Tarja Porkka-Heiskanen

Brain site-specificity of extracellular adenosine concentration changes during sleep deprivation and spontaneous sleep: an in vivo microdialysis study

Previous data suggested that increases in extracellular adenosine in the basal forebrain mediated the sleep-inducing effects of prolonged wakefulness. The present study sought to determine if the state-related changes found in basal forebrain adenosine levels occurred uniformly throughout the brain. In vivo microdialysis sample collection coupled to microbore high-performance liquid chromatography measured extracellular adenosine levels in six brain regions of the cat: basal forebrain, cerebral cortex, thalamus, preoptic area of hypothalamus, dorsal raphe nucleus and pedunculopontine tegmental nucleus. In all these brain regions extracellular adenosine levels showed a similar decline of 15 to 20% during episodes of spontaneous sleep relative to wakefulness. Adenosine levels during non-rapid eye movement sleep did not differ from rapid eye movement sleep. In the course of 6h of sleep deprivation, adenosine levels increased significantly in the cholinergic region of the basal forebrain (to 140% of baseline) and, to a lesser extent in the cortex, but not in the other regions. Following sleep deprivation, basal forebrain adenosine levels declined very slowly, remaining significantly elevated throughout a 3-h period of recovery sleep, but elsewhere levels were either similar to, or lower than, baseline. The site-specific accumulation of adenosine during sleep deprivation suggests a differential regulation of adenosine levels by as yet unidentified mechanisms. Moreover, the unique pattern of sleep-related changes in basal forebrain adenosine level lends strong support to the hypothesis that the sleep-promoting effects of adenosine, as well as the sleepiness associated with prolonged wakefulness, are both mediated by adenosinergic inhibition of a cortically projecting basal forebrain arousal system.

Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic neuronal activity in the control of behavioral state.

This review describes a series of animal experiments that investigate the role of endogenous adenosine (AD) in sleep. We propose that AD is a modulator of the sleepiness associated with prolonged wakefulness. More specifically, we suggest that, during prolonged wakefulness, extracellular AD accumulates selectively in the basal forebrain (BF) and cortex and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting cholinergic and non-cholinergic wakefulness-promoting BF neurons at the AD A1 receptor. New in vitro data are also compatible with the hypothesis that, via presynaptic inhibition of GABAergic inhibitory input, AD may disinhibit neurons in the preoptic/anterior hypothalamus (POAH) that have SWS-selective activity and Fos expression. Our in vitro recordings initially showed that endogenous AD suppressed the discharge activity of neurons in the BF cholinergic zone via the AD A1 receptor. Moreover, in identified mesopontine cholinergic neurons, AD was shown to act post-synaptically by hyperpolarizng the membrane via an inwardly rectifying potassium current and inhibition of the hyperpolarization-activated current, I(h). In vivo microdialysis in the cat has shown that AD in the BF cholinergic zone accumulates during prolonged wakefulness, and declines slowly during subsequent sleep, findings confirmed in the rat. Moreover, increasing BF AD concentrations to approximately the level as during sleep deprivation by a nucleoside transport blocker mimicked the effect of sleep deprivation on both the EEG power spectrum and behavioral state distribution: wakefulness was decreased, and there were increases in SWS and REM sleep. As predicted, microdialyis application of the specific A1 receptor antagonist cyclopentyltheophylline (CPT) in the BF produced the opposite effects on behavioral state, increasing wakefulness and decreasing SWS and REM. Combined unit recording and microdialysis studies have shown neurons selectively active in wakefulness, compared with SWS, have discharge activity suppressed by both AD and the A1-specific agonist cyclohexyladenosine (CHA), while discharge activity is increased by the A1 receptor antagonist, CPT. We next addressed the question of whether AD exerts its effects locally or globally. Adenosine accumulation during prolonged wakefulness occurred in the BF and neocortex, although, unlike in the BF, cortical AD levels declined in the 6th h of sleep deprivation and declined further during subsequent recovery sleep. Somewhat to our surprise, AD concentrations did not increase during prolonged wakefulness (6 h) even in regions important in behavioral state control, such as the POAH, dorsal raphe nucleus, and pedunculopontine tegmental nucleus, nor did it increase in the ventrolateral/ventroanterior thalamic nucleii. These data suggest the presence of brain region-specific differences in AD transporters and/or degradation that become evident with prolonged wakefulness, even though AD concentrations are higher in all brain sites sampled during the naturally occurring (and shorter duration) episodes of wakefulness as compared to sleep episodes in the freely moving and behaving cat. Might AD also produce modulation of activity of neurons that have sleep selective transcriptional (Fos) and discharge activity in the preoptic/anterior hypothalamus zone? Whole cell patch clamp recordings in the in vitro horizontal slice showed fast and likely GABAergic inhibitory post-synaptic potentials and currents that were greatly decreased by bath application of AD. Adenosine may thus disinhibit and promote expression of sleep-related neuronal activity in the POAH. In summary, a growing body of evidence supports the role of AD as a mediator of the sleepiness following prolonged wakefulness, a role in which its inhibitory actions on the BF wakefulness-promoting neurons may be especially important.

Sleep restriction increases the risk of developing cardiovascular diseases by augmenting proinflammatory responses through IL-17 and CRP

Background Sleep restriction, leading to deprivation of sleep, is common in modern 24-h societies and is associated with the development of health problems including cardiovascular diseases. Our objective was to investigate the immunological effects of prolonged sleep restriction and subsequent recovery sleep, by simulating a working week and following recovery weekend in a laboratory environment. Methods and Findings After 2 baseline nights of 8 hours time in bed (TIB), 13 healthy young men had only 4 hours TIB per night for 5 nights, followed by 2 recovery nights with 8 hours TIB. 6 control subjects had 8 hours TIB per night throughout the experiment. Heart rate, blood pressure, salivary cortisol and serum C-reactive protein (CRP) were measured after the baseline (BL), sleep restriction (SR) and recovery (REC) period. Peripheral blood mononuclear cells (PBMC) were collected at these time points, counted and stimulated with PHA. Cell proliferation was analyzed by thymidine incorporation and cytokine production by ELISA and RT-PCR. CRP was increased after SR (145% of BL; p<0.05), and continued to increase after REC (231% of BL; p<0.05). Heart rate was increased after REC (108% of BL; p<0.05). The amount of circulating NK-cells decreased (65% of BL; p<0.005) and the amount of B-cells increased (121% of BL; p<0.005) after SR, but these cell numbers recovered almost completely during REC. Proliferation of stimulated PBMC increased after SR (233% of BL; p<0.05), accompanied by increased production of IL-1β (137% of BL; p<0.05), IL-6 (163% of BL; p<0.05) and IL-17 (138% of BL; p<0.05) at mRNA level. After REC, IL-17 was still increased at the protein level (119% of BL; p<0.05). Conclusions 5 nights of sleep restriction increased lymphocyte activation and the production of proinflammatory cytokines including IL-1β IL-6 and IL-17; they remained elevated after 2 nights of recovery sleep, accompanied by increased heart rate and serum CRP, 2 important risk factors for cardiovascular diseases. Therefore, long-term sleep restriction may lead to persistent changes in the immune system and the increased production of IL-17 together with CRP may increase the risk of developing cardiovascular diseases.

Adenosine, energy metabolism and sleep homeostasis

Adenosine is directly linked to the energy metabolism of cells. In the central nervous system (CNS) an increase in neuronal activity enhances energy consumption as well as extracellular adenosine concentrations. In most brain areas high extracellular adenosine concentrations, through A1 adenosine receptors, decrease neuronal activity and thus the need for energy. Adenosine may be a final common pathway for various sleep factors. We have identified a relatively specific area, the basal forebrain (BF), which appears to be central in the regulation/execution of recovery sleep after sleep deprivation (SD), or prolonged wakefulness. Adenosine concentration increases in this area during SD, and this increase induces sleep while prevention of the increase during SD abolishes recovery sleep. The increase in adenosine is associated with local changes in energy metabolism as indicated by increases in levels of pyruvate and lactate and increased phosphorylation of AMP-activated protein kinase. The increases in adenosine and sleep are associated with intact cholinergic system since specific lesion of the BF cholinergic cells abolishes both. Whether adenosine during SD is produced by the cholinergic neurons or astrocytes associated with them remains to be explored. An interesting, but so far unexplored question regards the relationship between the local, cortical regulation of sleep homeostasis and the global regulation of the state of sleep as executed by lower brain mechanisms, including the BF. The increase in adenosine concentration during SD also in cortical areas suggests that adenosine may have a role in the local regulation of sleep homeostasis. The core of sleep need is probably related to primitive functions of life, like energy metabolism. It can be noted that this assumption in no way excludes the possibility that later in evolution additional functions may have developed, e.g., related to complex neuronal network functions like memory and learning.

Short Sleep Duration and Behavioral Symptoms of Attention-Deficit/Hyperactivity Disorder in Healthy 7- to 8-Year-Old Children

OBJECTIVE. It has been hypothesized that sleep deprivation may manifest in children as behavioral symptoms rather than as tiredness, but only a few studies have investigated this hypothesis. The objective of our study was to evaluate whether short sleep is associated with behavioral symptoms of attention-deficit/hyperactivity disorder in 7- to 8-year-old children. METHODS. We performed a cross-sectional study of children born in 1998 in Helsinki, Finland. The participants included 280 (146 girls, 134 boys) children with a mean age of 8.1 years (SD: 0.3; range: 7.4–8.8). Sleep quality was measured by using actigraphs. The Sleep Disturbance Scale for Children and the Attention-Deficit/Hyperactivity Disorder Rating Scale IV were administered to parents. RESULTS. Children whose average sleep duration as measured by actigraphs was short (<10th percentile, ie, <7.7 hours) and had a higher hyperactivity/impulsivity score (9.7 vs 7.8 or 7.5) and a higher attention-deficit/hyperactivity disorder total score (17.3 vs 14.5 or 13.1) but a similar inattention score (7.6 vs 6.7 or 5.6) compared with children sleeping 7.7 to 9.4 hours or >9.4 hours. In multivariate statistical models, short sleep duration remained a statistically significant predictor of hyperactivity/impulsivity, and sleeping difficulties were associated with hyperactivity/impulsivity, inattention, and the total score. There were no significant interactions between short sleep and sleeping difficulties. CONCLUSIONS. Childrens short sleep duration and sleeping difficulties increase the risk for behavioral symptoms of attention-deficit/hyperactivity disorder.

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.

Adenosine in sleep and wakefulness

Sleep propensity increases in the course of wakefulness: the longer the previous wakefulness period is, the longer and deeper (measured as delta power in EEG recordings) is the following sleep. The mechanisms that regulate the need of sleep at the cellular level are largely unknown. The inhibitory neuromodulator, adenosine, is a promising candidate for a sleep-inducing factor: its concentration is higher during wakefulness than during sleep, it accumulates in the brain during prolonged wakefulness, and local perfusions as well as systemic administration of adenosine and its agonists induce sleep and decrease wakefulness. Adenosine receptor antagonists, caffeine and theophylline, are widely used as stimulants of the central nervous system to induce vigilance and increase the time spent awake. Our hypothesis is that adenosine accumulates in the extracellular space of the basal forebrain during wakefulness, increasing the sleep propensity. The increase in extracellular adenosine concentration decreases the activity of the wakefulness-promoting cell groups, especially the cholinergic cells in the basal forebrain. When the activity of the wakefulness-active cells decreases sufficiently sleep is initiated. During sleep the extracellular adenosine concentrations decrease, and thus the inhibition of the wakefulness-active cells also decreases allowing the initiation of a new wakefulness period.

Adenosine and behavioral state control: adenosine increases c-Fos protein and AP1 binding in basal forebrain of rats

In several brain areas, extracellular adenosine (AD) levels are higher during waking than sleep and during prolonged wakefulness AD levels in the basal forebrain increase progressively. Similarly, c-Fos levels in several brain areas are higher during waking than sleep and remain elevated during prolonged wakefulness. In the present study, we investigated the effect of extracellular AD levels on c-Fos protein and activator protein-1 (AP1) binding in the basal forebrain of rats. Increased levels of extracellular AD were induced either by keeping the animals awake, or by local perfusion of AD into the basal forebrain. During prolonged wakefulness extracellular AD concentration was monitored using in vivo microdialysis. The effect of AD perfusion on the behavioral states was recorded using polysomnography. At the end of the perfusion period the basal forebrain tissue was analyzed for the levels of c-Fos protein and AP1 binding. In vivo microdialysis measurements showed an increase in AD levels with prolonged wakefulness. Unilateral perfusion of AD (300 microM) increased non-REM sleep and delta power (0.5 to 4 Hz) when compared to rats perfused with artificial CSF. The levels of c-Fos protein and the AP1 DNA binding were high in the basal forebrain of both sleep-deprived animals and in animals perfused with AD. The results suggest that AD might mediate, at least in part, the long term effects of sleep deprivation by inducing c-Fos protein and subsequent AP1 binding.

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.

Opposite changes in adenosine A1 and A2A receptor mRNA in the rat following sleep deprivation.

Extracellular levels of adenosine increase in basal forebrain following prolonged wakefulness. Moreover, perfusion of adenosine into basal forebrain increases sleep. In this study we have examined the adenosine receptor subtypes, A1 and A2A, for changes in the levels of mRNA using RT-PCR and in situ hybridization and the receptor ligand binding efficiency using autoradiography following 3 and 6 h of sleep deprivation. We observed that A1 receptor mRNA levels increased in basal forebrain with no changes in other forebrain areas examined. A1 receptor binding was not affected. A2A receptor mRNA and ligand binding were undetectable in basal forebrain. However, in the olfactory tubercle, A2A mRNA and receptor binding decreased significantly. Based on the significant increase in the A1 but not in A2A receptor, we hypothesize that the effects of sleep deprivation-induced increased adenosine are mediated by A1 receptor in basal forebrain of rats.