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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.

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.

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.

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.

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.

Many small mammals, such as the laboratory mouse, utilize the hypometabolic state of torpor in response to caloric restriction. The signals that relay the lack of fuel to initiate a bout of torpor are not known. Because the mouse will only enter a torpid state when calorically challenged, it may be that one of the inputs for initiation into a bout of torpor is the lack of the primary fuel (glucose) used to power brain metabolism in the mouse. Using glucose telemetry in mice, we tested the hypotheses that 1) circulating glucose (GLC), core body temperature (Tb), and activity are significantly interrelated; and 2) that the level of GLC at the onset of torpor differs from both GLC during arousal from torpor and during feeding when there is no torpor. To test these hypotheses, six C57Bl/6J mice were implanted with glucose telemeters and exposed to different feeding conditions (ad libitum, fasting, limited food intake, and refeeding) to create different levels of GLC and Tb. We found a strong positive and linear correlation between GLC and Tb during ad libitum feeding. Furthermore, mice that were calorically restricted entered torpor bouts readily. GLC was low during torpor entry but did not drop precipitously as Tb did at the onset of a torpor bout. GLC significantly increased during arousal from torpor, indicating the presence of endogenous glucose production. While low GLC itself was not predictive of a bout of torpor, hyperactivity and low GLC preceded the onset of torpor, suggesting that this may be involved in triggering torpor.

Restless legs syndrome (RLS) is a neurological disorder that entails an urge to move with a circadian pattern during the evening/night. RLS may be accompanied by decreased sleep time and increased occurrence of periodic leg movements during sleep (PLMS), which involve bursts of tibialis anterior (TA) muscle electromyogram (EMG). Mild hypoxia and non-anemic iron deficiency, a highly prevalent nutritional deficiency, are relatively unexplored factors in RLS pathophysiology. We tested whether mice exposed to mild hypoxia, alone or in combination with non-anemic iron deficiency, show decreased sleep time particularly in the light (rest) period and increased occurrence of TA EMG phasic events similar to human PLMS. Female C57BL/6J mice were fed diets with low or normal iron for 6 months from weaning and instrumented with electrodes to record the electroencephalogram and the EMG of both TA muscles. Mice were recorded in a whole-body plethysmograph while breathing a normoxic or mildly hypoxic (15% O2) gas mixture for 48 h. Hypoxia increased minute ventilation during sleep. The low-iron diet decreased liver and serum iron, leaving blood hemoglobin and brainstem iron levels unaffected. Hypoxia, either alone or in combination with non-anemic iron deficiency, decreased non-rapid-eye-movement (non-REM) sleep time, but this occurred irrespective of the light/dark period and was not associated with increased occurrence of TA EMG events during non-REM sleep. These results do not support the hypothesis that mild hypoxia is sufficient to cause signs of RLS, either alone or in combination with non-anemic iron deficiency, pointing to the necessity of further susceptibility factors.

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.

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

Summary Hypocretin/orexin peptides are known for their role in the control of the wake–sleep cycle and narcolepsy–cataplexy pathophysiology. Recent studies suggested that hypocretin peptides also have a role in pregnancy. We tested this hypothesis by conducting a retrospective analysis on pregnancy complications in two different mouse models of hypocretin deficiency. We recorded 85 pregnancies of mice lacking either hypocretin peptides (knockout) or hypocretin-releasing neurons (transgenic) and their wild-type controls. Pregnancy was associated with unexplained dam death before delivery in 3/15 pregnancies in knockout mice, and in 3/23 pregnancies in transgenic mice. No casualties occurred in wild-type pregnant dams (P

Cannabinoid type 1 (CB1) receptors are highly expressed in the brain and play a role in behavior control. Endogenous cannabinoid signaling is modulated by high-fat diet (HFD). We investigated the consequences of congenital lack of CB1 receptors on sleep in mice fed standard diet (SD) and HFD. CB1 cannabinoid receptor knock-out (KO) and wild-type (WT) mice were fed SD or HFD for 4 months (n = 9-10 per group). Mice were instrumented with electroencephalographic (EEG) and electromyographic electrodes. Recordings were performed during baseline (48 hours), sleep deprivation (gentle handling, 6 hours), sleep recovery (18 hours), and after cage switch (insomnia model paradigm, 6 hours). We found multiple significant effects of genotype on sleep. In particular, KO spent more time awake and less time in non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) than WT during the dark (active) period but not during the light (rest) period, enhancing the day-night variation of wake-sleep amounts. KO had slower EEG theta rhythm during REMS. REMS homeostasis after sleep deprivation was less effective in KO than in WT. Finally, KO habituated more rapidly to the arousing effect of the cage-switch test than WT. We did not find any significant effects of diet or of diet x genotype interaction on sleep. The occurrence of multiple sleep alterations in KO indicates important roles of CB1 cannabinoid receptors in limiting arousal during the active period of the day, in sleep regulation, and in sleep EEG in mice.

Cannabinoid type 1 (CB1) receptors are expressed in the nervous and cardiovascular systems. In mice, CB1 receptor deficiency protects from metabolic consequences of a high-fat diet (HFD), increases sympathetic activity to brown fat, and entails sleep anomalies. We investigated whether sleep-wake and diet-dependent cardiorespiratory control is altered in mice lacking CB1 receptors. CB1 receptor knock-out (KO) and intact wild-type (WT) mice were fed standard diet or a HFD for 3 months, and implanted with a telemetric arterial pressure transducer and electrodes for sleep scoring. Sleep state was assessed together with arterial pressure and heart rate (home cage), or breathing (whole-body plethysmograph). Increases in arterial pressure and heart rate on passing from the light (rest) to the dark (activity) period in the KO were significantly enhanced compared with the WT. These increases were unaffected by cardiac (β1) or vascular (α1) adrenergic blockade. The breathing rhythm of the KO during sleep was also more irregular than that of the WT. A HFD increased heart rate, impaired cardiac vagal modulation, and blunted the central autonomic cardiac control during sleep. A HFD also decreased cardiac baroreflex sensitivity in the KO but not in the WT. In conclusion, we performed the first systematic study of cardiovascular function in CB1 receptor deficient mice during spontaneous wake-sleep behavior, and demonstrated that CB1 receptor KO alters cardiorespiratory control particularly in the presence of a HFD. The CB1 receptor signaling may thus play a role in physiological cardiorespiratory regulation and protect from some adverse cardiovascular consequences of a HFD.

The central neural pathways underlying the physiological coordination between thermoregulation and the controls of the wake-sleep behavior and cardiovascular function remain insufficiently understood. Growing evidence supports the involvement of hypocretin (orexin) peptides in behavioral, cardiovascular, and thermoregulatory functions. We investigated whether the effects of ambient temperature on wake-sleep behavior and cardiovascular control depend on the hypothalamic neurons that release hypocretin peptides. Orexin-ataxin3 transgenic mice with genetic ablation of hypocretin neurons (n = 11) and wild-type controls (n = 12) were instrumented with electrodes for sleep scoring and a telemetric blood pressure transducer. Simultaneous sleep and blood pressure recordings were performed on freely-behaving mice at ambient temperatures ranging between mild cold (20°C) and the thermoneutral zone (30°C). In both mouse groups, the time spent awake and blood pressure were higher at 20°C than at 30°C. The cold-related increase in blood pressure was significantly smaller in rapid-eye-movement sleep (REMS) than either in non-rapid-eye-movement sleep (NREMS) or wakefulness. Blood pressure was higher in wakefulness than either in NREMS or REMS at both ambient temperatures. This effect was significantly blunted in orexin-ataxin3 mice irrespective of ambient temperature and particularly during REMS. These data demonstrate that hypocretin neurons are not a necessary part of the central pathways that coordinate thermoregulation with wake-sleep behavior and cardiovascular control. Data also support the hypothesis that hypocretin neurons modulate changes in blood pressure between wakefulness and the sleep states. These concepts may have clinical implications in patients with narcolepsy with cataplexy, who lack hypocretin neurons.

A daily rhythm of blood pressure (BP), with maximum values in the activity period, carries important prognostic information. The extent to which this rhythm depends on behavioral factors remains debated. Mice are the species of choice for functional genomics. In mice, episodes of wakefulness and sleep are not restricted to particular daily periods, allowing BP in each wake-sleep state to be measured at each time of day. The aim of this study was to investigate whether a circadian rhythm of BP is manifest in each wake-sleep state in mice. Mice with B6 genetic background (n = 26) were implanted with a telemetric BP transducer and electrodes to discriminate wake-sleep states and recorded while housed under a 12:12 h light-dark period. For each mouse, 8 values of BP were obtained in each wake-sleep state (wakefulness, non-rapid-eye-movement sleep, and rapid-eye-movement sleep) by averaging over successive 3-h time bins. Analysis of variance evidenced a significant time effect in each wake-sleep state as well as a significant wake-sleep state × time interaction effect. In an additional group of mice (n = 3) recorded in constant darkness, the Lomb-Scargle periodogram also revealed a significant circadian rhythm of BP in each wake-sleep state. These findings demonstrate that during each wake-sleep state, mice show daily and circadian rhythms of BP in conditions of entrainment to the light-dark cycle and in free-running conditions of constant darkness, respectively.

STUDY OBJECTIVES: sleep deeply affects cardiac autonomic control, the impairment of which is associated with cardiovascular mortality. Obesity entails increased cardiovascular risk and derangements in sleep and cardiac autonomic control. We investigated whether cardiac autonomic control is impaired during sleep in ob/ob mice with morbid obesity caused by congenital leptin deficiency. DESIGN: indexes of cardiac autonomic control based on spontaneous cardiovascular fluctuations were compared between ob/ob and lean wild-type (+/+) mice during wakefulness, non-rapid eye movement sleep (NREMS), and rapid eye movement sleep (REMS). SETTING: N/A PATIENTS OR PARTICIPANTS: 7 ob/ob and 11 +/+ male mice. INTERVENTIONS: instrumentation with electrodes for sleep recordings and a telemetric transducer for measuring blood pressure and heart period. MEASUREMENTS AND RESULTS: In ob/ob mice, the variability of heart period and cardiac baroreflex sensitivity (sequence technique) were significantly lower than in +/+ mice during each wake-sleep state. The vagal modulation of heart period was significantly weaker in ob/ob than in +/+ mice during NREMS and REMS. In ob/ob mice, the cross-correlation function between heart period and blood pressure suggested that the baroreflex contribution to cardiac control was lower than in +/+ mice during wakefulness and NREMS, whereas the contribution of central autonomic commands was lower than in +/+ mice during NREMS and REMS. CONCLUSIONS: These data indicate a dysregulation of cardiac autonomic control during sleep in ob/ob mice. Ob/ob mice may represent a useful tool to understand the molecular pathways that lead to cardiac autonomic dysregulation during sleep in obesity.

Leptin increases sympathetic activity, possibly contributing to hypertension in obese subjects. Hypertension increases cardiovascular mortality, with nighttime (sleep) blood pressure having a substantial prognostic value. We measured blood pressure in male leptin-deficient obese mice (ob/ob; n=7) and their lean wild-type littermates (+/+; n=11) during wakefulness, non–rapid-eye-movement sleep, and rapid-eye-movement sleep to investigate whether, in the absence of leptin, derangements of blood pressure are still associated with obesity and depend on the wake-sleep state. Mice were implanted with a telemetric pressure transducer and electrodes for discriminating wake-sleep states. Mean blood pressure was significantly higher in ob/ob than in +/+ mice during wakefulness (7.3±2.6 mm Hg) and non–rapid-eye-movement sleep (6.7±2.8 mm Hg) but not during rapid-eye-movement sleep (2.6±2.6 mm Hg). In ob/ob and +/+ mice, mean blood pressure was substantially higher during wakefulness than during non–rapid-eye-movement sleep. On passing from non–rapid-eye-movement sleep to rapid-eye-movement sleep, mean blood pressure decreased significantly in ob/ob but not in +/+ mice. The time spent during wakefulness was lower in ob/ob than in +/+ mice during the dark (active) period, whereas the opposite occurred during the light (rest) period. Consequently, mean blood pressure was significantly higher in ob/ob than in +/+ mice during the light (8.2±2.4 mm Hg) but not during the dark (3.0±2.9 mm Hg) period. These data suggest that, in the absence of leptin, obesity may entail hypertensive derangements of blood pressure, which are substantially modulated by the cardiovascular effects of the wake-sleep states.

We compared blood-brain barrier (BBB) permeability to glucose between quiet wakefulness and rapid-eye-movement (REM) sleep to assess whether changes in BBB permeability play a role in coupling glucose supply to the physiologic metabolic needs of the brain. Male Sprague-Dawley rats were prepared with electrodes for wake-sleep state scoring and with arterial and venous catheters. Using the single-pass, dual-label indicator method, unidirectional glucose extraction by the brain and cerebral blood flow (CBF) were simultaneously measured during states of quiet wakefulness ( n = 12) or REM sleep ( n = 7). The product of BBB surface area and permeability to glucose (PS product) was computed in each state. During REM sleep, CBF significantly exceeded that during quiet wakefulness in all regions but the cerebellum, whereas the difference in the PS product between quiet wakefulness and REM sleep was not statistically significant in any brain region. In the brain as a whole, CBF significantly increased 29% from quiet wakefulness to REM sleep, while a nonsignificant 0.8% increase occurred in the PS product. During REM sleep, the increase in CBF indicates a higher rate of brain glucose consumption than in quiet wakefulness, given the tight flow-metabolism coupling in the brain. Therefore, these data show that modulation of BBB permeability to glucose is not a mechanism that provides ‘energy on demand’ during the physiologic brain activation characterising REM sleep.

We read with interest the recent paper by Li et al. (2013) concerning the cardiovascular effects of the dual orexin receptor antagonist Almorexant on the spontaneously hypertensive rat (SHR) model of essential hypertension. The finding that Almorexant caused a significant reduction in blood pressure (BP) in SHR, but not in normotensive control rats of the Wistar–Kyoto (WKY) strain (Li et al. 2013) is of clear pathophysiological interest and is supported by a recent independent report (Lee et al. 2013). Our comment concerns the claim by Li et al. (2013) that the effect of Almorexant on BP did not differ significantly between wakefulness and non-rapid eye movement sleep (NREMS) in SHR in either the light (rest) or dark (activity) period of the day. This led Li et al. (2013) to suggest that modulation of BP by orexin in SHR is not dependent on the state of arousal or diurnal cycle. We would like to draw attention to a few issues which need to be addressed before this suggestion can be accepted. First, the recording protocol employed by Li et al. (2013) may not have been sufficiently sensitive to detect changes in BP related to sleep states and the day–light cycle. The mean value of BP robustly decreases on passing from wakefulness to NREMS in human subjects and animal models (Silvani, 2008), including SHR and WKY rats (Kuo & Yang, 2005; Berteotti et al. 2007). At least during wakefulness, BP is also higher during the dark than during the light period in normotensive rats (Sei et al. 1997) and mice (Bastianini et al. 2011). However, Li et al. (2013) reported that, before Almorexant treatment, BP in SHR was lower during NREMS than during wakefulness in the light period, but not in the dark period, and did not differ between the light and dark periods. Second, Li et al. (2013) did not report the effects of Almorexant on sleep structure in SHR and WKY rats. This is of relevance because changes in sleep structure may exert a significant impact on BP (Silvani, 2008; Silvani et al. 2009). Li et al. (2013) appropriately discussed this issue with reference to a previous study of theirs on normotensive Sprague-Dawley rats (Li & Nattie, 2010). However, SHR show distinct derangements in sleep phenotype, and their poor sleep quality may be linked to sympathetic overdrive via α1 adrenergic receptors (Kuo et al. 2012). Thus, the effects of Almorexant on sleep in SHR may have differed from those in normotensive WKY or Sprague-Dawley rats, potentially confounding the observed results on BP. Third, even with the caveats just mentioned, the paper by Li et al. (2013) did not indicate whether Almorexant modified the difference in BP between wakefulness and NREMS in SHR and WKY rats. Sleep-related differences in BP are preserved in rats with a partial postnatal loss of orexin neurons (Schwimmer et al. 2010). Conversely, the BP reduction on passing from wakefulness to NREMS is significantly attenuated in either knock-out mice with congenital orexin deficiency (Bastianini et al. 2011) or transgenic mice with complete postnatal loss of orexin neurons (Bastianini et al. 2011; Lo Martire et al. 2012; Silvani et al. 2013) compared with wild-type controls. The changes in BP on falling asleep and those on awakening from sleep are also attenuated in patients with narcolepsy with cataplexy (NC), which is a debilitating sleep disorder associated with virtually complete loss of orexin neurons (Grimaldi et al. 2012). Moreover, patients with NC are at high risk of having a non-dipper BP profile (Dauvilliers et al. 2012; Grimaldi et al. 2012). Fourth, Li et al. (2013) did not report the effects of Almorexant on BP during rapid-eye-movement sleep (REMS) in SHR and WKY rats. REMS represents a minor fraction of total sleep time but in different species including humans, it entails an increase in BP towards waking levels compared with NREMS (Silvani, 2008). The increase in BP during REMS compared with NREMS may be detected also in SHR and WKY rats (Berteotti et al. 2007). Importantly, such an increase in BP during REMS was found to be significantly enhanced in knock-out and transgenic mice with congenital orexin deficiency (Bastianini et al. 2011; Lo Martire et al. 2012; Silvani et al. 2013) and in human NC patients (Grimaldi et al. 2012) compared with the respective controls. In conclusion, we have raised a few issues concerning the interpretation of data recently reported by Li et al. (2013) in this journal. In general, these issues are expansions of points recognized by the authors, but with a focus on the role of orexins in sleep-related cardiovascular control. This focus may be of clinical as well as physiological relevance, because growing evidence links cardiovascular risk to high BP values during sleep and to a reduced difference in BP between night-time sleep and daytime wakefulness (i.e. a non-dipping BP profile) (Ben-Dov et al. 2007; Hermida et al. 2011, 2013). However, orexin neurons fire almost solely during wakefulness (Mileykovskiy et al. 2005), yet extracellular orexin concentration remains substantial during sleep (Kiyashchenko et al. 2002), thus potentially continuing to exert physiological effects. The very finding by Li et al. (2013) that Almorexant is effective in SHR also during sleep is at least consistent with this view.

Hypertension is accompanied by circulatory changes in the brain and in other vascular districts; at disease onset, these changes may be largely functional and dependent on metabolic and vegetative drives. The wake-sleep cycle is a major physiological source of ultradian variability in autonomic function and in cerebral blood flow and metabolism. Aim of the study was to investigate whether sleep induces functional changes in the brain microcirculation in the developing hypertensive state. The fraction of brain capillaries perfused by plasma (perfused/ anatomical capillaries) was assessed in young (8-10 weeks) spontaneously hypertensive rats (SHR) during quiet wakefulness, quiet sleep and active sleep. The density of anatomical capillaries was assessed in two groups of animals using both a histochemical method (alkaline phosphatase, AP, for morphometric measurements) and an immunofluorescence method (anti-fibronectin antibodies, FN, to detect all existing capillaries). The density of perfused capillaries was determined by intravascular injection of a fluorescent marker. The fraction of anatomical capillaries perfused by plasma was always close to maximal (0.96-0.97), without significant variations among the states of the wake-sleep cycle, and was the same for AP-stained and FN-stained sections. Data thus indicate that in this model of essential hypertension no functional changes in plasma perfusion of cerebral capillaries occur in the early stages of the disease.

Objectives. Blood pressure (BP) physiologically has higher and lower values during the active and rest period, respectively. Subjects failing to show the appropriate BP decrease (10-20%) on passing form diurnal activity to nocturnal rest and sleep have increased risk of target organ damage at the cardiac, vascular and cerebrovascular levels. Hypocretin (HCRT) releasing neurons, mainly located in the lateral hypothalamus, project widely to the central nervous system. Thus HCRT neurons are involved in several autonomic functions, including BP regulation. HCRT neurons also play a key role in wake-sleep cycle regulation, the lack of which becomes evident in HCRT-deficient narcoleptic patients. I investigated whether chronic lack of HCRT signaling alters BP during sleep in mouse models of narcolepsy. Methods. The main study was performed on HCRT-ataxin3 transgenic mice (TG) with selective post-natal ablation of HCRT neurons, HCRT gene knockout mice (KO) with preserved HCRT neurons, and Wild-Type control mice (WT) with identical genetic background. Experiments where replicated on TG and WT mice with hybrid genetic background (hTG and hWT, respectively). Mice were implanted with a telemetric pressure transducer (TA11PA-C10, DSI) and electrodes for discriminating wakefulness (W), rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Signals were recorded for 3 days. Mean BP values were computed in each wake-sleep state and analyzed by ANOVA and t-test with significance at p