Dag Stenberg

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 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.

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.

Visual and spectral EEG analysis in the evaluation of the outcome in patients with ischemic brain infarction

Serial EEGs were recorded in 15 patients with acute cerebral infarctions in order to study clinical and prognostic correlations. The EEG was recorded within 48 h from the first symptoms and thereafter weekly for 4 weeks. The EEGs were analyzed both visually and with a computerized spectral analysis. Eight of the patients recovered fully and seven had permanent neurological deficits. On admission, 87% of the patients had an abnormal EEG by visual analysis. The spectral parameters correlated well with visual findings, especially the delta and alpha bands. The spectral analysis was superior to visual in predicting the correct laterality of the lesion. It showed the correct side of the lesion in 87%, while the visual did it in only 54% of the cases. The first EEG records reliably predicted the outcome of the patients. The degree of background abnormality was most important in visual EEG analysis. In spectral analysis, parameters from single derivations were superior to the average of all derivations. A high proportion of delta or low proportion of alpha power were reliable indicators of poor outcome.

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, Nω‐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.

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.

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.

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.

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

Abstract: 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.