The role of cholinergic basal forebrain neurons in adenosine-mediated homeostatic control of sleep: lessons from 192 IgG-saporin lesions.

A topic of high current interest and controversy is the basis of the homeostatic sleep response, the increase in non-rapid-eye-movement (NREM) sleep and NREM-delta activity following sleep deprivation (SD). Adenosine, which accumulates in the cholinergic basal forebrain (BF) during SD, has been proposed as one of the important homeostatic sleep factors. It is suggested that sleep-inducing effects of adenosine are mediated by inhibiting the wake-active neurons of the BF, including cholinergic neurons. Here we examined the association between SD-induced adenosine release, the homeostatic sleep response and the survival of cholinergic neurons in the BF after injections of the immunotoxin 192 immunoglobulin G (IgG)-saporin (saporin) in rats. We correlated SD-induced adenosine level in the BF and the homeostatic sleep response with the cholinergic cell loss 2 weeks after local saporin injections into the BF, as well as 2 and 3 weeks after i.c.v. saporin injections. Two weeks after local saporin injection there was an 88% cholinergic cell loss, coupled with nearly complete abolition of the SD-induced adenosine increase in the BF, the homeostatic sleep response, and the sleep-inducing effects of BF adenosine infusion. Two weeks after i.c.v. saporin injection there was a 59% cholinergic cell loss, correlated with significant increase in SD-induced adenosine level in the BF and an intact sleep response. Three weeks after i.c.v. saporin injection there was an 87% cholinergic cell loss, nearly complete abolition of the SD-induced adenosine increase in the BF and the homeostatic response, implying that the time course of i.c.v. saporin lesions is a key variable in interpreting experimental results. Taken together, these results strongly suggest that cholinergic neurons in the BF are important for the SD-induced increase in adenosine as well as for its sleep-inducing effects and play a major, although not exclusive, role in sleep homeostasis.

Glutamatergic stimulation of the basal forebrain elevates extracellular adenosine and increases the subsequent sleep.

A prolonged period of waking accumulates sleep pressure, increasing both the duration and the intensity of the subsequent sleep period. Delta power, which is calculated from the slow range electroencephalographic (EEG) oscillations (0.1-4 Hz), is regarded as the marker of sleep intensity. Recent findings indicate that not only the duration but also the quality of waking, determines the level of increase in the delta activity during the subsequent sleep period. Elevated levels of extracellular adenosine in the basal forebrain (BF) during prolonged waking have been proposed to act as the molecular signal of increased sleep pressure, but the role of BF neuronal activity in elevating adenosine has not been previously explored. We hypothesized that an increase in neuronal discharge in the BF would lead to increase in the extracellular adenosine and contribute to the increase in the subsequent sleep. To experimentally increase neuronal activity in the rat BF, we used 3 h in vivo microdialysis application of glutamate or its receptor agonists N-methyl-D-aspartate (NMDA) or AMPA. Samples for adenosine measurement were collected during the drug application and the EEG was recorded during and after the treatment, altogether for 24 h. All treatments increased the duration of the subsequent sleep following the application. In contrast, delta power was elevated only if both the waking EEG theta (5-9 Hz) power (which can be regarded as a marker of active waking) and the extracellular adenosine in the BF were increased during the application. These results indicate that increased neuronal activity in the BF, and particularly the type of neuronal activity coinciding with active waking, is one of the factors contributing to the buildup of the sleep pressure.

Neuroanatomy and neurochemistry of sleep.

Sleep is regulated by homeostatic and circadian factors, and the regulation of sleep of mammals shares many molecular properties with the rest state of submammalian species. Several brain structures take part in waking: the basal forebrain, posterior and lateral hypothalamus, and nuclei in the tegmentum and pons. Active sleep mechanisms are located to the preoptic/anterior hypothalamic area. In addition to acetylcholine and monoamines, glutamate and hypocretin/orexin are important waking factors. Gamma-aminobutyric acid and several peptide factors, including cytokines, growth hormone-releasing hormone and prolactin, are related to sleep promotion. Adenosine is an important homeostatic sleep factor acting in basal forebrain and preoptic areas through A1 and A2A receptors. Prolonged waking activates inducible nitric oxide synthase in the basal forebrain, which through energy depletion causes adenosine release and recovery sleep. Numerous genes have been found differentially displayed in waking compared with sleep, and they relate to neural transmission, synaptic plasticity, energy metabolism and stress protection. The genetic background of a few sleep disorders has been solved.

Nitric oxide production in the basal forebrain is required for recovery sleep.

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. Here, we assessed the role of the intercellular gaseous signaling agent NO in sleep homeostasis. We measured the concentration of nitrite and nitrate, indicative of NO production, in the basal forebrain (BF) of rats during sleep deprivation (SD), and found the level increased by 100 +/- 51%. To test whether an increase in NO production might play a causal role in recovery sleep, we administered compounds into the BF that increase or decrease concentrations of NO. Infusion of either a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or a NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), completely abolished non-rapid eye movement (NREM) recovery sleep. Infusion of a NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate (DETA/NO), produced an increase in NREM that closely resembled NREM recovery after prolonged wakefulness. The effects of inhibition of NO synthesis and the pharmacological induction of sleep were effective only in the BF area. Indicators of energy metabolism, adenosine, lactate and pyruvate increased during prolonged wakefulness and DETA/NO infusion, whereas L-NAME infusion during SD prevented the increases. We conclude that an increase in NO production in the BF is a causal event in the induction of recovery sleep.

Inducible and neuronal nitric oxide synthases (NOS) have complementary roles in recovery sleep induction.

Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. We have previously shown that nitric oxide (NO) generation increases in the basal forebrain (BF) during sleep deprivation (SD). Moreover, both NO synthase (NOS) inhibition and a NO scavenger prevented recovery sleep induction, while administration of a NO donor during the spontaneous sleep-wake cycle increased sleep, indicating that NO is necessary and sufficient for the induction of recovery sleep. Next we wanted to know which NOS isoform is involved in the production of recovery sleep. Using in vivo microdialysis we infused specific inhibitors of NOS into the BF of rats during SD, and found that an inhibitor of inducible NOS (iNOS), 1400W, prevented non-rapid eye movement (NREM) recovery, while an inhibitor of neuronal NOS (nNOS), L-N-propyl-arginine, decreased REM recovery but did not affect NREM recovery. Using immunoblot analysis we found that iNOS was not expressed during the spontaneous sleep-wake cycle, but was induced by prolonged wakefulness (increased by 278%). A known iNOS inducer, lipopolysaccharide, evoked an increase in sleep that closely resembled recovery sleep, and its effects were abolished by 1400W. These results suggest that the elevation of NO produced by induction of iNOS in the BF during prolonged wakefulness is a specific mechanism for producing NREM recovery sleep and that the two NOS isoforms have a complementary role in NREM and REM recovery induction.

Orexin A and B levels in the hypothalamus of female rats: the effects of the estrous cycle and age.

OBJECTIVE: Orexins have been implicated in the regulation of several physiological functions including reproduction, energy balance and vigilance state. For successful reproduction, the precisely timed hormonal secretions of the estrous cycle must be combined with appropriate nutritional and vigilance states. The steroid- and nutritional state-dependent modulation of LH release by orexins, as well as an increase of vigilance, suggest that orexins may co-ordinate these functions in the course of the estrous cycle. DESIGN: We studied the brain tissue levels of orexins in the course of the estrous cycle in young and middle-aged rats. Young cycling rats (3 months old) and irregularly/non-cycling (7-9 months old) female rats were inspected for vaginal smears and serum hormone levels. METHODS: Tissue concentrations of orexin A and B were measured in the hypothalamus and lateral hypothalamus on different days of the estrous cycle. RESULTS: Orexin A concentration in the hypothalamus of young cycling rats was higher on the day of proestrus 5-6 h after the lights were switched on than on the other days of the estrous cycle at the same circadian time. Orexin B concentration was higher on both the day of proestrus and the day of estrus as compared with the days of diestrus. The hypothalamic concentrations of both orexin A and B in the non-cycling middle-aged rats were lower than those in cycling rats on the days of proestrus and estrus. CONCLUSIONS: We have concluded that the high hypothalamic concentration of orexins on the day of proestrus may contribute to the LH and prolactin surges. High orexin A levels may also contribute to the decreased amount of sleep on the day of proestrus.

Intracerebroventricular and locus coeruleus microinjections of somatostatin antagonist decrease REM sleep in rats.

In order to study the role of endogenous somatostatin in the physiologic modulation of REM sleep (REMS), we measured the effect of intracerebroventricular (ICV) injection of somatostatin antagonist (SA) cyclo-(7-aminoheptanoyl-phe-d-trp-lys-thr(bzl)) on sleep in rats. The effect of ICV SA was also tested after 24-h REMS deprivation with the platform method. To study the role of locus coeruleus (LC) as a site of the sleep inducing action for somatostatin and galanin we microinjected SA, somatostatin, and galanin locally into LC. In all experiments, vigilance state was analyzed visually from 6 h post-injection EEG/EMG recording. Injection of 0.5 and 2 nmol of SA ICV reduced spontaneous REMS and 2 nmol dose reduced also rebound REMS after REMS deprivation when compared with controls (artificial cerebrospinal fluid vehicle). Microinjection of 0.25 nmol of SA into LC reduced REMS, whereas microinjection of somatostatin, galanin, and a combined injection of them were not effective to induce REMS. The results suggest that endogenous somatostatin may contribute to facilitation of REMS. Somatostatin receptors in the LC may be one possible mediator of this effect.

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.

Sleep deprivation increases somatostatin and growth hormone-releasing hormone messenger RNA in the rat hypothalamus.

We studied the effect of sleep deprivation (SD) on the amount of somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) mRNA in rat hypothalamic nuclei. According to earlier studies SRIF possibly facilitates REM sleep and GHRH slow-wave sleep. Adult male rats were sleep deprived by the gentle handling method either for 6 h during the first half of the light phase or for 12 h during the dark phase. Undisturbed rats sacrificed at the same time as the SD rats served as controls. After oligonucleotide in situ hybridization the amount of SRIF and GHRH mRNA was measured in brain sections by image analysis and cell count. SD increased the amount of SRIF mRNA in the arcuate nucleus (ARC). In the periventricular nucleus (PE) there was no effect. The amount of GHRH mRNA increased in the paraventricular nucleus (PA) in the 6 h SD group but no effect was detected in ARC. In the periventromedial hypothalamic area (pVMH) the amount of GHRH mRNA was higher in the control rats sacrificed in the morning (09.00 hours) than in the afternoon (15.00 hours), and SD had no effect. We conclude that SRIF cells in ARC and GHRH cells in PA are modulated by sleep loss, which is in accordance with the possible sleep regulatory function of these neuropeptides.

Sleep deprivation increases brain serotonin turnover in the rat.

IN order to study possible time-dependent changes in serotonin metabolism in rat brain, male Wistar rats were subjected to 3, 6 or 12 h total sleep deprivation (SD) by gentle handling. In addition two groups of rats subjected first to 6 h SD were allowed 2 or 4 h rebound sleep. Tissue concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured from several brain areas using HPLC/ECD. SD significantly increased the 5-HIAA/5-HT ratio in frontal cortex, hippocampus, hypothalamus and brain stem, indicating increased 5-HT turnover in those areas. After 2 and 4 h rebound sleep, the 5-HIAA/5-HT ratio was similar to that in controls. We conclude that a short SD increases 5-HT turnover in the rat brain for the duration of SD only.

The effect of REM sleep deprivation on somatostatin and growth hormone-releasing hormone gene expression in the rat hypothalamus.

Growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) have been implicated as sleep factors. We studied how the hypothalamic SRIF/GHRH system is affected by possible feedback regulation resulting from REM sleep deprivation at the level of gene expression and how this is reflected in serum growth hormone (GH) content. Male rats were deprived of REM sleep on small platforms for 24 or 72 h, and one group was allowed a rebound sleep of 24 h after 72 h deprivation. Animals maintained on large platforms and animals taken directly from their home cages served as controls. In situ hybridization was made from 20 microm cryosections through the periventricular, paraventricular and arcuate hypothalamic nuclei using oligonucleotide probes for GHRH and SRIF. The number of cells expressing SRIF or GHRH was counted. Serum GH was measured by means of radioimmunoassay in similarly treated rats. Fewer cells expressed GHRH in the paraventricular nucleus of animals subjected to 24 and 72 h of REM sleep deprivation than in home control animals. A similar trend was observed in the arcuate nucleus. The number of cells expressing SRIF was elevated in the arcuate nucleus after 24 h of REM sleep deprivation but not after 72 h. In the periventricular nucleus the number of cells expressing SRIF was higher after 72 h of deprivation when compared to expression in animals maintained on large platforms. Serum GH levels were decreased in animals maintained on either small or large platforms. It is concluded that the expression of the SRIF and GHRH genes is modulated by REM sleep deprivation.

Sleep deprivation increases brain serotonin turnover in the Djungarian hamster.

Djungarian hamsters well adapted to a short photoperiod were subjected to 4 h of total sleep deprivation (SD) by gentle handling. Tissue concentrations of monoamines and of their metabolites were measured from several brain areas using HPLC with electrochemical detection. The 5-hydroxyindoleacetic acid/5-hydroxytryptamine (5-HIAA/5-HT) ratio was significantly increased after SD in the hippocampus, hypothalamus and brain stem, indicating increased serotonin (5-HT) turnover in those areas, while no changes were found in the frontal cortex and olfactory bulb. Dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations were elevated in the hypothalamus, while the noradrenaline concentrations did not change in any of the measured areas. We conclude that a short SD, which has been shown to elevate EEG slow-wave activity during recovery sleep, specifically increases 5-HT turnover in the brain.

Noradrenergic activity in rat brain during rapid eye movement sleep deprivation and rebound sleep.

Noradrenergic locus ceruleus neurons are most active during waking and least active during rapid eye movement (REM) sleep. We expected REM sleep deprivation (REMSD) to increase norepinephrine utilization and activate the tyrosine hydroxylase (TH) gene critical for norepinephrine production. Male Wistar rats were deprived of REM sleep with the platform method. Rats were decapitated after 8, 24, or 72 h on small (REMSD) or large (control) platforms or after 8 or 24 h of rebound sleep after 72 h of the platform treatment. During the first 24 h, norepinephrine concentration, measured by high-performance liquid chromatography/electrochemical detection, was lower in the neocortex, hippocampus, and posterior hypothalamus in REMSD rats than in large-platform controls. After 72 h of REMSD, TH mRNA, measured by in situ hybridization, was increased in the locus ceruleus and norepinephrine concentrations were increased. Polygraphy showed that small-platform treatment caused effective and selective REMSD. Serum corticosterone measurement by radioimmunoassay indicated that the differences found in norepinephrine and TH mRNA were not due to differences in stress between the treatments. The novel finding of sleep deprivation-specific increase in TH gene expression indicates an important mechanism of adjusting to sleep deprivation.

REM sleep deprivation induces galanin gene expression in the rat brain.

Rats were deprived of REM sleep for 24 h by keeping them on small platforms that were placed in a water bath (the platform method). Galanin coding mRNA was visualized using in situ hybridization, and cells expressing galanin mRNA were counted. In REM sleep-deprived animals the cell count was higher in the preoptic area and periventricular nucleus. Lesions of this area have been reported to induce wakefulness in cats and rats. Galanin administered into the lateral ventricle had no effect on sleep. We conclude that REM sleep deprivation can induce galanin gene expression in some brain areas, but galanin alone does not modify spontaneous sleep.

The effect of REM sleep deprivation on histamine concentrations in different brain areas.

Rats were deprived of REM sleep (REMS) for 72 h with the platform method and decapitated in the morning immediately after the deprivation or in the afternoon after having been allowed 5 hours of rebound sleep. The histamine concentrations of the anterior and posterior hypothalamus, the cortex, the hippocampus and the pineal gland were measured, as well as the tele-methylhistamine concentrations of the anterior and posterior hypothalamus. Histamine concentrations were no different after REMS deprivation compared to large platform or dry cage controls, but in the anterior hypothalamus histamine levels increased during rebound sleep only in the REMS deprived rats. tele-Methylhistamine/histamine ratios were higher after 72 h of both REMS deprivation and the large platform treatment compared to dry cage controls, indicating increased histamine utilization during the platform treatment procedure.

Twenty-four-hour rhythms in relation to the natural photoperiod: a field study in humans.

The daily rhythms of salivary melatonin, salivary cortisol, and axillary body temperature were measured in nine healthy volunteers in midsummer, around the autumn equinox, and in midwinter, at a latitude of 60 degrees N. The aim was to find out whether these rhythms were dependent on variations of the natural daylength. The samples were collected every 2 hr during 24-hr periods in everyday conditions. The individual rhythms were characterized with the acrophase estimates of the best-fitting cosine curve models and with the half-rise and half-decline times calculated from the raw data. The melatonin and cortisol rhythms were delayed significantly (about 1 hr) in midwinter as compared with summer and autumn. The most advanced rhythms were found in autumn. The shifts of the melatonin and cortisol rhythms could be explained as a result of the changes of natural illumination. The overt temperature rhythms did not differ significantly among the sampling months. The lack of seasonal patterns in temperature rhythms probably primarily reflected the socially determined rest-activity cycles of the subjects.

One-hour exposure to moderate illuminance (500 lux) shifts the human melatonin rhythm.

Salivary melatonin levels were measured in 12 healthy volunteers in order to determine whether a moderate light intensity, which suppresses the nocturnal rise of melatonin, was able to shift the melatonin rhythm. The samples were collected at 1-hr intervals under lighting of < 100 lux (experiment 1) or < 10 lux (experiment 2). The control melatonin profiles were determined during the first night. In the second night the subjects were exposed to light of 500 lux for 60 min during the rising phase of melatonin synthesis. The third series of samples was collected during the third night. The mean decrease of melatonin levels by the exposure to light was 56% of the prelight concentrations. The melatonin onset times were delayed significantly (about 30 min) the night after the exposure to light. The melatonin offset times tended to be delayed in experiment 2. The shifts of the melatonin offset correlated positively with the amount of the melatonin suppression. The results suggest that a relatively small and short lasting light-induced interruption of melatonin synthesis may affect the melatonin rhythm in humans.

Alpha 2-adrenoceptors and vigilance in cats: antagonism of medetomidine sedation by atipamezole.

In order to evaluate the effect of a specific alpha 2-adrenoceptor antagonist, atipamezole, on vigilance, adult cats with implanted electrodes for polygraphy were tested in a double-blind Latin square design. The standard clinical dose (0.1 mg/kg i.m.) of the specific alpha 2-adrenoceptor agonist, medetomidine, promptly induced stuporous sedation. Atipamezole, given 30 min later at 0.2, 0.4 or 0.8 mg/kg i.m., reversed the sedation within 3 min, resulting in complete awareness of the animal. After the small dose of atipamezole, arousal with some motor excitation continued for 6 h, whereas after the larger doses, the physiological sleep-wake cycle returned earlier. Used alone, the preferred dose, 0.4 mg/kg atipamezole i.m., allowed physiological sleep within 33 +/- 9 min, compared to 22 +/- 3 min after saline. Atipamezole thus proved to be a most effective antagonist to sedation with alpha 2-adrenoceptor agonist drugs, without disturbing excitatory effects. Specific alpha 2-adrenoceptor modulating drugs have evident clinical application, as antidotes to overdosage of alpha 2-adrenoceptor agonists, or to terminate their effect after surgical procedures.

Pineal melatonin and locomotor activity of rats under gradual illuminance transitions.

The locomotor activity and pineal melatonin patterns of adult male rats were compared under two different lighting regimes. The animals were kept 8 days under 12/12 h light/dark cycles with abrupt or slowly decreasing and increasing transitions (twilight periods about 2 h). The onsets of high activity and melatonin rise were phase-locked in the two conditions and related to about half-maximal illuminance level of the gradual dusk. The high activity of the control rats stopped 30-60 min before the abrupt light onset and the rats under the gradual lighting transitions ceased the locomotor activity at about 1 hour before the half-maximal illuminance. The melatonin peak levels were found 4 h before the abrupt lights-on time. Under the slow illuminance transitions the average melatonin peak was related to the illuminance level between maximum and minimum in the morning. Thus, both the melatonin rhythm and the rest-activity rhythm under the gradual dawn and dusk were adjusted according to about half-maximal illuminances in the present conditions.