Cholinergic basal forebrain structures are involved in the mediation of the arousal effect of noradrenaline.

Cholinergic basal forebrain structures are implicated in cortical arousal and regulation of the sleep-wake cycle. Cholinergic neurones are innervated by noradrenergic terminals, noradrenaline excites them via alpha-1 receptors and microinjection of noradrenaline into the basal forebrain enhances wakefulness. However, it is not known to what extent the cholinergic versus non-cholinergic basal forebrain projection neurones contribute to the arousing effects of noradrenaline. To elucidate the roles of cholinergic basal forebrain structures we administered methoxamine, an alpha-1-adrenergic agonist into the basal forebrain, in intact animals and again after selective destruction of the basal forebrain cholinergic cells by 192 IgG-saporin. In eight male Han-Wistar rats implanted with electroencephalogram/electromyogram electrodes, a microdialysis probe targeted into the basal forebrain was perfused with artificial cerebrospinal fluid for 6 h on a baseline day, and with cerebrospinal fluid in the first and with methoxamine in the second 3-h period of the subsequent day. The sleep-wake activity was recorded for 24 h on both days. Saporin was then injected into the basal forebrain and 2 weeks later the same experimental schedule (with cerebrospinal fluid and methoxamine) was repeated. In the intact animals, methoxamine exhibited a robust arousing effect and non-rapid eye movement (NREM) and REM sleep was suppressed. Lesioning of the basal forebrain cholinergic neurones abolished almost completely the NREM sleep-suppressing effect of methoxamine, whereas the REM sleep-suppressing effect remained intact. Thus, the basal forebrain cholinergic neurones mediate, at least in part, cortical arousal and non-REM sleep-suppression, but they are not involved in the REM sleep-suppressing effects of noradrenaline.

Effect of sleep deprivation on multi-unit discharge activity of basal forebrain.

The basal forebrain (BF) is an important wakefulness/arousal-promoting structure involved in homeostatic responses to sleep deprivation (SD). However, the effects of SD and subsequent sleep recovery on the BF discharge have not been investigated. Multi-unit BF activity was recorded on freely moving rats during 8 h of baseline (BL) and, on the following day, during 4 h of SD by gentle handling followed by 4 h of recovery. The effect of SD on the waking discharge was evaluated during the last 10 min of each hour when attentive waking was induced. The wakefulness level was defined based on the ratio between theta and delta electroencephalogram (EEG) powers, and epochs with ratios >or=1 but <2 (T/D-1) and >or=2 but <4 (T/D-2) were analysed separately. During T/D-1 wakefulness, the BF multi-unit discharge rate increased significantly during the second and third hours of SD and decreased during the third hour of recovery when compared with corresponding hours of BL. Non-rapid eye movement sleep discharge rate during recovery decreased significantly in the second and third versus the first and last hours. The results suggest that maintenance of the level of vigilance necessary for adequate performance during SD requires increased activation of BF neurones when compared with the BL, whereas the same level of vigilance after several hours of recovery can be maintained with lesser activation of BF neurones.

Nitric oxide modulates the discharge rate of basal forebrain neurones: a study in freely moving rats.

In urethane-anaesthetized rats the infusion of a nitric oxide (NO)-donor [NOC-18, 1 mM (DETA/NO); 2,2'-(hydroxynitrosohydrazino)bis-ethanamine)] into the basal forebrain (BF) inhibited the discharge rate of most neurones, suggesting that NO may promote sleep via inhibition of wake-promoting neurones in the BF. However, this hypothesis still needs to be confirmed in freely moving rats. The objective of this study was to examine whether NO modulates the discharge rate of BF neurones in freely moving rats in a similar manner to anaesthetized rats. We measured the discharge rates of BF neurones in freely moving rats during microdialysis infusion of a NO-donor (1 mm; NOC-18) in different vigilance states. Neurones were characterized as wake (W)-on (51.8%), W-off (28.6%) and W/non-rapid eye movement (REM)-independent (21.4%) based on their discharge profiles during wakefulness (W) and non-REM sleep. The NO-donor affected the discharge rate of most BF neurones during quiet wakefulness (QW; 55%) and non-REM sleep (64%). The most prominent response in all neuronal groups was a decrease in the discharge rate during QW and non-REM sleep. A small subpopulation of neurones increased the discharge rate. The increase in NO in the BF during prolonged wakefulness may facilitate sleep via inhibition of wake-promoting neurones.

Nitric oxide modulates the discharge rate of basal forebrain neurons.

RATIONALE: During prolonged wakefulness, the concentrations of nitric oxide (NO) and adenosine (AD) increase in the basal forebrain (BF). AD inhibits neuronal activity via adenosine (A1) receptors, thus providing a potential mechanism for sleep facilitation. Although NO in the BF increases adenosine and promotes sleep, it is not clear whether the sleep promotion by NO is mediated through adenosine increase, or NO independently of adenosine could modulate sleep. OBJECTIVE: The objective of the study was to clarify whether NO modulates the discharge rate of BF neurons and whether this effect is mediated via AD. MATERIALS AND METHODS: We measured the discharge rates of BF neurons in anesthetized rats during microdialysis infusion of NO donor alone or in combination with A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine. RESULTS: NO dose dependently modulated the discharge rate of BF neurons. NO donor (0.5 mM) increased the discharge rates in 48% of neurons and decreased it in 22%. A 1-mM dose decreased it in 55% and increased in 18%. Tactile stimulus affected the discharge rates of most neurons: 60% increased (stimulus-on) it and 14% decreased it (stimulus-off). A 1-mM NO donor predominantly inhibited neurons of both stimulus related types. A small proportion of stimulus-on (23%) neurons but none of the stimulus-off neurons were activated by NO donor. The blockade of A1 receptors partly prevented the inhibitory effect of NO on most of the neurons. This response was more prominent in stimulus-on than in stimulus-off neurons. CONCLUSION: NO modulates the BF neuronal discharge rates in a dose-dependent manner. The inhibitory effect is partly mediated via adenosine A1 receptors.

Preliminary findings of proton magnetic resonance spectroscopy in occipital cortex during sleep deprivation.

Proton magnetic resonance spectroscopy ((1)H MRS) has revealed biochemical alterations in various psychiatric disorders. Changes in brain metabolites may be caused not only by the disease's progression or response to treatment, but also by physiological variability. The aim of this study was to use (1)H MRS to assess the effects of specific short-term physiological states on major metabolites. Eight healthy women underwent (1)H MRS at the beginning and end of a 40-h period of sleep deprivation. The ratios of N-acetyl-aspartate (NAA), total creatine (tCr), and choline-containing compounds (Cho) to water (H(2)O) were determined from the occipital cortex during both baseline and photic stimulation conditions. During sleep deprivation, NAA/H(2)O decreased by 7% and Cho/H(2)O by 12%. Photic stimulation had no effect on the measured metabolites in the alert state, but in the sleep-deprived state the level of Cho/H(2)O increased during neuronal activation. The results suggest that NAA/H(2)O and Cho/H(2)O may depend on the state of alertness.

Adenosine A1 receptor-dependent G-protein activity in the rat brain during prolonged wakefulness.

Adenosine accumulates in the basal forebrain during prolonged wakefulness and induces sleep. There is abundant evidence showing that the sleep-inducing effects are mediated locally in the basal forebrain through the adenosine A1 receptor. In previous studies an increase in the mRNA expression but no apparent change in the ligand binding of the A1 receptors have been found. In the present study we used [(35)S]GTPgammaS autoradiography to assess regional A1 receptor dependent G-protein activity in rat brain during prolonged wakefulness and recovery sleep. We found that the G-protein activity was increased in the cortex but not in the basal forebrain during the first hours of sleep deprivation, suggesting different A1 receptor mediated responses to increasing adenosine concentrations in different brain areas.

Stimulus-induced brain lactate: effects of aging and prolonged wakefulness.

Both aging and sleep deprivation disturb the functions of the frontal lobes. Deficits in brain energy metabolism have been reported in these conditions. Neurons use not only glucose but also lactate as their energy substrate. The physiological response to elevated neuronal activity is a transient increase in lactate concentrations in the stimulated area. We have previously shown that cognitive stimulation increases brain lactate. To study the effect of prolonged wakefulness on the lactate response we designed an experiment to assess brain lactate levels during a 40-h sleep deprivation period in young (19-24 years old; n = 13) and in aged (60-68 years old; n = 12) healthy female volunteers. Brain lactate levels were assessed with proton MR-spectroscopy ((1)H MRS) during the performance of a silent word generation task. The (1)H MRS voxel location was individually selected, using functional magnetic resonance imaging, to cover the activated area in the left frontal lobe. The degree of sleepiness was verified using vigilance tests and self-rating scales. In the young alert subjects, the silent word generation test induced a 40% increase in lactate, but during the prolonged wakefulness period this response disappeared. In the aged subjects, the lactate response could not be detected even in the alert state. We propose that the absence of the lactate response may be a sign of malfunctioning of normal brain energy metabolism. The behavioral effects of prolonged wakefulness and aging may arise from this dysfunction.

The effect of age on prepro-orexin gene expression and contents of orexin A and B in the rat brain.

Orexin A and B (hypocretin 1 and 2) are hypothalamic peptides, which are synthesized in the lateral hypothalamus. Orexins participate in the regulation energy balance, food intake, vigilance and several endocrine and autonomic functions. The widespread projections of the orexin neurons suggest that they may have a role in coordination of different brain activities. The effects of ageing on the orexin system have not been studied previously. Prepro-orexin gene expression in the lateral hypothalamus, and the contents of orexin A and B peptides in the lateral hypothalamus and hypothalamus were measured in young, middle-aged and old (3, 12 and 24 months) rats. In the course of ageing, the expression of the prepro-orexin gene and the levels of orexin A and B decreased; the main decrease occurred by 12 months. Sleep deprivation for 6h increased slightly the expression of prepro-orexin gene in young rats. Deterioration of the orexin system may play a role in the phenomenon associated with aging, e.g. decreased consolidation of vigilance states, endocrine changes and dysfunctions of autonomic nervous system.

Sleep and its homeostatic regulation in mice lacking the adenosine A1 receptor.

Sleep deprivation (SD) increases extracellular adenosine levels in the basal forebrain, and pharmacological manipulations that increase extracellular adenosine in the same area promote sleep. As pharmacological evidence indicates that the effect is mediated through adenosine A1 receptors (A1R), we expected A1R knockout (KO) mice to have reduced rebound sleep after SD. Male homozygous A1R KO mice, wild-type (WT) mice, and heterozygotes (HET) from a mixed 129/C57BL background were implanted during anesthesia with electrodes for electroencephalography (EEG) and electromyography (EMG). After 1 week of recovery, they were allowed to adapt to recording leads for 2 weeks. EEG and EMG were recorded continuously. All genotypes had a pronounced diurnal sleep/wake rhythm after 2 weeks of adaptation. We then analyzed 24 h of baseline recording, 6 h of SD starting at light onset, and 42 h of recovery recording. Neither rapid eye movement sleep (REM sleep) nor non-REM sleep (NREMS) amounts differed significantly between the groups. SD for 6 h induced a strong NREMS rebound in all three groups. NREMS time and accumulated EEG delta power were equal in WT, HET and KO. Systemic administration of the selective A1R antagonist 8-cyclopentyltheophylline (8-CPT) inhibited sleep for 30 min in WT, whereas saline and 8-CPT both inhibited sleep in KO. We conclude that constitutional lack of adenosine A1R does not prevent the homeostatic regulation of sleep.

Nitrobenzylthioinosine (NBMPR) binding and nucleoside transporter ENT1 mRNA expression after prolonged wakefulness and recovery sleep in the cortex and basal forebrain of rat.

We have previously shown that extracellular adenosine levels increase locally in the basal forebrain (BF) during prolonged wakefulness, yet the cellular mechanisms of this local accumulation have remained unknown. The extracellular adenosine levels are strictly regulated by adenosine metabolism and its transport through cell membrane by the nucleoside transporters. As we previously showed that the key adenosine metabolizing enzymes were not affected by prolonged wakefulness, we now focussed on potential changes in the nucleoside transporters. In the present study, we measured the binding of nitrobenzylthioinosine (NBMPR), an ENT1 transporter inhibitor, and the ENT1 transporter mRNA after prolonged wakefulness and recovery sleep. Rats were sleep-deprived for 3 or 6 h using gentle handling. After 6 h one group was allowed to sleep for 2 h. NBMPR binding was determined from BF and cortex by incubating tissue extracts with [3H] NBMPR. The in situ hybridization was carried out on 20 microm cryosections using [35S]dATP-labelled oligonucleotide probe for ENT1 mRNA. The NBMPR binding was significantly decreased in the BF, but not in the cortex, after 6 h sleep deprivation when compared with the time-matched controls, suggesting a decline in adenosine transport. The expression of ENT1 mRNA did not change during prolonged wakefulness or recovery sleep in either cortex or the BF, although circadian variations were measured in both areas. We conclude that the regional decrease in adenosine transport could contribute to the gradual accumulation of extracellular adenosine in the basal forebrain during prolonged wakefulness.

Adenosine, energy metabolism, and sleep.

While the exact function of sleep remains unknown, it is evident that sleep was developed early in phylogenesis and represents an ancient and vital strategy for survival. Several pieces of evidence suggest that the function of sleep is associated with energy metabolism, saving of energy, and replenishment of energy stores. Prolonged wakefulness induces signs of energy depletion in the brain, while experimentally induced, local energy depletion induces increase in sleep, similarly as would a period of prolonged wakefulness. The key molecule in the induction of sleep appears to be adenosine, which induces sleep locally in the basal forebrain.

Metabolic imaging of human cognition: an fMRI/1H-MRS study of brain lactate response to silent word generation.

Proton magnetic resonance spectroscopy (1H-MRS) allows in vivo assessment of the metabolism related to human brain functions. Visual, auditory, tactile, and motor stimuli induce a temporary increase in the brain lactate level, which may act as a rapid source of energy for the activated neurons. The authors studied the metabolism of the frontal lobes during cognitive stimulation and measured local lactate levels with standard 1H-MRS, after localizing the activated area by functional MRI. Lactate levels were monitored while the subjects either silently listed numbers (baseline) or performed a silent word-generation task (stimulus-activation). The cognitive stimulus-activation produced a 50% increase in the brain lactate level in the left inferior frontal gyrus. The results show that metabolic imaging of neuronal activity related to cognition is possible using 1H-MRS.

Sleep among habitually violent offenders with antisocial personality disorder.

The aim of the present study was to characterize the subjective and objective sleep and sleep quality in habitually violent offenders with DSM-IV diagnosis of antisocial personality disorder using a sleep questionnaire, actigraphy, polysomnography and power spectral analysis. Subjects for the study were 19 drug-free males (mean age +/- SEM 30.7 +/- 2.58 years) recruited from a forensic psychiatric examination in a special ward of a university psychiatric hospital. The most striking finding was the high amount of slow-wave sleep, particularly the deepest S4 stage (17% as compared with 6% in healthy controls), in males with antisocial personality disorder. Moreover, in the spectral power analysis, both the delta and the theta power were significantly elevated. Whether this increase in persons with antisocial personality disorder reflects a specific brain pathology, or a delay in the normal development of sleep patterns in the course of ageing needs to be clarified with further experiments.

Local energy depletion in the basal forebrain increases sleep.

Sleep saves energy, but can brain energy depletion induce sleep? We used 2,4-dinitrophenol (DNP), a molecule which prevents the synthesis of ATP, to induce local energy depletion in the basal forebrain of rats. Three-hour DNP infusions induced elevations in extracellular concentrations of lactate, pyruvate and adenosine, as well as increases in non-REM sleep during the following night. Sleep was not affected when DNP was administered to adjacent brain areas, although the metabolic changes were similar. The amount and the timing of the increase in non-REM sleep, as well as in the concentrations of lactate, pyruvate and adenosine with 0.5-1.0 mM DNP infusion, were comparable to those induced by 3 h of sleep deprivation. Here we show that energy depletion in localized brain areas can generate sleep. The energy depletion model of sleep induction could be applied to in vitro research into the cellular mechanisms of prolonged wakefulness.

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat.

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.

Adenosine and sleep.

Adenosine is directly linked to the energy metabolism of cells. In the central nervous system an increase in neuronal activity enhances energy consumption as well as extracellular adenosine concentrations. In most brain areas high extracellular adenosine concentrations, through A(1) adenosine receptors, decrease neuronal activity and thus the need for energy. Adenosine seems to act as a direct negative feed-back inhibitor of neuronal activity. Hypoxia and ischemia induce very high extracellular adenosine levels, which may limit further brain damage. In brain areas that regulate cortical vigilance, particularly in the basal forebrain, high extracellular adenosine concentrations, induced by prolonged wakefulness, decrease the activity of presumably cholinergic cells and via this mechanism promote sleep. Our hypothesis is that in the cholinergic basal forebrain prolonged wakefulness induces local energy depletion that generates increases in extracellular adenosine concentrations in this area. In addition to the immediate effects, high extracellular adenosine concentrations also induce intracellular changes in signal transduction and transcription, e.g. increase in A(1) receptor expression and NF-kappaB binding activity. These changes may at least partially mediate the long term effects of prolonged wakefulness. Adenosine may also be a common mediator of the effects of several other sleep-inducing factors.