Relation of pontine choline acetyltransferase immunoreactive neurons with cells which increase discharge during REM sleep

The purpose of this study was to determine whether neurons in the medial pontine reticular formation with high discharge rates during REM sleep could be localized in regions of the brainstem having neurons displaying choline acetyltransferase immunoreactivity. Six cats were implanted with sleep recording electrodes and microwires to record extracellular potentials of neurons in the pontine reticular formation. Single-units with a S:N ratio greater than 2:1 were recorded for at least two REM sleep cycles. A total of 49 units was recorded from the pontine reticular formation at medial-lateral planes ranging from 0.8 to 3.7 mm. The greatest proportion of the units (28.6%) showed highest discharge during active waking and phasic REM sleep compared to quiet waking, non-REM sleep, transition into REM sleep or quiet REM sleep periods. A percentage (20.4%) of the cells had high discharge associated with phasic REM sleep periods while 8.2% of the cells showed a progressive increase in discharge from waking to REM sleep. Subsequent examination of the distribution of choline acetyltransferase immunoreactive cells in the PRF revealed that cells showing high discharge during REM sleep were not localized near presumed cholinergic neurons. Indeed, we did not find any ChAT immunoreactive somata in the medial PRF, an area which has traditionally been implicated in the generation of REM sleep. These results suggest that while increased discharge of PRF cells may be instrumental to REM sleep generation, these cells are not cholinergic.     

Hypothalamic control of sleep

A sleep-promoting function for the rostral hypothalamus was initially inferred from the presence of chronic insomnia following damage to this brain region. Subsequently, it was determined that a unique feature of the preoptic hypothalamus and adjacent basal forebrain is the presence of neurons that are activated during sleep compared to waking. Preoptic area "sleep-active" neurons have been identified by single and multiple-unit recordings and by the presence of the protein product of the c-Fos gene in the neurons of sleeping animals. Sleep-active neurons are located in several subregions of the preoptic area, occurring with high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Neurons in the vlPOA contain the inhibitory neuromodulator, galanin, and the inhibitory neurotransmitter, GABA. A majority of MnPN neurons activated during sleep contain GABA. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior and lateral hypothalamus and the rostral brainstem. Cumulative evidence indicates that preoptic area neurons function to promote sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent studies suggest a role for preoptic area neurons in the homeostatic aspects of the regulation of both rapid eye movement (REM) and non-REM (NREM) sleep and as a potential target for endogenous somnongens, such as cytokines and adenosine.     

c-fos proto-oncogene change in relation to REM sleep duration

Auditory stimulation has been shown to increase REM sleep periods in cats and humans. This effect has been attributed to an elevation of the level of excitability in a variety of brain stem neuronal groups. Fos-like immunostaining (FLI) has been useful in constructing maps of post-synaptic neuronal activity with single cell resolution, and has been suggested to be tightly correlated with ongoing neuronal activity. This study used FLI to quantify neurons from structures expressing c-fos in brain stem areas in animals with normal REMs and compared them with those showing extended REM periods. The results basically indicated that brain stem areas which in other studies have been described as having REM-ON cells, showed an increase in FLI, while no FLI changes occurred in areas described as having REM-OFF cells. These results are discussed in terms of the possibility that REM maintenance is related to a widespread increase in brain stem excitability.     

Enhancement of desynchronized sleep signs after pontine microinjection of the muscarinic agonist bethanechol

The question of which brainstem neuronal receptors can mediate cholinergic REM sleep induction was investigated by injecting the pure muscarinic agonist bethanechol via glass micropipettes in the pontine tegmentum of cats. The REM sleep enhancement was observed to be equally potent, equally dose-dependent and its appearance equally site-dependent as that previously observed with carbachol, a mixed muscarinic/nicotinic agonist. The results suggest that the pharmacological activation of muscarinic receptors in pontine neurons is sufficient to trigger REM sleep.     

c-fos proto-oncogene changes in relation to REM sleep duration.

Auditory stimulation has been shown to increase REM sleep periods in cats and humans. This effect has been attributed to an elevation of the level of excitability in a variety of brain stem neuronal groups. Fos-like immunostaining (FLI) has been useful in constructing maps of post-synaptic neuronal activity with single cell resolution, and has been suggested to be tightly correlated with ongoing neuronal activity. This study used FLI to quantify neurons from structures expressing c-fos in brain stem areas in animals with normal REMs and compared them with those showing extended REM periods. The results basically indicated that brain stem areas which in other studies have been described as having REM-ON cells, showed an increase in FLI, while no FLI changes occurred in areas described as having REM-OFF cells. These results are discussed in terms of the possibility that REM maintenance is related to a widespread increase in brain stem excitability.     

Sleep suppression following kainic acid-induced lesions of the basal forebrain

We have described elsewhere neurons in the ventral basal forebrain of cats that have elevated discharge rates during sleep and during transitions from waking to sleep, yet have comparatively low discharge rates during waking. These sleep-active neurons may mediate the hypnogenic properties of the basal forebrain. To further evaluate their role in the control of sleep, we examined the effects of basal forebrain lesions produced by microinjections of the relatively cell-selective neurotoxin, kainic acid, on sleep. Lesions were made bilaterally in two regions that contain high densities of sleep-active neurons: the horizontal limb of the diagonal bands of Broca and the lateral preoptic area-substantia innominata. Twelve-hour polygraph recordings were made before and at various intervals after basal forebrain damage in a total of eight cats. The lesions resulted in reduced time spent in deep, nonrapid eye-movement sleep and REM sleep, and increased time spent awake. These abnormalities persisted through 6 to 7 weeks postlesion. Reductions in deep non-REM sleep were due to decreases in bout number, particularly in the number of extended deep non-REM episodes (i.e., those greater than 5 min in duration). The number of REM sleep episodes was also significantly reduced. The average duration of epochs of waking was elevated throughout the postlesion period. Thus, in the postlesion period, cats exhibited an impaired ability to initiate and maintain consolidated periods of sleep, particularly of deeper sleep stages. Lesions were also associated with reduced EEG spindling during sleep. These results are consistent with our hypothesis that sleep-active neurons are a component of a basal forebrain sleep- and EEG-regulating mechanism.     

Brain structures and mechanisms involved in the generation of NREM sleep: focus on the preoptic hypothalamus

Four lines of research have greatly increased our understanding of the hypothalamic preoptic area (POA) sleep-promoting system. First, sleep-active neurons within the POA have been identified using both electrophysiological recording and immediate early gene protein (c-Fos) staining methods. Segregated sleep-active neurons were found in ventrolateral and median POA (VLPO and MnPN). Additional sleep-active neurons may be intermixed with non-sleep specific neurons in other POA regions and the adjacent basal forebrain. Second, the putative sleep factors, adenosine and prostaglandin D2, were found to excite sleep-active neurons. Other sleep factors may also modulate these sleep-active populations. Third, many sleep-active neurons are warm-sensitive neurons (WSNs). WSNs are identified by excitatory responses to small increases in local POA temperature. The same local POA thermal stimuli strongly modulate sleep propensity and EEG delta activity within sleep. Interactions between sleep regulation and thermoregulation are consistent with studies of circadian sleep propensity, prolonged sleep deprivation in rats, and species differences in sleep amounts. Fourth, sleep-active neurons were found to co-localize the inhibitory neurotransmitter, gamma-aminobutyric acid and to have projections to arousal-related neuronal subgroups in the posterior hypothalamus and midbrain. Sleep-active and arousal-related neurons exhibit reciprocal changes in discharge across the wake-NREM-REM cycle, and activation of WSNs suppresses the neuronal activity of some arousal-related neuronal groups. These studies establish mechanisms by which POA hypnogenic neurons can inhibit EEG and behavioral arousal. In addition, there is evidence that arousal-related neurotransmitters inhibit VLPO sleep-active neurons. Mutually inhibitory interactions between sleep-promoting and the arousal system provide a substrate for a . 2001 Harcourt Publishers Ltd     

Chronic low-amplitude electrical stimulation of the laterodorsal tegmental nucleus of freely moving cats increases REM sleep

While cholinergic stimulation of the PRF evokes a REM-like state, electrical stimulation of LDT/PPT neurons has not been used to test the hypothesis of mesopontine cholinergic control of REM sleep. Adult cats were implanted for electrographic recording and with bipolar unilateral stimulating electrodes, either in the LDT or within the PRF (stimulation control). Baseline recordings of the normal sleep-wake cycle were carried out for 5 h. On the next day, continuous stimulation of the LDT or mPRF was carried out during the same time period (0.5 ms pulses, 1 μA, 8 Hz) and with post-stimulation recording for 3 h. A second baseline recording day followed with same protocol as the first baseline day. This 3-day sequence, separated by 3 days, was repeated three times in each of the three LDT and the three medial PRF cats. Five hours of chronic low-amplitude stimulation of the LDT induced a highly significant increase in total REM and in the duration of REM sleep bouts. Stimulation of the mPRF did not affect any of the behavioral states. This study, the first to our knowledge to use low-amplitude stimulation of LDT in freely moving cats, indicates the importance of mesopontine cholinergic neurons in REM sleep.     

Activity of medial mesopontine units during cataplexy and sleep-waking states in the narcoleptic dog.

Narcolepsy has been hypothesized to be a disease of rapid eye movement (REM) sleep. According to this hypothesis, cataplexy is a result of the triggering during waking of the mechanism that normally serves to suppress muscle tone in REM sleep. REM sleep control mechanisms have been localized to the pons. Narcoleptic dogs have increased numbers of cholinergic receptors in the medial pons. These findings suggest that neurons mediating the triggering of cataplexy might be located in medial pontine regions. In the present study, this hypothesis has been investigated by recording the discharge of units in the medial mesopontine region of the narcoleptic dog. Unit activity was examined in the nucleus reticularis pontis oralis, caudalis, and central gray, with each cell being recorded during both cataplexy and sleep states. Maximal discharge rates were observed, in all of these regions, during active waking states (mean rate, 45.3/sec) and REM sleep (16.0/sec), with minimal discharge rates in non-REM sleep (8.3/sec). Unit discharge was reduced in cataplexy relative to precataplexy periods. Cataplexy discharge rates were 8.3/sec, 52% of the mean REM sleep rate. Cataplexy discharge rates were also significantly lower than those at REM sleep onset. Cataplexy discharge rates were comparable to rates in quiet waking and non-REM sleep. While medial mesopontine neurons discharge at high rates in REM sleep, they have little or no activity in cataplexy. We interpret the lack of activation of medial mesopontine units in cataplexy as indicating that the characteristic phasic motor activation of REM sleep does not occur in this state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brainstem function in rapid eye movement sleep behavior disorder: the evaluation of brainstem function by proton MR spectroscopy (1H-MRS)

Brainstem function was evaluated by proton magnetic resonance (MR) spectroscopy (1H-MRS) in a 69-year-old man with idiopathic rapid eye movement (REM) sleep behavior disorder. An analysis of spectral peak area ratios revealed an increase in the choline/creatine ratio. This change suggests that brainstem neurons have functional impairment at the cell membrane level. Further, our results suggest that 1H-MRS may provide for non-invasive, metabolic evaluation of brainstem neuronal function in REM sleep behavior disorder and find application in the differentiation of secondary REM sleep behavior disorders with neurodegenerative disorders from idiopathic disorders.     

Brain and sleep mechanism]

It is now accepted that sleep is induced by biological clock located in the suprachiasmatic nucleus and/or sleep promoting substances, which influence ventrolateral preoptic (VLPO) neurons. The VLPO neurons affects more caudally situated posterior hypothalamic ones containing orexine and/or histamine, reciprocally. When these neurons inhibit lower brainstem aminergic ones, sleep is induced. REM (Rapid Eye Movement) sleep can be induced mainly by brainstem cholinergic neurons, when aminergic ones are completely inhibited. During this stage, tonic activities and phasic Ponto-Geniculate-Occipital (PGO) ones originated within brainstem cholinergic neurons activate irregularly many parts of the brain such as the cerebral cortex and limbic system to produce dream-like activity. Muscle atonia is also observed during REM sleep. This atonia is caused by neurons in the pontine reticular inhibitory area (PIA), which is normally inhibited by aminergic inputs. The PIA affects medullary neurons of the paramedian and/or magnocelullar nuclei to regulate motoneurons in the ventral horn. Therefore. muscle atonia is produced when these PPT cells are active during REM sleep. In addition, based upon many recent data, sleep is not a passive state but rather an active state, during which recuperation of neuronal system is promoted and information processing is executed.     

Preoptic area unit activity during sleep and wakefulness in the cat

The spontaneous discharge of 86 preoptic area (POA) neurons was recorded extracellularly in chronically prepared cats during wakefulness (W), slow-wave sleep (SWS), and REM sleep. Of these, the percentage of units exhibiting maximal discharge rates in SWS and REM sleep (84%) was significantly greater than that of those exhibiting a maximal discharge rate in W (16%). Furthermore, those neurons that discharged rapidly in sleep (fast units) generally had a reduced discharge rate in W. Sixteen of the 86 units showed a strong tendency to discharge in bursts during SWS but not during W or REM sleep. The mean coefficient of variation and the mean discharge rate for these bursting cells in SWS were significantly greater than the corresponding values for the same cells in W and REM sleep, and for the nonbursting cells in SWS. Because POA stimulation is known to initiate behavioral and electrocortical signs of sleep, it is suggested that "fast units" in SWS with reduced discharge rates in W, may be "hypnogenic" cells.     

Responses of preoptic neurons to stimulation of caudal and rostral brain stem reticular structures

Effect of stimulation (1 Hz) of rostral and caudal brain stem reticular formation was studied on 41 neurons of preoptic area in encéphale isolé cats. Primary excitation was seen on almost all the 25 neurons influenced by stimulation of either of the areas. Many of these influenced neurons received inputs from both areas and showed poststimulatory oscillations in excitability. The two brain stem reticular structures, which have antagonistic influence on cortical EEG, cortical and subcortical neuronal activity, had identical influence on preoptic area neurons when stimulated at 1 Hz.     

Sleep-related neuronal discharge in the basal forebrain of cats

Although evidence suggests that the basal forebrain contains a hypnogenic mechanism, putative sleep-promoting neural elements within this area have not been identified. We examined basal forebrain neuronal activity during waking, non-rapid-eye-movement (NREM) sleep, REM sleep and various transition states. Based on state-related discharge rates. 3 cell types were defined. Thirty-nine of 83 cells were classified as waking-active, i.e. waking discharge rates were greater than 2 times NREM sleep rates. Twenty-three of 82 cells were classified as state-indifferent (waking and NREM rates differed by a factor of less than 2). NREM sleep discharge rates of the remaining 20 cells were greater than 2 times waking rates. These were labeled sleep-active cells. Discharge rates of these cells during epochs of alert waking were low, averaging less than 1 spike/s. Maximal discharge rates occurred during NREM sleep, averaging 9.44 spikes/s. Increased discharge of sleep-active cells anticipated sleep onset; cells had an average discharge rate of 6.60 spikes/s during transitions between waking and NREM sleep. Sleep-active cells were confined to the ventral basal forebrain, in the horizontal limb of the diagonal bands of Broca, substantia innominata, entopeduncular nucleus and ventral globus pallidus. These areas overlap, in part, with those where chemical, thermal and electrical stimulations evoke sleep, and where lesions suppress sleep. Based on location and discharge pattern we consider sleep-active cells candidates for mediating some of the sleep-promoting functions of the basal forebrain.     

Local cerebral glucose utilization non-selectively elevated in rapid eye movement sleep of the fetus

The [14C]deoxyglucose method for measuring local cerebral glucose utilization was employed in an effort to identify regions of the brain which participate in the increased neuronal activity of rapid eye movement (REM) sleep. The study was conducted in near term fetal sheep in which REM periods are of sufficient duration to obtain reliable data with this method. Neither the postulated executive centers of REM sleep nor those structures in the brainstem known to participate in the electrical activity peculiar to this sleep phase were found to have selectively elevated rates of glucose utilization. Rather, these regions shared equally with virtually all other structures in having rates higher than those which accompany non-REM sleep.     

Regulation of sleep and wakefulness through the monoaminergic and cholinergic systems

Sleep and wakefulness are regulated in the brainstem and hypothalamus. Classical brain dissecting or stimulating studies have proposed the concept of an ascending reticular activating system, presently known as the wakefulness center, located in the caudal midbrain/rostral pontine (mesopontine) areas, comprising the serotonergic, noradrenergic and cholinergic neural populations. These neural groups, in association with the histaminergic and orexinergic neurons in the hypothalamus, activate the cerebral the cortex through the thalamus or basal forebrain. This activating (waking) system is controlled by the slow wave sleep (SWS) generating system in the preoptic area, which receives inhibitory signals from the waking center. The mesopontine area is also involved in the regulation of rapid eye movement (REM) sleep. Reciprocal interactions between the cholinergic/glutamatergic excitatory systems and the aminergic/GABAergic inhibitory systems are crucial for the regulation of REM sleep. In the REM activating system, mutual excitatory interactions between cholinergic and glutamatergic neurons serve to maintain the state of REM sleep. The REM activating system in the mesopontine area receives GABAergic inhibitory signals from several neural groups in the periaqueductal gray and the medulla. Thus, sleep and wakefulness are controlled by the interplay of various neural populations located in several areas in the central nervous system.     

REM sleep behaviour disorder: clinical profiles and pathophysiology

Rapid eye movement (REM) sleep behaviour disorder (RBD) is a parasomnia characterized by the intermittent loss of electromyographic atonia normally present during REM sleep and the emergence of purposeful complex motor activity associated with vivid dreams. Rapid eye movement sleep behaviour disorder usually affects older males and can be either idiopathic or symptomatic of various underlying disorders, in particular neurodegenerative diseases; in the latter case, RBD may be a prodromal symptom of the neurological disease. Several brainstem regions have been implicated in RBD pathophysiology, although the exact mechanism of the disorder in humans remains to be clarified. On clinical grounds, differentiation of RBD should be made from several non-REM parasomnias and other aberrant behaviours occurring during sleep. Rapid eye movement sleep behaviour disorder can be diagnosed on the basis of a systematic medical, neurological and psychiatric evaluation of the patient, assisted by a standard polysomnographic recording that includes continuous overnight videotaping; a brain imaging study is mandatory when an underlying brain disease is being suspected. Clonazepam at bedtime is the treatment of choice for RBD; alternatively, melatonin or pramipexole can be administered when clonazepam is contraindicated.     

Heterogeneous distribution of the serotonin 5‐HT1A receptor mRNA in chemically identified neurons of the mouse rostral brainstem: Implications for the role of serotonin in the regulation of wakefulness and REM sleep

The 5‐HT_1A receptor (5‐HT_1AR) plays a key role in the inhibitory influence of serotonin (5‐HT) on rapid eye movement (REM) sleep in rodents. However, the neuronal networks mediating such influence are mostly unknown, notably in the mouse. This led us to map 5‐HT_1AR mRNA, by in situ hybridization histochemistry (ISHH), and to characterize the neuronal phenotype of 5‐HT_1AR mRNA‐positive neurons by dual ISHH and ISHH combined with immunohistochemistry, throughout the mouse rostral brainstem, a pivotal region for the generation of REM sleep and cortical activation. 5‐HT_1AR mRNA was found in most 5‐HT neurons in the dorsal raphe (DR), the median raphe (MnR), the B9, and the interpeduncular (IP) nuclei. 5‐HT_1AR mRNA‐positive neurons were also identified in individualized clusters of γ‐aminobutyric acid (GABA)ergic neurons in the DR and in neurons of an undetermined phenotype in the MnR. In addition, 1) GABAergic neurons of the ventral portion of Gudden's dorsal tegmental nucleus (DTg), the IP, and the caudal portion of the deep mesencephalic nucleus (DpMe), and 2) glutamatergic neurons scattered in the caudal pontine reticular nucleus (PnC) and densely packed in the internal lateral parabrachial subnucleus (PBil) also expressed 5‐HT_1AR mRNA. In contrast, no specific 5‐HT_1AR‐related ISHH signal was generally detected in brainstem cholinergic and catecholaminergic neurons. These results emphasize the role of 5‐HT_1AR as an autoreceptor and the phenotypical heterogeneity of 5‐HT_1AR‐expressing neurons within the DR and the MnR in the mouse brain. They also provide a neuroanatomical basis for understanding the influence of 5‐HT_1AR on REM sleep and wakefulness. J. Comp. Neurol. 518:2744–2770, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of electrical stimulation in the amygdala on ponto-geniculo-occipital waves in rats

We examined the role of the amygdala in the modulation of sleep and ponto-geniculo-occipital (PGO) waves in the rat. The amygdala projects massively, via its central nucleus, into brainstem regions involved in alerting and in the generation of rapid-eye movement (REM) sleep and PGO waves. Electrical stimulation of the central nucleus of the amygdala during REM sleep increased PGO wave amplitude. Stimulation during non-REM sleep decreased PGO wave frequency. The results indicate that the amygdala has a role in modulating brainstem neural mechanisms underlying alerting during sleep.