The cellular substrates of sleep oscillations have recently been investigated by means of multi-site, intracellular and extracellular recordings under anesthesia, and these data have been validated during natural sleep in cats and humans. Although various rhythms occurring during the state of resting sleep (spindle, 7-14 Hz; delta, 1-4 Hz; and slow oscillation, <1 Hz) are conventionally described by using their different frequencies, they are coalesced within complex wave-sequences due to the synchronizing power of the cortically generated slow oscillation (main peak around 0.7 Hz). In intracellular recordings from anesthetized animals, the slow oscillation is characterized by a biphasic sequence consisting of a prolonged hyperpolarization and depolarization. Basically similar patterns are observed by means of extracellular discharges and/or field potentials in naturally sleeping animals and humans. The depolarizing component of the slow oscillation is transferred to the thalamus where it contributes to the synchronization of spindles over widespread territories. The association between the depolarizing component of the slow oscillation and the subsequent sequence of spindle waves forms what is termed the K-complex. The slow oscillation also groups cortically generated delta waves. At variance with previous assumptions that the brain lies for the most part in the dark and a global inhibition occurs in resting sleep, cortical cells are quite active in this behavioral state. This unexpectedly rich activity raises the possibility that, during sleep, the brain is occupied to specify/reorganize circuits and to consolidate memory traces acquired during wakefulness.

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Cellular responses to many extracellular signals occur through phosphorylation or dephosphorylation of intracellular proteins. To determine whether changes in protein phosphorylation accompany the electrophysiological changes occurring during the sleep-waking cycle, immunocytochemical mapping of cells labeled with anti-phosphoserine and anti-phosphothreonine antibodies was performed on brain sections of sleeping and waking rats. Animals implanted for chronic polysomnographic recordings were sacrificed after either 3h of sleep or 3h of sleep deprivation by gentle handling. Anti-phosphoserine and anti-phosphothreonine staining was mainly localized in neurons and was high in some brain regions, such as cerebral cortex and hypothalamus, and low in others, such as the thalamus. In all cases, the number of cells labeled with either antibody in the cerebral cortex was markedly higher in rats sacrificed after 3h of waking than in rats sacrificed after 3h of sleep. Unilateral lesions of the locus coeruleus by local injection of 6-hydroxydopamine were performed in other animals to determine whether the increase in protein phosphorylation during waking was influenced by the activity of the noradrenergic system, which is higher in waking than in sleep. In animals sacrificed after 3h of spontaneous or forced waking, the number of labeled neurons in the cerebral cortex was decreased on the side in which noradrenergic fibers had been lesioned. These results suggest that 1) neurons exist physiologically in different states of phosphorylation, ranging from a state of very high phosphorylation (e.g., in the cerebral cortex) to a state of very low phosphorylation (e.g., in many thalamic nuclei); 2) the fraction of highly phosphorylated neurons in cerebral cortex is higher in waking than in sleep and 3) part of the immunoreactive phosphorylation present in highly labeled cortical neurons is controlled by the locus coeruleus.

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The effects of both REM sleep deprivation and its recovery on pontine and hippocampus muscarinic M₂ receptors were investigated in synaptosomes using [³H]-AF-DX 384 as a ligand. Animals were divided into three groups: REM sleep deprivation group (small platforms 6.5 cm of diameter); stress group (large platforms 14 cm of diameter) and cage control group. In a second experiment REM sleep-deprived animals were allowed 48 h of recovery. REM sleep-deprived rats showed a reduction in M₂ receptors compared with both intact and stress groups. Changes in M₂ receptors were also observed after 48 h of recovery from REM sleep deprivation only in hippocampus. The enhancement of acetylcholine release during both REM sleep deprivation and recovery could explain the present findings.

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Muscular pharyngeal structural changes, as fibre type disproportion, have been described in patients affected by Obstructive Sleep Apnea (OSA) and in an animal experimental OSA model. The unsolved question is whether these muscular abnormalities are either secondary to a compensatory increased activity or due to a constitutionally determined reduction of slow-alpha motor neurons. In the present study Medium Pharyngeal Constrictor Muscles (MPCM) of OSA (n = 13) and non-OSA (n = 9) patients have been morphologically evaluated. In addition a needle biopsy of Vastus Lateralis Muscle (VLM) was performed in 5 randomly selected patients of each group. Our results confirmed a specific fibre type disproportion of MPCM of OSA patients compared to non-OSA ones with a type II predominance and aspecific myopathic changes such as fibrosis and central nuclei. No difference was found in the VLM of the two groups. This finding could be explained by a secondary adaptive transformation consequent to nocturnal upper airway resistance in OSA. In fact, it has been demonstrated in human muscle that heavy-resistance training may produce preferential type II fibre hypertrophy in stimulated muscle.

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The interaction of cholinergic and catecholaminergic mechanisms in the mesopontine region has been hypothesized as being critical for the generation and maintenance of active (REM) sleep. To further examine this hypothesis, we sought to determine the pattern of neuronal activation (via c-fos expression) of catecholaminergic and cholinergic neurons in this region during active sleep induced by the pontine microapplication of carbachol (designated as active sleep-carbachol). Accordingly, we used two sets of double-labeling techniques; the first to identify tyrosine hydroxylase-containing neurons (putative catecholaminergic cells) which also express the c-fos protein product Fos, and the second to reveal choline acetyltransferase-containing neurons (putative cholinergic cells) which also express Fos. Compared to control cats, active sleep-carbachol cats exhibited a significantly greater number of Fos-expressing neurons in the dorsolateral region of the pons, which encompasses the locus coeruleus, the lateral pontine reticular formation, the peribrachial nuclei and the latero-dorsal and pedunculo-pontine tegmental nuclei. However, both control and active sleep-carbachol cats exhibited a similar number of catecholaminergic and cholinergic neurons in those regions that expressed Fos (i.e., double-labeled cells). A large number of c-fos-expressing neurons in the active sleep-carbachol cats whose neurotransmitter phenotype was not identified suggests that non-catecholaminergic, non-cholinergic neuronal populations in mesopontine regions are involved in the generation and maintenance of active sleep. The lack of increased c-fos expression in catecholaminergic neurons during active sleep-carbachol confirms and extends previous data that indicate that these cells are silent during active sleep-carbachol and naturally-occurring active sleep. The finding that cholinergic neurons of the dorsolateral pons were not activated either during wakefulness or active sleep-carbachol raises questions regarding the synaptic mechanisms of activation of these cells during these behavioral states.

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Dorsal mesopontine cholinergic neurons control rapid eye movement sleep (REMS) and wakefulness and contain nitric oxide (NO) synthase. To assess whether local inhibition of NO synthase has distinct effects on sleep, N-nitro-L-arginine methyl ester, an NO synthesis inhibitor (L-NAME, 80 mM), carbachol, a cholinergic agonist (2, 10 or 50 mM), or saline were microinjected (120-200 nl) into the dorsal mesopontine tegmentum in rats. Sleep-wake cycles were monitored during the subsequent 6 h periods. Compared to control injections, L-NAME changed the pattern of REMS by prolonging individual episodes with a small increase in the percentage time of REMS and no change in slow wave sleep (SWS). Carbachol, at 50 mM, enhanced wakefulness and suppressed both SWS and REMS, especially during the first 2 h post-injection. At the two lower concentrations, carbachol moderately enhanced REMS 2-6 h post-injection by increasing the frequency, rather than duration, of individual episodes. Thus, a reduced NO release in the dorsal pontine tegmentum has a powerful consolidating effect on REMS episodes, whereas the direction of the effect of carbachol on the amount of sleep, and REMS in particular, depends on the magnitude of cholinergic stimulation. The REMS-consolidating effects of NO synthase inhibition in the pons may result from modulatory effects of NO on the release of acetylcholine and other neurotransmitters within the dorsal mesopontine tegmentum.

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Amphetamine-like stimulants are commonly used to treat sleepiness in narcolepsy. These compounds have little effect on rapid eye movement (REM) sleep-related symptoms such as cataplexy, and antidepressants (monoamine uptake inhibitors) are usually required to treat these symptoms. Although amphetamine-like stimulants and antidepressants enhance monoaminergic transmission, these compounds are non-selective for each monoamine, and the exact mechanisms mediating how these compounds induce wakefulness and modulate REM sleep is not known. In order to evaluate the relative importance of dopaminergic and noradrenergic transmission in the mediation of these effects, five dopamine (DA) uptake inhibitors (mazindol, GBR-12909, bupropion, nomifensine and amineptine), two norepinephrine (NE) uptake inhibitors (nisoxetine and desipramine), d-amphetamine, and modafinil, a non-amphetamine stimulant, were tested in control and narcoleptic canines. All stimulants and dopaminergic uptake inhibitors were found to dose-dependently increase wakefulness in control and narcoleptic animals. The in vivo potencies of DA uptake inhibitors and modafinil on wake significantly correlated with their in vitro affinities to the DA and not the NE transporter. DA uptake inhibitors also moderately reduced REM sleep, but this effect was most likely secondary to slow wave sleep (SWS) suppression, since selective DA uptake inhibitors reduced both REM sleep and SWS proportionally. In contrast, selective NE uptake inhibitors had little effect on wakefulness, but potently reduced REM sleep. These results suggest that presynaptic activation of DA transmission is critical for the pharmacological control of wakefulness, while that of the NE system is critical for that of REM sleep regulation. Our results also suggest that presynaptic activation of DA transmission is a key pharmacological property mediating the wake-promoting effects of currently available CNS stimulants.

We previously reported that the P1 or P50 midlatency evoked potential underwent decreased habituation or disinhibition in patients with Parkinson's Disease. This sleep state-dependent response appears to be generated by cholinergic elements of the reticular activating system. We attempted to determine if the decreased habituation or disinhibition of the P1 potential would be altered by bilateral pallidotomy. Twenty-three patients who met inclusion criteria for surgery underwent pre- and post-operative evaluation using a Modified United Parkinson's Disease Rating Scale (UPDRS) and P1 potential recordings. Decreased habituation of the P1 potential was determined using a paired stimulus paradigm in which click stimuli were presented at 250, 500 and 1000 msec interstimulus intervals (ISI). Pre-operatively, patients showed disinhibition of the P1 potential at the 250 msec ISI (60 ± 37% vs 21 ± 20%) and 500 msec ISI (78 ± 47% vs 43 ± 31%) compared to age-matched control subjects. Post-operatively, the same patients showed a significant improvement in habituation of the P1 potential at the same ISIs (250 msec 37 ± 21%; 500 msec 43 ± 32%). UPDRS scores for these patients pre-operatively were 59 ± 18 and 24 ± 11 post-operatively, resulting in a significant reduction in symptom severity. We conclude that bilateral pallidotomy resulted in a significant improvement in symptom ratings and reduced the disinhibition of the P1 midlatency evoked response.

Because Obstructive Sleep Apnea Syndrome (OSAS) patients may be treated for comorbidities prior to OSAS diagnosis, we examined the health care utilization records of 181 OSAS patients and those of matched controls. We compared OSA patient health care utilization for a ten-year interval prior to diagnosis to those of randomized age-, gender-, and geographically-matched controls from the general population. We found that OSAS patients used approximately twice the resources (as defined by physician claims and stays in hospital) in the ten years prior to their diagnosis. Physician claims for cases totaled $686,365 ($3,972 per patient) compared to $356,376 ($1,969 per patient) for the controls for the length of the study. Utilization was significantly higher in 7 of 10 years prior to diagnosis. OSAS patients also had more hospitalizations: they had 1,118 nights (6.2 per patient) in hospital versus 676 nights (3.7 per patient) for controls over the ten-year period. Thus OSA patients are heavy users of health care resources ten years prior to diagnosis.

Our aim was to determine the effects of intrauterine compromise, induced by maternal anemia, on ventilatory responsiveness of the sleeping newborn to progressive asphyxia. We induced anemia in 6 sheep for the final third of pregnancy and studied their offspring for 2-3 weeks after birth. Lambs from anemic ewes were growth-restricted at birth; they and 6 control lambs were chronically instrumented soon after birth and underwent studies during which we determined ventilatory and arousal responsiveness to a progressive asphyxic stimulus during sleep. During quiet wakefulness, active sleep and quiet sleep, lambs from anemic ewes had elevated end-tidal CO₂ levels (FECO₂,%) compared to controls. Ventilatory responsiveness (i.e., gradient of relationship between minute ventilation and FECO₂) was greater in quiet sleep than in active sleep for both groups of lambs but did not differ between the two groups in either active or quiet sleep. Lambs from anemic ewes had significantly higher FECO₂ values than controls before arousing from either active or quiet sleep. Other indices of arousability (time to arousal, percent hemoglobin saturation at arousal) were not different between the two groups. Our results indicate that prenatal exposure to maternal anemia induces fetal growth restriction and elevates the CO₂ 'set-point' for normal ventilation. It does not, however, produce significant abnormalities in ventilatory responsiveness to progressive asphyxia during sleep.

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