Cholinergic neurons of the basal forebrain mediate biochemical and electrophysiological mechanisms underlying sleep homeostasis

The tight coordination of biochemical and electrophysiological mechanisms underlies the homeostatic sleep pressure (HSP) produced by sleep deprivation (SD). We have reported that during SD the levels of inducible nitric oxide synthase (iNOS), extracellular nitric oxide (NO), adenosine [AD]_ex, lactate [Lac]_ex and pyruvate [Pyr]_ex increase in the basal forebrain (BF). However, it is not clear whether all of them contribute to HSP leading to increased electroencephalogram (EEG) delta activity during non‐rapid eye movement (NREM) recovery sleep (RS) following SD. Previously, we showed that NREM delta increase evident during RS depends on the presence of BF cholinergic (ChBF) neurons. Here, we investigated the role of ChBF cells in coordination of biochemical and EEG changes seen during SD and RS in the rat. Increases in low‐theta power (5–7 Hz), but not high‐theta (7–9 Hz), during SD correlated with the increase in NREM delta power during RS, and with the changes in nitrate/nitrite [NO_x]_ex and [AD]_ex. Lesions of ChBF cells using IgG 192‐saporin prevented increases in [NO_x]_ex, [AD]_ex and low‐theta activity, during SD, but did not prevent increases in [Lac]_ex and [Pyr]_ex. Infusion of NO donor DETA NONOate into the saporin‐treated BF failed to increase NREM RS and delta power, suggesting ChBF cells are important for mediating NO homeostatic effects. Finally, SD‐induced iNOS was mostly expressed in ChBF cells, and the intensity of iNOS induction correlated with the increase in low‐theta activity. Together, our data indicate ChBF cells are important in regulating the biochemical and EEG mechanisms that contribute to HSP.     

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.     

Sleep and sleep regulation in the ferret (Mustela putorius furo)

We investigated sleep-wake (S-W) architecture and sleep regulation in the ferret: a phylogenetically primitive mammal increasingly used in neurobiological studies. Twenty-four hour S-W baseline data were collected in eight adult ferrets. Seven ferrets were then sleep deprived for 6h at the beginning of the light period. Like other placental mammals, ferrets exhibited the main vigilance states of wakefulness, rapid-eye-movement (REM) sleep and non-REM (NREM) sleep. Interestingly, the amount of REM sleep in the ferret was considerably higher (24.01+/-1.61% of total recording time) than typically reported in placental mammals. Ferret sleep was homeostatically regulated as sleep deprivation produced a significant increase in NREM EEG delta power during the recovery period. Therefore, ferret sleep in most respects is comparable to sleep in other placental mammals. However, the large amount of REM sleep in this phylogenetically more ancient species suggests that REM sleep may have been present in greater amounts in early stages of mammalian evolution.     

Single cell activity patterns of pedunculopontine tegmentum neurons across the sleep-wake cycle in the freely moving rats

Microinjections of the excitatory amino acid, L-glutamate into the cholinergic cell compartment of the pedunculopontine tegmentum (PPT) of the rat induces both wakefulness and/or rapid eye movement (REM) sleep depending on the glutamate dosage. However, no studies have systematically recorded the electrical activity of these cells in the freely moving rat across the sleep-wake cycle. In this study, we have recorded the spontaneous activity patterns of single PPT cells (n = 70) in the freely moving rat across the sleep-wake cycle. PPT neurons were classified into three groups based on patterns in their spontaneous activity. The first group of cells (12.86%) was more active during REM sleep than they were during wakefulness or slow-wave sleep (SWS). The second group of cells (60.0%) was more active during REM and wakefulness than during SWS. The firing rate of the third group of cells (27.14%) did not change as a function of behavioral state. This study also demonstrated that the level of activity within the cholinergic cell compartment of the PPT during SWS drops to 7.4% of that observed during wakefulness and that during REM sleep it changes to 65.5% of wakefulness levels. These findings indicate that in the freely moving rat, the discharging of PPT neurons correlates with wakefulness and REM sleep. Additionally, these neurons may be an integral part of the brainstem wakefulness and REM sleep-generating mechanisms in the rat.     

Varying expressions of alerting mechanisms in wakefulness and across sleep states.

Alerting stimuli, such as intense tones, presented to cats in wakefulness (W) elicit the orienting response (OR) and/or the acoustic startle reflex (ASR) in conjunction with elicited ponto-geniculo-occipital waves (PGOE) from the lateral geniculate body (LGB) and elicited waves from the thalamic central lateral nucleus (CLE). Alerting stimuli presented during rapid eye movement sleep (REM) and non-rapid eye movement sleep (NREM) also elicit PGOE. We presented tones in W, REM and NREM to determine whether CLE could be obtained in sleep and to examine the patterns of responsiveness of PGOE and CLE across behavioral states. Also, we recorded ASR and OR and compared the response patterns of behavioral and central correlates of alerting. The subjects were 7 cats; all exhibited spontaneously occurring waves in LGB and CL. All cats exhibited PGOE and 5 cats exhibited CLE in W, REM and NREM. PGOE and CLE showed less evidence of habituation than did ASR and OR. The pattern of responsiveness of CLE across behavioral states was different from that found for PGOE, and spontaneous CL waves were much rarer than the LGB waves. ASR was elicited in 5 cats during W trials, and in 3 cats during REM trials. OR habituated rapidly in W and did not occur in REM and NREM. The data indicate that central mechanisms of alerting function in sleep states as well as in W and suggest that CLE and PGOE reflect activity in mechanisms underlying cortical desynchronization and visual processes which may act in concert during alerting.     

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.     

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.     

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.     

Extended sleep in humans in 14 hour nights (LD 10:14): relationship between REM density and spontaneous awakening

The sleep patterns of 8 normal subjects living in a winter-type photoperiod (10 h light and 14 h darkness; LD 10:14)_for 4 weeks were characterized by the presence of periods of spontaneous wakefulness alternating with periods of spontaneous sleep. Transitions from sleep to wakefulness occurred much more frequently out of REM sleep than out of NREM sleep (P<0.002). REM periods that terminated in wakefulness showed shorter REM durations (P<0.0005) and higher REM densities (P<0.0005) than REM periods that did not terminate in wakefulness. The authors discuss these results in terms of possible relationship between REM density and arousal level. The higher REM density preceding wakefulness and the increased number of REM periods terminating in spontaneous awakenings could reflect an enhanced level of a brain arousing process, resulting from reduced sleep pressure in the extended nights.     

Sleep stage-related changes in sympathetic sudomotor and vasomotor skin responses in man.

OBJECTIVES: The aim of the study was to evaluate the characteristics of the spontaneous and evoked sympathetic skin responses (SSR) during sleep and wakefulness in comparison with the skin vasomotor responses (SVR). METHODS: Five healthy subjects underwent a night of videopolysomnographic recording. Spontaneous SSR were recorded via surface electrodes placed on the dorsal and ventral aspect of the hand while SVR were evaluated by means of an infrared photoelectric transducer placed on the index finger. SSR and SVR were evoked via electrical stimuli applied to the left supraorbital nerve. RESULTS: Spontaneous SSR frequency was highest during stage 4 of NREM sleep and lowest during REM phases. On the contrary, spontaneous SVR frequency reached its lowest value during stage 4 and its highest value during stage 2 of NREM sleep, remaining at levels above waking values during REM. SSR could be elicited by stimuli inducing arousal during light sleep but it was absent during deep NREM and REM sleep. SVR could be evoked throughout NREM and REM sleep. CONCLUSIONS: Spontaneous SSR and SVR act differently during physiological modifications of vigilance. Evoked SSR is strictly dependent upon the state of vigilance, whereas evoked SVR shows no modifications during the different stages of the wake-sleep cycle.     

The effects of changing state on elicited ponto-geniculo-occipital (PGO) waves.

Waves similar to ponto-geniculo-occipital (PGO) waves occurring spontaneously in the lateral geniculate body (LGB), pons, and occipital cortex during rapid eye movement (REM) sleep can be elicited in the LGB and the cortex by tones in waking (W), non-rapid eye movement sleep (NREM), and REM. In W, the elicited waves (PGOE) sometimes accompany orienting responses (OR). We have hypothesized that REM is a state resembling exaggerated "orienting" in part because spontaneous PGO waves similar to PGOE accompanying OR are constantly observed in REM. The present experiment tested whether: (1) PGOE and OR were strongly correlated in W across a large number of tone presentations as might be predicted if PGOE were central wave form markers for a state of orienting; and (2) recovery of responsiveness of PGOE to tones would then be greater in REM than NREM, as might be expected if REM but not NREM were a state in which central mechanisms of orienting were highly active. Tones were presented in W and then in REM and NREM to six cats in order to measure the degree of habituation of OR and PGOE simultaneously. PGOE and OR exhibited a degree of independence: the former were readily produced in W despite the rapid decline in OR across trials. Recovery in the amplitude of PGOE occurred in both NREM and REM. The recovery tended to be greater in REM than NREM, although this was not statistically significant. Refinements of the theory that REM represents a state of exaggerated internal orienting are discussed.     

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.     

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.     

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.     

Remote long-term registrations of sleep-wake rhythms, core body temperature and activity in marmoset monkeys

Initial studies in the day active marmoset monkey (Callithrix jacchus) indicate that the sleep-wake cycle of these non-human primates resembles that of humans and therefore conceivably represent an appropriate model for human sleep. The methods currently employed for sleep studies in marmosets are limited. The objective of this study was to employ and validate the use of specific remote monitoring system technologies that enable accurate long-term recordings of sleep-wake rhythms and the closely related rhythms of core body temperature (CBT) and locomotor activity in unrestrained group-housed marmosets. Additionally, a pilot sleep deprivation (SD) study was performed to test the recording systems in an applied experimental setup. Our results show that marmosets typically exhibit a monophasic sleep pattern with cyclical alternations between NREM and REM sleep. CBT displays a pronounced daily rhythm and locomotor activity is primarily restricted to the light phase. SD caused an immediate increase in NREM sleep time and EEG slow-wave activity as well as a delayed REM sleep rebound that did not fully compensate for REM sleep that had been lost during SD. In conclusion, the combination of two innovative technical approaches allows for simultaneous measurements of CBT, sleep cycles and activity in multiple subjects. The employment of these systems represents a significant refinement in terms of animal welfare and will enable many future applications and longitudinal studies of circadian rhythms in marmosets.     

Electrical activity of the human amygdala during all-night sleep and wakefulness

Objective

The aim of the present work was to characterize the dynamics of the human amygdala across the different sleep stages and during wakefulness.

Glutamatergic Neurons in the Preoptic Hypothalamus Promote Wakefulness,Destabilize NREM Sleep,Suppress REM Sleep,and Regulate Cortical Dynamics

Clinical and experimental data from the last nine decades indicate that the preoptic area of the hypothalamus is a critical node in a brain network that controls sleep onset and homeostasis. By contrast, we recently reported that a group of glutamatergic neurons in the lateral and medial preoptic area increases wakefulness, challenging the long-standing notion in sleep neurobiology that the preoptic area is exclusively somnogenic. However, the precise role of these subcortical neurons in the control of behavioral state transitions and cortical dynamics remains unknown. Therefore, in this study, we used conditional expression of excitatory hM3Dq receptors in these preoptic glutamatergic (Vglut2⁺) neurons and show that their activation initiates wakefulness, decreases non-rapid eye movement (NREM) sleep, and causes a persistent suppression of rapid eye movement (REM) sleep. We also demonstrate, for the first time, that activation of these preoptic glutamatergic neurons causes a high degree of NREM sleep fragmentation, promotes state instability with frequent arousals from sleep, decreases body temperature, and shifts cortical dynamics (including oscillations, connectivity, and complexity) to a more wake-like state. We conclude that a subset of preoptic glutamatergic neurons can initiate, but not maintain, arousals from sleep, and their inactivation may be required for NREM stability and REM sleep generation. Further, these data provide novel empirical evidence supporting the hypothesis that the preoptic area causally contributes to the regulation of both sleep and wakefulness.SIGNIFICANCE STATEMENT Historically, the preoptic area of the hypothalamus has been considered a key site for sleep generation. However, emerging modeling and empirical data suggest that this region might play a dual role in sleep-wake control. We demonstrate that chemogenetic stimulation of preoptic glutamatergic neurons produces brief arousals that fragment sleep, persistently suppresses REM sleep, causes hypothermia, and shifts EEG patterns toward a “lighter” NREM sleep state. We propose that preoptic glutamatergic neurons can initiate, but not maintain, arousal from sleep and gate REM sleep generation, possibly to block REM-like intrusions during NREM-to-wake transitions. In contrast to the long-standing notion in sleep neurobiology that the preoptic area is exclusively somnogenic, we provide further evidence that preoptic neurons also generate wakefulness.     

Effect of sleep deprivation on spike-wave discharges in idiopathic generalised epilepsy: a 4 x 24 h continuous long term EEG monitoring study

The aim of the study was to investigate the effect of sleep and sleep deprivation on spike-wave discharges (SWD) in an idiopathic generalised epileptic patient population by continuous long term cassette EEG monitoring for 4 x 24 h. In ten patients with idiopathic generalised epilepsy, showing SWD during awake state and in sleep as well, EEG and sleep polygraphy was recorded for 4 x 24 h. Sleep was deprived during the second 24 h. Awake state, NREM1, NREM2, NREM3+4 and REM sleep duration and number of SWD episodes were evaluated in 4 min intervals by visual scoring. For analysing the effect of 'day' and 'night', 'vigilance' and 'sleep deprivation' (SD) on the density variables multi-way ANOVAs were carried out in different designs. SWD densities for different vigilance states were not significantly different during 'day' and 'night' in 24 h without SD or rebound after SD. Sleep had an activating effect SWD densities being the highest during NREM1 and NREM2. There was an increase of SWD densities in all vigilance states after SD, but SWD densities remained the highest in NREM1 andNREM2. Our results support the view that sleep dependent rather than sleep independent mechanisms cause activation of SWD after SD in generalised epilepsy. We assume that fine graded vigilance fluctuations, that are more frequent after SD, have an essential role in SWD activation in superficial sleep or even in wakefulness.