Knockdown of orexin type 2 receptor in the lateral pontomesencephalic tegmentum of rats increases REM sleep

Dysfunction of the orexin/hypocretin neurotransmitter system causes the sleep disorder narcolepsy, characterized by intrusion of rapid eye movement (REM) sleep‐like events into normal wakefulness. The sites where orexins act to suppress REM sleep are incompletely understood. Previous studies suggested that the lateral pontomesencephalic tegmentum (lPMT) contains an important REM sleep inhibitory area, and proposed that orexins inhibit REM sleep via orexin type 2 receptors (OxR2) in this region. However, this hypothesis has heretofore not been tested. We thus performed bilateral injection of small interfering RNAs (siRNAs) targeting Ox2R into the lPMT on two consecutive days. This led to a approximately 30% increase of time spent in REM sleep in both the dark and light periods for the first 2 days after injection, with a return to baseline over the next two post‐injection days. This increase was mainly due to longer (> 120 s) REM episodes. Cataplexy‐like episodes were not observed. The percentage of time spent in wakefulness and non‐(N)REM sleep, as well as the power spectral profile of NREM and REM sleep, were unaffected. Control animals injected with scrambled siRNA had no sleep changes post‐injection. Quantification of the knockdown revealed that unilateral microinjection of siRNAs targeting OxR2 into the lPMT induced a approximately 40% reduction of OxR2 mRNA 2 days following the injections when compared with the contralateral side receiving control (scrambled) siRNA. Orexin type 1 receptor mRNA level was unaffected. Our results indicate that removal of OxR2 neurotransmission in the lPMT enhances REM sleep by increasing the duration of REM episodes.     

REM sleep changes in rats induced by siRNA-mediated orexin knockdown

Short interfering RNAs (siRNA) targeting prepro-orexin mRNA were microinjected into the rat perifornical hypothalamus. Prepro-orexin siRNA-treated rats had a significant (59%) reduction in prepro-orexin mRNA compared to scrambled siRNA-treated rats 2 days postinjection, whereas prodynorphin mRNA was unaffected. The number of orexin-A-positive neurons on the siRNA-treated side decreased significantly (23%) as compared to the contralateral control (scrambled siRNA-treated) side. Neither the colocalized dynorphin nor the neighbouring melanin-concentrating hormone neurons were affected. The number of orexin-A-positive neurons on the siRNA-treated side did not differ from the number on the control side 4 or 6 days postinjection. Behaviourally, there was a persistent (approximately 60%) increase in the amount of time spent in rapid eye movement (REM) sleep during the dark (active) period for 4 nights postinjection, in rats treated with prepro-orexin siRNA bilaterally. This increase occurred mainly because of an increased number of REM episodes and decrease in REM-to-REM interval. Cataplexy-like episodes were also observed in some of these animals. Wakefulness and NREM sleep were unaffected. The siRNA-induced increase in REM sleep during the dark cycle reverted to control values on the 5th day postinjection. In contrast, the scrambled siRNA-treated animals only had a transient increase in REM sleep for the first postinjection night. Our results indicate that siRNA can be usefully employed in behavioural studies to complement other loss-of-function approaches. Moreover, these data suggest that the orexin system plays a role in the diurnal gating of REM sleep.     

Participation of histaminergic H1 and noradrenergic alpha 1 receptors in orexin A-induced wakefulness in rats

The participation of histaminergic H(1) and noradrenergic alpha(1) receptors in orexin A-induced wakefulness was studied by examining the sleep-wakefulness cycle in rats. Intracerebroventricular infusion of orexin A (1 nmol) caused an increase in the wakefulness state, while non-rapid eye movement sleep (NREM sleep) and rapid eye movement sleep (REM sleep) states were decreased. Prazosin (150 nmol) showed no significant antagonistic effect on the orexin A-induced increase in the wakefulness state and decrease in NREM and REM sleep. On the contrary, pyrilamine (150 nmol) was effective in antagonizing orexin A-induced increase in wakefulness and decrease in NREM sleep. When prazosin (150 nmol) and pyrilamine (150 nmol) were simultaneously perfused into the lateral ventricle, an almost complete antagonistic effect was observed with the increase in the wakefulness state and decrease in NREM sleep. Orexin A (1 nmol) caused a significant decrease in the histamine contents of the cortex, hippocampus and hypothalamus, whereas noradrenaline contents were decreased only in the hypothalamus. From these results, we concluded that the arousal effect induced by orexin A occurs through histaminergic H(1) and noradrenergic alpha(1) receptors, although participation of the H(1) receptor was more important than the alpha(1) receptor.     

Locus Coeruleus Acid-Sensing Ion Channels Modulate Sleep–Wakefulness and State Transition from NREM to REM Sleep in the Rat

The locus coeruleus (LC) is one of the essential chemoregulatory and sleep–wake (S–W) modulating centers in the brain. LC neurons remain highly active during wakefulness, and some implicitly become silent during rapid eye movement (REM) sleep. LC neurons are also involved in CO₂-dependent modulation of the respiratory drive. Acid-sensing ion channels (ASICs) are highly expressed in some brainstem chemosensory breathing regulatory areas, but their localization and functions in the LC remain unknown. Mild hypercapnia increases the amount of non-REM (NREM) sleep and the number of REM sleep episodes, but whether ASICs in the LC modulate S–W is unclear. Here, we investigated the presence of ASICs in the LC and their role in S–W modulation and the state transition from NREM to REM sleep. Male Wistar rats were surgically prepared for chronic polysomnographic recordings and drug microinjections into the LC. The presence of ASIC-2 and ASIC-3 in the LC was immunohistochemically characterized. Microinjections of amiloride (an ASIC blocker) and APETx2 (a blocker of ASIC-2 and -3) into the LC significantly decreased wakefulness and REM sleep, but significantly increased NREM sleep. Mild hypercapnia increased the amount of NREM and the number of REM episodes. However, APETx2 microinjection inhibited this increase in REM frequency. These results suggest that the ASICs of LC neurons modulate S–W, indicating that ASICs could play an important role in vigilance-state transition. A mild increase in CO₂ level during NREM sleep sensed by ASICs could be one of the determinants of state transition from NREM to REM sleep.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12264-020-00625-0.     

Evidence for excessive sleepiness in canine narcoleptics

Six genetically narcoleptic dogs, as well as 6 age- and breed-matched control dogs, were recorded continuously for 24 h to compare sleep/wake patterns and to determine whether narcoleptic dogs exhibit evidence of excessive sleepiness. Compared with controls, the affected animals showed a substantial reduction in wakefulness and a significant increase in time spent in the drowsy state. Total NREM sleep and total sleep time (NREM and REM sleep) in the 2 groups, however, were very similar. Results suggest that, whereas genetically narcoleptic dogs show less wakefulness than controls, they are not hypersomnolent. However, the greater amounts of drowsiness in the affected animals suggest that they are 'sleepier' than controls. These results are highly consonant with those found in human narcolepsy.     

Effects on sleep and wakefulness of the injection of hypocretin-1 (orexin-A) into the laterodorsal tegmental nucleus of the cat

Anatomical data demonstrate a dense projection, in the cat, from hypocretin (orexin) neurons in the hypothalamus to the laterodorsal tegmental nucleus (LDT), which is a critical pontine site that is involved in the regulation of the behavioral states of sleep and wakefulness. The present study was therefore undertaken to explore the hypocretinergic control of neurons in the LDT vis-à-vis these behavioral states. Accordingly, hypocretin-1 was microinjected into the LDT of chronic, unanesthetized cats and its effects on the percentage, latency, frequency and duration of wakefulness, quiet (non-REM) sleep and active (REM) sleep were determined. There was a significant increase in the time spent in wakefulness following the microinjection of hypocretin-1 into the LDT and a significant decrease in the time spent in active sleep. The increase in the percentage of wakefulness was due to an increase in the duration of episodes of wakefulness; the reduction in active sleep was due to a decrease in the frequency of active sleep episodes, but not in their duration. These data indicate that hypocretinergic processes in the LDT play an important role in both of the promotion of wakefulness and the suppression of active sleep.     

Sleep during neuromuscular blockade in cats

The purpose of the experiment was to determine whether normal sleep patterns can occur during neuromuscular blockade. Electrographic variables for determining the states of sleep and wakefulness, the electrocorticogram, lateral geniculate nucleus potentials, and dorsal hippocampal potentials, were recorded before, during and after the administration of gallamine triethiodide to cats with chronically implanted electrodes. When respiratory muscles became paralyzed, artificial ventilation commenced through a chronic tracheal fistula. The electrographic wave forms of the states (wakefulness, NREM sleep and REM sleep) in paralyzed cats were indistinquishable by visual observation from those of freely moving animals. As compared to freely moving cats, paralyzed cats had more wakefulness at the expense of both states of sleep (about 33% NREM and 3% REM compared to 45% NREM and 15% REM respectively). REM sleep wasdemonstrated to occur, albeit increase across repeated session in the same cats nor was the distribution uneven within the average session. Large percentages of REM sleep with respect to total recording time were associated with large percentages of NREM sleep (correlation coefficient = 0.58). The sequence of sleep states was like that of freely behaving animals. The main conclusion is that this preparation, depsite low amounts of REM sleep, is useful in neural studies of sleep and wakefulness.     

Influence of sleep stage and wakefulness on spectral EEG activity and heart rate variations around periodic leg movements.

OBJECTIVE: Typical changes in spectral electroencephalographic (EEG) activity and heart rate (HR) have been described in periodic leg movements (PLM) associated with or without microarousals (MA). We aimed to determine the effects of sleep stage and wakefulness on these responses to ascertain whether a common pattern of EEG and HR activation takes place. METHODS: The time course of EEG spectral activity and HR variability associated with PLM was analysed in 13 patients during light NREM sleep, rapid-eye-movement (REM) sleep and wakefulness. The same analysis was also conducted for PLM without MA occurring in stage 2. RESULTS: A significant EEG and electrocardiogram (ECG) activation was found associated with PLM during sleep, but not during wakefulness. While in light NREM sleep, an increase in delta and theta bands was detected before the PLM onset, in REM sleep the EEG activation occurred simultaneously with the PLM onset. Moreover, during stage 1 and REM sleep, alpha and fast frequencies tended to remain sustained after the PLM onset. In contrast, during wakefulness, a small and not significant increase in cerebral activity was present, starting at the PLM onset and persisting in the post-movement period. A typical pattern of cardiac response was present during NREM and REM sleep, the autonomic activation being lesser and prolonged during wakefulness. CONCLUSIONS: We conclude that the EEG and HR responses to PLM differ between sleep stages and wakefulness with lesser changes found during wakefulness. SIGNIFICANCE: These findings suggest that specific sleep state-dependent mechanisms may underlie the occurrence of PLM.     

Reappraisal of Interictal Electroencephalograms in Infantile Spasms

Summary: To delineate interictal electroencephalographic (EEC) features before treatment of patients with clinically defined infantile spasms, EEGs of 82 infants having tonic spasms in clusters were analyzed by type of paroxysmal abnormalities, continuity, interhemispheric synchrony, topography, and wave component of hypsarrhythmia during wakefulness and sleep. Hypsarrhythmia occurred less frequently in wakefulness than in non-rapid eye movement (NREM) sleep at any age, least frequently in wakefulness after 1 year of age, and disappeared in rapid eye movement (REM) sleep at any age. The continuity of hypsarrhythmia changed with states, but did not change with age, and was greatest in wakefulness and stage 1 and decreased in stage 2–3. Interhemispheric synchrony increased with increasing age but decreased with advancing sleep stage. The term modified hypsarrhythmia should be discarded, and unusual features, if present, should be specified.     

Changes in the thermal characteristics of hypothalamic neurons during sleep and wakefulness

The characteristics of the mammalian thermoregulatory system are dependent upon arousal state. During NREM sleep thermoregulatory mechanisms are intact but body temperature is regulated at a lower level than during wakefulness. In REM sleep thermoregulatory effector mechanisms are inhibited and thermal homeostasis is severely disrupted. Thermosensitivity of neurons in the preoptic/anterior hypothalamus (POAH) was determined for behaving kangaroo rats (Dipodomys deserti) during electrophysiologically defined wakefulness, NREM sleep and REM sleep to elucidate possible neural mechanisms for previous findings of state-dependent changes in thermoregulation. Thirty cells were tested during at least two arousal states. During wakefulness, 70% of the recorded cells were sensitive to changes in local temperature, with the number of warm-sensitive (W) cells outnumbering cold-sensitive (C) cells by 1.6:1. In NREM sleep, 43% of the cells were thermally sensitive, with the ratio of W:C remaining the same as in wakefulness. In REM sleep only two cells were thermosensitive (both W). The decrease in neuronal thermosensitivity of POAH cells during REM sleep parallels findings of inhibition of thermoregulatory effector responses during REM, although further work is necessary to determine the source and nature of the inhibition.     

Eye Closure Sensitivity Without Photosensitivity in Juvenile Myoclonic Epilepsy: Polysomnographic Study of Electroencephalographic Epileptiform Discharge Rates

Two cases of juvenile myoclonic epilepsy (JME) presented with myoclonic jerks and EEG activation after eye closure, without sensitivity to intermittent photic stimulation. The effect of eye closure was computed by comparing discharge rates of polyspike-and-wave (PSW) complexes after eye closure and after eye opening. For one patient, never treated pharmacologically, a nocturnal polysomnograph was performed to study the variation of discharge rates of PSW complexes during wakefulness and sleep. The rate of PSW complexes was high during wakefulness before sleep onset, increased during spontaneous nocturnal awakenings, and became maximal during final morning awakening. Among nonrapid eye movement (NREM) sleep stages, EEG epileptiform activity was maximal during stages III and IV. Discharges were completely suppressed by rapid eye movement (REM) sleep. Awakenings following deep NREM sleep were very activating if no REM sleep was interposed. Awakenings from light NREM sleep were much less activating. There were no EEG abnormalities in awakenings immediately following REM sleep. Results suggest that REM sleep, similarly to eye opening, plays a role in inhibiting EEG manifestations of JME with eye closure sensitivity.     

Sleep and activity rhythms in mice: a description of circadian patterns and unexpected disruptions in sleep.

Studies on daily and circadian rhythms in wheel running and electrographically defined wakefulness, NREM sleep, and REM sleep in M. musculus were done to gather data on the temporal distribution of activity and sleep. Generally, peaks in NREM and sleep tended to coincide and to alternate with the coincident peaks of wakefulness and wheel running. However, during the active phase of the circadian wheel running cycle some NREM and REM sleep did occur; conversely, during its rest phase, wakefulness was often present. The most striking finding was that in mice with clearly entrained or free-running activity onsets, the circadian peak-through patterns in wakefulness, NREM, and REM sleep were not always distinct--they could be damped and/or polyphasic. Several explanations of these phenomena are considered.     

Ischemic stroke selectively inhibits REM sleep of rats

Sleep disorders are important risk factors for stroke; conversely, stroke patients suffer from sleep disturbances including disruptions of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep and a decrease in total sleep. This study was performed to characterize the effect of stroke on sleep architecture of rats using continuous electroencephalography (EEG) and activity monitoring. Rats were implanted with transmitters which enabled continuous real time recording of EEG, electromyography (EMG), and locomotor activity. Baseline recordings were performed prior to induction of either transient middle cerebral artery (MCA) occlusion or sham surgery. Sleep recordings were obtained for 60 h after surgery to identify periods of wakefulness, NREM, and REM sleep before and after stroke. Spectral analysis was performed to assess the effects of stroke on state-dependent EEG. Finally, we quantified the time in wake, NREM, and REM sleep before and after stroke. Delta power, a measure of NREM sleep depth, was increased the day following stroke. At the same time, there was a significant shift in theta rhythms to a lower frequency during REM and wake periods. The awake EEG slowed after stroke over both hemispheres. The EEG of the ischemic hemisphere demonstrated diminished theta power specific to REM in excess of the slowing seen over the contralateral hemisphere. In contrast to rats exposed to sham surgery which had slightly increased total sleep, rats undergoing stroke experienced decreased total sleep. The decrease in total sleep after stroke was the result of dramatic reduction in the amount of REM sleep after ischemia. The suppression of REM after stroke was due to a decrease in the number of REM bouts; the length of the average REM bout did not change. We conclude that after stroke in this experimental model, REM sleep of rats is specifically and profoundly suppressed. Further experiments using this experimental model should be performed to investigate the mechanisms and consequences of REM suppression after stroke.     

Effects of social stimuli on sleep in mice: non-rapid-eye-movement (NREM) sleep is promoted by aggressive interaction but not by sexual interaction

Sleep is generally considered to be a process of recovery from prior wakefulness. In addition to being affected by the duration of the waking period, sleep architecture and sleep EEG also depend on the quality of wakefulness. In the present experiment, we examined how sleep is affected by different social stimuli (social conflict and sexual interaction). Male C57BL/6J mice were placed in the cage of an aggressive dominant male or an estrous female for 1 h in the middle of the light phase. The conflict with an aggressive male had a pronounced NREM sleep-promoting effect. EEG slow wave activity, a measure of NREM sleep intensity, was increased for about 6 h and NREM sleep time was significantly increased for 12 h. REM sleep was strongly suppressed during the remainder of the light phase after the conflict, followed by a rebound later in the recovery phase. The sexual interaction, in contrast, had only mild effects. Both NREM sleep and REM sleep were somewhat suppressed shortly after the interaction. In a separate group of mice, blood samples were taken to measure prolactin and corticosterone. The results suggest that the temporary suppression of REM sleep following the social stimuli may be partly due to elevated corticosterone. The different effects of the social stimuli on NREM sleep are not easily explained by differences in the hormone responses. In conclusion, although both social conflict and sexual interaction induce a strong physiological activation, only social conflict has a strong stimulatory effect on NREM sleep mechanisms.     

Complexity measures of the central respiratory networks during wakefulness and sleep

Since sleep is known to influence respiratory activity we studied whether the sleep state would affect the complexity value of the respiratory network output. Specifically, we tested the hypothesis that the complexity values of the diaphragm EMG (EMGdia) activity would be lower during REM compared to NREM. Furthermore, since REM is primarily generated by a homogeneous population of neurons in the medulla, the possibility that REM-related respiratory output would be less complex than that of the awake state was also considered. Additionally, in order to examine the influence of neuron vulnerabilities within the rostral ventral medulla (RVM) on the complexity of the respiratory network output, we inhibited respiratory neurons in the RVM by microdialysis of GABA(A) receptor agonist muscimol. Diaphragm EMG, nuchal EMG, EEG, EOG as well as other physiological signals (tracheal pressure, blood pressure and respiratory volume) were recorded from five unanesthetized chronically instrumented intact piglets (3-10 days old). Complexity of the diaphragm EMG (EMGdia) signal during wakefulness, NREM and REM was evaluated using the approximate entropy method (ApEn). ApEn values of the EMGdia during NREM and REM sleep were found significantly (p < 0.05 and p < 0.001, respectively) lower than those of awake EMGdia after muscimol inhibition. In the absence of muscimol, only the differences between REM and wakefulness ApEn values were found to be significantly different.     

Automated determination of wakefulness and sleep in rats based on non-invasively acquired measures of movement and respiratory activity

The current standard for monitoring sleep in rats requires labor intensive surgical procedures and the implantation of chronic electrodes which have the potential to impact behavior and sleep. With the goal of developing a non-invasive method to determine sleep and wakefulness, we constructed a non-contact monitoring system to measure movement and respiratory activity using signals acquired with pulse Doppler radar and from digitized video analysis. A set of 23 frequency and time-domain features were derived from these signals and were calculated in 10 s epochs. Based on these features, a classification method for automated scoring of wakefulness, non-rapid eye movement sleep (NREM) and REM in rats was developed using a support vector machine (SVM). We then assessed the utility of the automated scoring system in discriminating wakefulness and sleep by comparing the results to standard scoring of wakefulness and sleep based on concurrently recorded EEG and EMG. Agreement between SVM automated scoring based on selected features and visual scores based on EEG and EMG were approximately 91% for wakefulness, 84% for NREM and 70% for REM. The results indicate that automated scoring based on non-invasively acquired movement and respiratory activity will be useful for studies requiring discrimination of wakefulness and sleep. However, additional information or signals will be needed to improve discrimination of NREM and REM episodes within sleep.     

Differential effects of persistent nociceptive stimulation on sleep stages

The purpose of this work was to investigate the sequence of modifications of sleep and pain parameters in a condition of persistent nociceptive stimulation. In freely moving cats carrying implanted electrodes, continuous polygraphic and behavioral recordings were collected 24 h a day for several consecutive days before and after treatment. Injection of formalin (2 ml, 37%) elicited continuous wakefulness (1-6 h) associated with behavioral manifestations of pain. This insomnia was followed by the delayed appearance of LS (light, slow wave sleep) DS (deep slow wave sleep) and REM (rapid eye movement sleep). On days 1 and 2 after injection, pain manifestations displayed a gradual decrease, while total sleep time (LS + DS + REM) slowly returned to normal levels. On day 1, the amount of LS was not modified, but DS and REM were greatly decreased. For 12 h after the first REM episode, REM was decreased while DS was already at the basal levels. Formalin elicited a long-lasting increase in EMG activity of the tibialis anterior muscle which was suppressed during REM and returned to higher levels afterwards. Prolonged wakefulness and delay in sleep stage appearance were also recorded when a 24-h sleep deprivation preceded formalin injection. In this condition, LS, DS and REM amount were at basal levels from their first reappearance, and a rebound in total sleep time and DS occurred on day 2 after the injection. After injection of smaller doses of formalin (0.5 ml, 8%), the amount of LS, DS and REM was at control levels since day 1. The results suggest that (1) the amount of sleep depends on sleep debt and on the level of pain intensity and (2) sleep stages are differentially sensitive to persistent pain.     

Sleep phenotypes of intellectual disability: a polysomnographic evaluation in subjects with Down syndrome and Fragile-X syndrome.

OBJECTIVE: To analyze sleep architecture and NREM sleep alterations by means of the Cyclic Alternating Pattern (CAP) in children with Down syndrome (DS) and Fragile-X syndrome (fraX), the two most common causes of inherited mental retardation, in order to find out eventual alterations of their sleep microstructure related to their mental retardation phenotypes. METHODS: Fourteen patients affected by fraX (mean age 13.1 years) and 9 affected by Down syndrome (mean age 13.8 years) and 26 age-matched normal controls were included. All subjects underwent overnight polysomnography in the sleep laboratory, after one adaptation night and their sleep architecture and CAP were visually scored. RESULTS: FraX subjects showed a reduced time in bed compared to DS subjects, whereas DS subjects showed a lower sleep efficiency, a higher percentage of wakefulness after sleep onset, and a reduced percentage of stage 2 NREM compared to the other groups. Furthermore, DS and fraX subjects, compared to normal controls, showed a higher percentage of stage 1 NREM and a lower percentage of REM sleep. FraX subjects showed the most disrupted sleep microstructure with low total CAP rate and CAP rate in S2 NREM. Both patient groups showed a lower percentage of A1 and higher percentage of A2 and A3 compared to normal controls. CONCLUSIONS: The analysis of CAP might be able to disclose new important findings in the sleep architecture of children with mental retardation and might characterize sleep microstructural patterns of the different phenotypes of intellectual disability. SIGNIFICANCE: The NREM sleep microstructure alterations found in our subjects, associated with the reduction in REM sleep percentage, seem to be distinctive features of intellectual disability.     

Gabapentin-induced modulation of interictal epileptiform activity related to different vigilance levels.

OBJECTIVES: Gabapentin (GBP) is a novel antiepileptic drug (AED), currently used as add-on therapy in patients with partial seizures. Similar to other AEDs, little is known about its effects on nocturnal sleep, despite the strict relationship between sleep and epileptic discharges. The aim of our study was to evaluate the effects of chronic therapy with GBP on both nocturnal sleep and on interictal epileptiform abnormalities (IEA) in relation to the different sleep stages. METHODS: Eighteen patients affected by partial seizures resistant to common AEDs were submitted to nocturnal polygraphic recordings under baseline conditions and after 4 months of add-on GBP treatment. RESULTS: We observed a significant increase in unilateral/focal IEA during light NREM sleep and a significant reduction in bilateral/diffuse IEA during wakefulness after sleep onset (WASO) with respect to the baseline condition. A significant increase in REM sleep and slow wave sleep (SWS) associated with a reduction in the number of awakenings and Stage 1 was also observed after GBP chronic therapy. CONCLUSIONS: GBP therapy improves the sleep pattern of epileptic patients and it seems to modulate the expression of IEA with different effects in relation to the various vigilance levels.