Firing of 'possibly' cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness.

To clarify functional roles of mesopontine cholinergic neurons as a component of an activating system, single neuronal activity in the laterodorsal tegmental nucleus (LDT) of undrugged rats, whose head was fixed painlessly, was recorded along with cortical EEG and neck EMG. Activity of some dorsal raphe (DR) neurons was also recorded for comparison. Most of the animals had been sleep-deprived for 24 h. Observation was made only on neurons generating broad spikes, presumed from previous studies to be cholinergic or monoaminergic. The position of recorded neurons was marked by Pontamine sky blue ejected from the glass pipette microelectrode, and was identified on sections processed for NADPH diaphorase histochemistry which specifically stained cholinergic neurons. According to their firing rates during wakefulness (AW), slow-wave sleep (SWS) and paradoxical sleep (PS), 46 broad-spike neurons in the LDT were classified into 4 groups: (1) neurons most active during AW and silent during PS (some of these neurons might be serotonergic rather than cholinergic, as all the 9 neurons in the DR); (2) neurons most active during PS and silent during AW; (3) neurons equally more active during AW and PS than SWS; and (4) others mainly characterized by transiently facilitated activity at awakening and/or onset of PS. Neurons of groups 2 and 3 were the major constituents of the LDT. In most neurons change in firing preceded EEG change, except at awakening from PS. These results suggest that: (1) the LDT is composed of cholinergic neurons with heterogenous characteristics in relation to sleep/wakefulness; and (2) some tegmental cholinergic neurons play a privotal role in induction and maintenance of PS.     

Sleep and wakefulness in the southern sea lion

We recorded an electroencephalogram from the two hemispheres, a neck musculature electromyogram, an electrooculogram, and respiratory acts during sleep and wakefulness on land in three 1-year-old sea lion females for 3 or 4 consecutive days. On average active wakefulness (AW) occupied 20.4+/-2.0% of the 24-h period; quiet wakefulness (QW) 54.9+/-2.5%; slow wave sleep (SWS) 15.0+/-2.5% and paradoxical sleep (PS) 9.7+/-2.0%. Between 30 and 50% (average 39.1+/-3.4%) of total sleep time was spent in PS. From 8 to 31 episodes of PS were recorded per day (average 17+/-6 per day), with the longest episode lasting 20 min (average 5.6+/-0.5 min). Episodes of interhemispheric EEG asymmetry accounted for 5.5+/-1.3% of total SWS time. Respiratory pauses in these animals varied in QW between 4 and 36 s (average 15.7+/-0.4 s), in SWS between 11 and 37 s (20.9+/-0.6 s) and in PS between 2 and 69 s (15.0+/-1.5 s). AW, QW, SWS and PS were approximately equally distributed between light (07:00-19:00) and dark time (19:00-07:00). The low amount of SWS with interhemispheric EEG asymmetry, the high proportion of PS in total sleep time and the nearly even distribution of sleep and wakefulness over the 24-h period could be both species-specific features and/or ontogenetic characteristics of the animals studied.     

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.     

The organization of the neuronal activity of the cortical cingulate gyrus in the waking-sleep cycle

Dynamics of the neuronal activity of the cingulate gyrus (CG) in the sleep-wakefulness cycle (SWC) was studied in free-moving cats. Most of neurons (65.4) discharged with high frequency during active wakefulness (AW) and emotional stage of paradoxical sleep (PS); the frequency of discharges decreased during the passive wakefulness (PW) and slow-wave sleep (SWS). 15% of neurons showed opposite dynamics of the activity. They fired more intensively during the SWS. 19.6% of neurons showed no statistically significant difference in the discharge frequency of different phases of the SWC. Most of neurons (75.2%) regularly changed the pattern of discharges at a chang of the phases of the SWC. In particular, those neurons discharged by single spikes, more or less uniformly distributed in time, against the background of AW and PS. With the development of the SWS neurons began to discharge according to the cluster-pause principle. During the development of the short fragments of the EEG arousal, most of neurons either decrease (42.6%) or did not change (50.4%) the activity. The involvement of the CG in the regulation of the SWC is discussed.     

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.     

Behavioural state‐specific neurons in the mouse medulla involved in sleep‐wake switching

The medullary reticular formation (RF) is involved in the maintenance of several vital physiological functions and level of vigilance. In this study, in nonanesthetised, head‐fixed mice, I examined the role of medullary RF neurons in the control of sleep‐wake states, that is, wakefulness (W), slow‐wave sleep (SWS) and paradoxical (or rapid eye movement) sleep (PS). I showed, for the first time, that the mouse medullary RF contains presumed SWS‐promoting, SWS‐on neurons that remain silent during W, display a sharp increase in discharge rate at sleep onset, and discharge tonically and selectively during SWS. In addition, I showed the presence in the medullary RF of both PS‐on and PS‐off neurons, which, respectively, commence discharging or cease firing selectively just prior to, and during, PS. PS‐off neurons were located in the raphe nuclei and ventral medulla, while PS‐on neurons were found in both the lateral part of the ventral gigantocellular reticular nucleus and the raphe nuclei, as were SWS‐on neurons. PS‐off and SWS‐on neurons appear to play an important role in both the W‐SWS and SWS‐PS switches, while PS‐on and PS‐off neurons play an important role in the PS‐W switch. The present findings on the trends in spike activity at the transitions from SWS to PS and from PS to W are in line with the reciprocal interaction hypothesis according to which PS occurs as a result of the cessation of discharge of PS‐off neurons, while PS ends as a result of the start of discharge of PS‐off neurons.     

Single unit activity of periaqueductal gray and deep mesencephalic nucleus neurons involved in sleep stage switching in the mouse

A total of 668 single units were recorded in the mouse periaqueductal gray (PAG) and adjacent deep mesencephalic nucleus (DpMe) to determine their role in the switching of sleep–wake states, that is, wakefulness (W), slow‐wave sleep (SWS) and paradoxical (or rapid eye movement) sleep (PS) in general, and, in particular, to determine whether PS‐on and PS‐off neurons involved in PS state switching are present in these structures and to identify neuronal substrates for the SWS‐PS switching mediated by DpMe neurons. Both structures were found to contain similar percentages of W/PS‐active neurons, which discharge at a higher rate during W and PS than during SWS, while W‐active neurons, which discharge maximally during W, were found mainly in the PAG. Both also contained similar percentages of SWS/PS‐active neurons, which discharge at higher rates during SWS and PS than during W, and PS‐active neurons, which discharge maximally during PS, while SWS‐active neurons, which discharge maximally during SWS, were found almost exclusively in the PAG. Both structures contained virtually no PS‐on or PS‐off neurons, which, respectively, discharge or cease firing selectively and tonically just prior to, and during, PS. Unlike the PAG, the DpMe contained many SWS/PS‐on neurons, which discharge selectively at high rates during SWS and PS, but show a decrease in discharge rate at the transition from SWS to PS. Analysis of discharge profiles and trends in spike activity at the state transitions strongly suggests that PAG and DpMe neurons play an important role in the W‐SWS, SWS‐PS and/or PS‐W switches.     

A potent non-monoaminergic paradoxical sleep inhibitory system: a reverse microdialysis and single-unit recording study

Using reverse microdialysis and polygraphic recordings in freely moving cats, we investigated the effects on sleep-waking states of application of excitatory and inhibitory amino acid agonists, cholinergic agonist and monoamines to the periaqueductal grey and adjacent mesopontine tegmentum. Single-unit recordings during behavioural states were further used to determine the neuronal characteristics of these structures. We found that muscimol, a GABAA receptor agonist, induced a significant increase in paradoxical sleep (PS) only when applied to a dorsocaudal central tegmental field (dcFTC) located just beneath the ventrolateral periaqueductal grey. In this structure, both kainic and N-methyl-aspartic acids caused a dose-dependent increase in wakefulness (W) and decrease in both slow-wave sleep (SWS) and PS. Norepinephrine and epinephrine, and to a lesser extent histamine, also increased W and decreased SWS and PS, whereas serotonin, dopamine and carbachol, a cholinergic agonist, had no effect. Two types of neurones were recorded in this structure, those exhibiting a higher rate of tonic discharge during both W and PS compared with during SWS, and those showing a phasic increase in firing rate during both active W and PS. Both types of neurones showed a gradual increase in unit activity during PS. Our study demonstrated for the first time that the ventrolateral periaqueductal grey and dcFTC play different roles in behavioural state control, that the dcFTC neurones are critically involved in the inhibitory mechanisms of PS generation, playing a central part in its maintenance, and that these neurones are under the control of GABAergic, glutamatergic, adrenergic and histaminergic systems.     

Neuronal activities in brain-stem cholinergic nuclei related to tonic activation processes in thalamocortical systems

This study was performed to examine the hypothesis that thalamic-projecting neurons of mesopontine cholinergic nuclei display activity patterns that are compatible with their role in inducing and maintaining activation processes in thalamocortical systems during the states of waking (W) and rapid-eye-movement (REM) sleep associated with desynchronization of the electroencephalogram (EEG). A sample of 780 neurons located in the peribrachial (PB) area of the pedunculopontine tegmental nucleus and in the laterodorsal tegmental (LDT) nucleus were recorded extracellularly in unanesthetized, chronically implanted cats. Of those neurons, 82 were antidromically invaded from medial, intralaminar, and lateral thalamic nuclei: 570 were orthodromically driven at short latencies from various thalamic sites: and 45 of the latter elements are also part of the 82 cell group, as they were activated both antidromically and synaptically from the thalamus. There were no statistically significant differences between firing rates in the PB and LDT neuronal samples. Rate analyses in 2 distinct groups of PB/LDT neurons, with fast (greater than 10 Hz) and slow (less than 2 Hz) discharge rates in W, indicated that (1) the fast-discharging cell group had higher firing rates in W and REM sleep compared to EEG-synchronized sleep (S), the differences between all states being significant (p less than 0.0005); (2) the slow-discharging cell group increased firing rates from W to S and further to REM sleep, with significant difference between W and S (p less than 0.01), as well as between W or S and REM sleep (p less than 0.0005). Interspike interval histograms of PB and LDT neurons showed that 75% of them have tonic firing patterns, with virtually no high-frequency spike bursts in any state of the wake-sleep cycle. We found 22 PB cells that discharged rhythmic spike trains with recurring periods of 0.8-1 sec. Autocorrelograms revealed that this oscillatory behavior disappeared when their firing rate increased during REM sleep. Dynamic analyses of sequential firing rates throughout the waking-sleep cycle showed that none of the full-blown states of vigilance is associated with a uniform level of spontaneous firing rate. Signs of decreased discharge frequencies of mesopontine neurons appeared toward the end of quiet W, preceding by about 10-20 sec the most precocious signs of EEG synchronization heralding the sleep onset. During transition from S to W, rates of spontaneous discharges increased 20 sec before the onset of EEG desynchronization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Waking selective neurons in the posterior hypothalamus and their response to histamine H3-receptor ligands: an electrophysiological study in freely moving cats

Neurons which discharge selectively during waking (waking selective) have been found in the tuberomamillary nucleus (TM) and adjacent areas of the posterior hypothalamus. Although they share some electrophysiological properties with aminergic neurons, there is no direct evidence that they are histaminergic. We have recorded from posterior hypothalamic neurons during the sleep-wake cycle in freely moving cats, and investigated the effects on waking selective neurons of specific ligands of histaminergic H3-receptors, which autoregulate the activity of histaminergic neurons. Two types of neurons were seen. Waking selective neurons, termed "waking-on (W-on)," were located exclusively within the TM and adjacent areas, and discharged at a low regular rate during waking (1.71-2.97 Hz), decreased firing during light slow wave sleep (SWS), became silent during deep SWS and paradoxical sleep (PS) and resumed their activity on, or a few seconds before, awakening. "Waking-related" neurons, located in an area dorsal to the TM, displayed a similar, although less regular, low rate of firing (1.74-5.41 Hz) and a similar discharge profile during the sleep-wake cycle; however, unlike "W-on" neurons, they did not completely stop firing during deep SWS and PS. Intramuscular (i.m.) injection of ciproxifan (an H3-receptor antagonist, 1mg/kg), significantly increased the discharge rate of W-on neurons and induced c-fos expression in histamine-immunoreactive neurons, whereas i.m. injection of imetit (an H3-receptor agonist, 1mg/kg) or microinjection of alpha-methylhistamine (another H3-receptor agonist, 0.025-0.1 microg/0.2 microl) in the vicinity of these cells significantly decreased their discharge rate. Moreover, the effect of the antagonist was reversed by the agonists and vice versa. In contrast, "waking-related" neurons were unaffected by these H3-receptor ligands. These data provide evidence for the histaminergic nature of "W-on" neurons and their role in cortical desynchronization during waking, and highlight the heterogeneity of posterior hypothalamic neuronal populations, which might serve different functions during the wakefulness.     

Selective Immunolesion of the Basal Forebrain Cholinergic Neurons: Effects on Hippocampal Activity During Sleep and Wakefulness in the Rat

Intracerebroventricular injection of the toxin 192 IgG-saporin (4μg) kills the cholinergic neurons of the basal forebrain bearing the low affinity NGF receptor (NGFr). The effect of this cholinergic denervation on the hippocampal and cortical electrical activity (EEG) was studied during sleep and wakefulness. EEG was recorded under freely-moving conditions in lesioned (n=10) and control (n=6) rats (8–16 days post-injection). In lesioned rats, active (AW) and quiet (QW) wakefulness episode durations were similar to those of controls whereas the REM sleep duration was reduced, 8 days post-lesion (P<0.01). Bouts of REM sleep were more numerous but shorter. The hippocampal theta activity was still present in lesioned-rats during AW (type 1 theta), QW (type 2 theta) and REM sleep. The frequency was unchanged but the amplitude of the three types of theta was significantly reduced (P<0.01). Type 2 theta occurred with shorter and less regular bouts (P<0.05). Abnormal slow waves (2–4 Hz) were observed during wakefulness. Histology showed a dramatic loss of NGFr-positive neurons in the basal forebrain and a decline in hippocampal and cortical acetylcholinesterase activity. These results suggest that the cholinergic septohippocampal input is not the primary pacemaker for the hippocampal theta rhythm.     

Associative and non-associative changes in unit activity of the rat hippocampus

Two main types of neurons of the dorsal hippocampus were recorded in chronic rats during the classical conditioning of an arousal phenomenon (neocortical EEG desynchrony). The relative importance of the associative and non-associative factors was assessed by a differentiation procedure. In naive rats, during the “acquisition” session most type-I neurons rapidly acquired an “inhibitory” response closely parallel to the EEG response. Neither of these responses showed differentiation. Some type-II neurons acquired an “excitatory” response which was independent of the EEG response and which had a significant tendency to differentiation. These data were confirmed in rats submitted to several “retention” sessions. All the type-I neurons were undifferentiated while 11 out of 41 type-II neurons were differentiated. Type-I neurons were characterized by a bursting mode of discharge. Their activity was higher during slow wave sleep (SWS) than during wakefulness (W) or paradoxical sleep (PS). Most of the differentiated type-II neurons were more active during W and/or PS than during SWS.     

Effects of hypnogenic vagal stimulation on thalamic neuronal activity in cats

Neuronal responses from the ventro-postero-medial (VPM) and reticular (NR) nuclei of cat thalamus to vagal stimulation was recorded during wakefulness (W), slow-wave-sleep (SWS), and paradoxical sleep (PS) using chronically implanted microelectrodes. Cellular firing was facilitated in NR and depressed in VPM when weak, hyponogenic stimuli were delivered to the vagal nerve during W and SWS. Higher intensity vagal stimulation increased firing frequency and duration of discharge in both nuclei. Vagally induced discharges of several VPM neurons were depressed by NR stimulation. We speculate that intrathalamic mechanisms play a role in the genesis of induced synchronization and sleep.     

Cholinergic neurons of the feline pontomesencephalon. I. Essential role in ‘Wave A’ generation

Wave A in the cat appears to be analogous to P1 in the human. Both are positive middle-latency auditory-evoked potentials, present at slow click rates during wakefulness and REM sleep but absent during slow-wave sleep. Wave A has been recorded in the parabrachial and medial tegmental areas of the midbrain and in thalamic target projections of the reticular activating system. Two nuclei in this system, the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei, contain cholinergic cells; the cholinergic antagonist scopolamine eliminates Wave A. To test whether PPT and LDT were important in Wave A generation, we attempted to lesion these nuclei bilaterally in 11 cats. Wave A was markedly diminished or absent in all but 2 cats, in which the lesions did not include PPT. Loss of choline acetyltransferase-positive cells in PPT, but not LDT, was correlated with effects on Wave A, i.e. greatest cell loss occurred in cats in which Wave A disappeared, and least cell loss in cats with no change in Wave A. We conclude that the PPT nucleus, and particularly its cholinergic cell component, is essential for Wave A generation and suggests that a similar substrate may be significant for generation of the human P1.     

Single neuron burst firing in the human hippocampus during sleep

Although there are numerous non-primate studies of the single neuron correlates of sleep-related hippocampal EEG patterns, very limited hippocampal neuronal data are available for correlation with human sleep. We recorded human hippocampal single neuron activity in subjects implanted with depth electrodes required for medical diagnosis and quantitatively evaluated discharge activity from each neuron during episodes of wakefulness (Aw), combined stage 3 and 4 slow-wave sleep (SWS), and rapid eye movement (REM) sleep. The mean firing rate of the population of single neurons was significantly higher during SWS and Aw compared with REM sleep (p = 0.002; p < 0.0001). In addition, burst firing was significantly greater during SWS compared with Aw (p = 0.001) and REM sleep (p < 0.0001). The synchronized state of SWS and associated high-frequency burst discharge found in human hippocampus may subserve functions similar to those reported in non-primate hippocampus that require burst firing to induce synaptic modifications in hippocampal circuitry and in hippocampal projections to neocortical targets that participate in memory consolidation.     

Activity of mesencephalic dopamine and non-dopamine neurons across stages of sleep and waking in the rat

Single unit activity of dopamine and non-dopamine neurons in the substantia nigra and ventral tegmental area was recorded across stages of sleep and waking in the rat. These stages consisted of slow wave sleep (SWS), rapid eye movement (REM) sleep, awake-quiet (AQ) and awake-moving (AM). The dopamine neurons showed no change in mean firing rate across the stages of sleep or waking. During REM sleep, however, the dopamine cells fired with a more variable interspike interval than during SWS. In contrast, non-dopamine neurons in the substantia nigra and ventral tegmental area showed large increases in firing rate in REM compared to SWS, and in AM compared to AQ, without showing changes in interspike interval variability. In conclusion, whereas other monoaminergic neurons and various cortical and subcortical neurons exhibit marked changes in firing rate across the stages of sleep and waking, the dopamine neurons are unique in their lack of change in firing rate across stages.     

Activity of mesencephalic dopamine and non-dopamine neurons across stages of sleep and walking in the rat

Single unit activity of dopamine and non-dopamine neurons in the substantia nigra and ventral tegmental area was recorded across stages of sleep and waking in the rat. These stages consisted of slow wave sleep (SWS), rapid eye movement (REM) sleep, awake-quiet (AQ) and awake-moving (AM). The dopamine neurons showed no change in mean firing rate across the stages of sleep or waking. During REM sleep, however, the dopamine cells fired with a more variable interspike interval than during SWS. In contrast, non-dopamine neurons in the substantia nigra and ventral tegmental area showed large increases in firing rate in REM compared to SWS, and in AM compared to AQ, without showing changes in interspike interval variability. In conclusion, whereas other monoaminergic neurons and various cortical and subcortical neurons exhibit marked changes in firing rate across the stages of sleep and waking, the dopamine neurons are unique in their lack of change in firing rate across stages.     

Acetylcholine and glutamate release during sleep–wakefulness in the pedunculopontine tegmental nucleus and norepinephrine changes regulated by nitric oxide

Cholinergic neurons in the pons appear to play a major role in generating rapid eye movement (REM) sleep. In the present study, acetylcholine and glutamate release in the pedunculopontine tegmental nucleus (PPT) during the sleep-waking cycle were investigated by in vivo microdialysis. Acetylcholine release during slow wave sleep (SWS) was significantly lower (P<0.05) than during REM sleep and wakefulness. On the other hand, glutamate release during wakefulness was higher (P<0.05) than during REM sleep and SWS. Furthermore, the application of N-methyl-D-aspartate (1 mM) induced a significant increase of nitric oxides (NOx) for 20 min (P<0.05) and a decrease of norepinephrine for the first 15 min (P=0.01), indicating NOx regulation on norepinephrine release in PPT.     

Differential c-Fos expression in cholinergic, monoaminergic, and GABAergic cell groups of the pontomesencephalic tegmentum after paradoxical sleep deprivation and recovery.

Multiple lines of evidence indicate that neurons within the pontomesencephalic tegmentum are critically involved in the generation of paradoxical sleep (PS). From single-unit recording studies, evidence suggests that unidentified but "possibly" cholinergic tegmental neurons discharge at higher rates during PS than during slow wave sleep or even waking and would thus play an active role, whereas "presumed" monoaminergic neurons cease firing during PS and would thus play a permissive role in PS generation. In the present study performed on rats, c-Fos immunostaining was used as a reflection of neuronal activity and combined with immunostaining for choline acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH), or glutamic acid decarboxylase (GAD) for immunohistochemical identification of active neurons during PS recovery ( approximately 28% of recording time) as compared with PS deprivation (0%) and PS control (approximately 15%) conditions. With PS recovery, there was a significant increase in ChAT+/c-Fos+ cells, a significant decrease in Ser+/c-Fos+ and TH+/c-Fos+ cells, and a significant increase in GAD+/c-Fos+ cells. Across conditions, the percent PS was correlated positively with tegmental cholinergic c-Fos+ cells, negatively with raphe serotonergic and locus coeruleus noradrenergic c-Fos+ cells, and positively with codistributed and neighboring GABAergic c-Fos+ cells. These results support the hypothesis that cholinergic neurons are active, whereas monoaminergic neurons are inactive during PS. They moreover indicate that GABAergic neurons are active during PS and could thus be responsible for inhibiting neighboring monoaminergic neurons that may be essential in the generation of PS.