (Cdkl5-KO) and wild-type (WT) mice. Using whole-body plethysmography, sleep and breathing were recorded non-invasively for 8 h during the light period. Sleep apneas occurred more frequently in Cdkl5-KO than in WT mice. A receiver operating characteristic (ROC) analysis discriminated Cdkl5-KO significantly from WT mice based on sleep apnea occurrence. These data demonstrate that sleep apneas are a core feature of CDKL5 disorder and a respiratory biomarker of CDKL5 deficiency in mice, and suggest that sleep-disordered breathing should be evaluated routinely in CDKL5 patients.

In Spontaneously Hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats, we quantified the extent to which spontaneous fluctuations of heart period (HP) may be determined from arterial pressure based on linear baroreflex properties. We analyzed time series (30-s length) of low-frequency (

We investigated whether the relative contribution of the baroreflex and central commands to the control of heart period differs between spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) during physiological behavior. Rats were instrumented with an arterial catheter and with electrodes for discriminating wakefulness, nonrapid eye movement sleep (NREMS), and rapid eye movement sleep (REMS). The cross-correlation function (CCF) between spontaneous fluctuations of heart period and mean arterial pressure was computed at frequencies

Narcolepsy type 1 is associated with loss of orexin neurons, sleep-wake derangements, cataplexy, and a wide spectrum of alterations in other physiological functions, including energy balance, cardiovascular, and respiratory control. It is unclear which narcolepsy signs are directly related to the lack of orexin neurons or are instead modulated by dysfunction of other neurotransmitter systems physiologically controlled by orexin neurons, such as the histamine system. To address this question, we tested whether some of narcolepsy signs would be detected in mice lacking histamine signaling (HDC-KO). Moreover, we studied double-mutant mice lacking both histamine signaling and orexin neurons (DM) to evaluate whether the absence of histamine signaling would modulate narcolepsy symptoms produced by orexin deficiency. Mice were instrumented with electrodes for recording the electroencephalogram and electromyogram and a telemetric arterial pressure transducer. Sleep attacks fragmenting wakefulness, cataplexy, excess rapid-eye-movement sleep (R) during the activity period, and enhanced increase of arterial pressure during R, which are hallmarks of narcolepsy in mice, did not occur in HDC-KO, whereas they were observed in DM mice. Thus, these narcolepsy signs are neither caused nor abrogated by the absence of histamine. Conversely, the lack of histamine produced obesity in HDC-KO and to a greater extent also in DM. Moreover, the regularity of breath duration during R was significantly increased in either HDC-KO or DM relative to that in congenic wild-type mice. Defects of histamine transmission may thus modulate the metabolic and respiratory phenotype of murine narcolepsy.

Sleep is a complex behavior that cyclically alternates with wakefulness and is fundamental for both mental and physical wellness. During normal sleep the body acquires a specific posture; any active behavior is absent and mental activity typically oscillates between a state of “loss of consciousness” and the experience of dreaming. Sleep is, by itself, a cyclical process characterized by the alternation of two phases, non-REM (NREM) sleep and “rapid eye movement” (REM) sleep, during which the typical synchronized electroencephalographic (EEG) pattern of NREM sleep is substituted by a “paradoxical” EEG desynchronization. NREM sleep is a state of minimal energy expenditure and motor activity, during which cardiorespiratory and thermoregulatory variables are driven by the autonomic nervous system at a lower and more stable level compared to wakefulness. During REM sleep, beyond the occurrence of REMs, posture control is lost and dreams become more vivid. Furthermore, autonomic activity is highly unstable leading to centrally driven surges in heart rate and blood pressure, breathing becomes irregular, and thermoregulation is suspended or depressed, suggesting a derangement of the integrative regulation of physiological functions. Overall sleep quality is one of the most important parameters considered for the subjective assessment of quality of life and general health status. However, although sleep function(s) is intuitively associated with some kind of rest that seems to be mostly required by the central nervous system, and many theories on sleep function have been proposed, a full comprehension of sleep function has not yet been achieved and is probably not imminent.

In 2012, high amplitude theta wave bursts (HATs) were originally described during REMS and cataplexy in ORX-deficient mice as a novel neurophysiological correlate of narcolepsy (Bastianini et al., 2012). This finding was replicated the following year by Vassalli et al. in both ORX-deficient narcoleptic mice and narcoleptic children during cataplexy episodes (Vassalli et al., 2013). The relationship between HATs and narcolepsy-cataplexy in mice and patients indicates that the lack of ORX peptides is responsible for this abnormal EEG activity, the physiological meaning of which is still unknown. This review aimed to explore different phasic EEG events previously described in the published literature in order to find analogies and differences with HATs observed in narcoleptic mice and patients. We found similarities in terms of morphology, frequency and duration between HATs and several physiological (mu and wicket rhythms, sleep spindles, saw-tooth waves) or pathological (SWDs, HVSs, bursts of polyphasic complexes EEG complexes reported in a mouse model of CJD, and BSEs) EEG events. However, each of these events also shows significant differences from HATs, and thus cannot be equaled to them. The available evidence thus suggests that HATs are a novel neurophysiological phenomenon. Further investigations on HATs are required in order to investigate their physiological meaning, to individuate their brain structure(s) of origin, and to clarify the neural circuits involved in their manifestation.