Chronic rapid eye movement sleep restriction during juvenility has long-term effects on anxiety-like behaviour and neurotransmission of male Wistar rats.

Modernity imposes a toll on the sleep time of young population, with concomitant increase in symptoms of anxiety and depression. Whether there is a causal relationship between these events are only now being experimentally tested in humans and rodents. In a previous study, we showed that chronic sleep deprivation in juvenile-adolescent male rats led to increased anxiety-like behaviour and changes in noradrenaline and serotonin in the amygdala and hippocampus. In the present study we investigated whether early chronic sleep restriction affects emotional behaviour, stress response and neurochemistry in adulthood. From 21 to 42 days of age, Wistar male rats were submitted to sleep restriction by the multiple platform method or allowed to sleep freely. Forty-five days after this period, rats were tested in the elevated plus maze (EPM) and blood samples were collected from non-tested rats or 30 and 60 min after the EPM for determination of plasma corticosterone levels. Levels of monoamines were determined in the frontal cortex, hippocampus, amygdala and hypothalamus 60 min after the EPM. Sleep restriction resulted in increased anxiety-like behaviour, decreased noradrenaline levels in the amygdala and dopamine levels in the ventral hippocampus. Anxiety index was positively correlated with increased serotonin metabolism in the frontal cortex and greater dopamine metabolism in the ventral hippocampus, and negatively correlated with dopamine levels in the ventral hippocampus. These results suggest that sleep restriction in juvenility and adolescence induces persistent changes in emotional behaviour in adult male rats and that levels of anxiety are correlated with increased serotonin and dopamine metabolism in specific brain areas.

Fish-oil supplementation decreases Indoleamine-2,3-Dioxygenase expression and increases hippocampal serotonin levels in the LPS depression model.

AIM: To test the hypothesis that the antidepressant-like effect of omega-3 polyunsaturated fatty acids is related to the Indoleamine-2,3-Dioxygenase (IDO) inhibition. METHODS: Animals were supplemented for 50 days with 3.0 g/kg of Fish Oil (FO) or received water (Control group - C), via gavage. At the end of this period, both groups were injected with LPS 24 h before the modified forced swim test (MFST) and the open field. To assess the possible involvement of IDO in the FO effects, we performed two independent experiments, using two IDO inhibitors: the direct inhibitor 1-methyl-DL-tryptophan (1-MT) and the anti-inflammatory drug minocycline (MINO), administered 23 h, 5 h and 1 h before the tests. After the tests, the animals' hippocampi were removed for quantification of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) by HPLC, and for IDO expression by western blot. RESULTS: LPS induced a depressive-like state in the animals, and this effect was blocked by 1-MT, MINO and FO. Regardless of IDO inhibition, FO supplemented animals displayed an antidepressant-like response by increasing swimming and decreasing immobility frequencies in the MFST when compared to the control group. The immune challenge induced an over-expression of IDO and reduced hippocampal 5-HT levels, both of which were reversed by MINO and FO. CONCLUSION: FO induced a pronounced antidepressant-like effect and prevented LPS-induced depressive-like behavior, and this effect was related to decreased IDO expression and increased 5-HT levels in the hippocampus.

Chronic REM Sleep Restriction in Juvenile Male Rats Induces Anxiety-Like Behavior and Alters Monoamine Systems in the Amygdala and Hippocampus.

Adolescence is marked by major physiological changes, including those in the sleep-wake cycle, such as phase delay, which may result in reduced sleep hours. Sleep restriction and/or deprivation in adult rats activate stress response and seem to be a risk factor for triggering emotional disorders. In the present study, we sought to evaluate the behavioral and neurobiological consequences of prolonged REM sleep restriction in juvenile male rats. Immediately after weaning, on postnatal day 21, three males from each litter were submitted to REM sleep deprivation and the other three animals were maintained in their home-cages. REM sleep restriction (REMSR) was accomplished by placing the animals in the modified multiple platform method for 18 h and 6 h in the home-cage, where they could sleep freely; the sleep restriction lasted 21 consecutive days, during which all animals were measured and weighed every 3 days. After the end of this period, all animals were allowed to sleep freely for 2 days, and then the behavioral tests were performed for evaluation of depressive and anxiety-like profiles (sucrose negative contrast test and elevated plus maze, EPM). Blood sampling was performed 5 min before and 30 and 60 min after the EPM for determination of corticosterone plasma levels. The adrenals were weighed and brains collected and dissected for monoamine levels and receptor protein expression. REMSR impaired the physical development of adolescents, persisting for a further week. Animals submitted to REMSR exhibited higher basal corticosterone levels and a greater anxiety index in the EPM, characteristic of an anxious profile. These animals also exhibited higher noradrenaline levels in the amygdala and ventral hippocampus, without any change in the expression of ß1-adrenergic receptors, as well as higher serotonin and reduced turnover in the dorsal hippocampus, with diminished expression of 5-HT1A. Finally, greater concentration of BDNF was observed in the dorsal hippocampus in chronically sleep-restricted animals. Chronic REMSR during puberty impaired physical development and induced anxiety-like behavior, attributed to increased noradrenaline and serotonin levels in the amygdala and hippocampus.

Brain prolactin is involved in stress-induced REM sleep rebound.

REM sleep rebound is a common behavioural response to some stressors and represents an adaptive coping strategy. Animals submitted to multiple, intermittent, footshock stress (FS) sessions during 96h of REM sleep deprivation (REMSD) display increased REM sleep rebound (when compared to the only REMSD ones, without FS), which is correlated to high plasma prolactin levels. To investigate whether brain prolactin plays a role in stress-induced REM sleep rebound two experiments were carried out. In experiment 1, rats were either not sleep-deprived (NSD) or submitted to 96h of REMSD associated or not to FS and brains were evaluated for PRL immunoreactivity (PRL-ir) and determination of PRL concentrations in the lateral hypothalamus and dorsal raphe nucleus. In experiment 2, rats were implanted with cannulas in the dorsal raphe nucleus for prolactin infusion and were sleep-recorded. REMSD associated with FS increased PRL-ir and content in the lateral hypothalamus and all manipulations increased prolactin content in the dorsal raphe nucleus compared to the NSD group. Prolactin infusion in the dorsal raphe nucleus increased the time and length of REM sleep episodes 3h after the infusion until the end of the light phase of the day cycle. Based on these results we concluded that brain prolactin is a major mediator of stress-induced REMS. The effect of PRL infusion in the dorsal raphe nucleus is discussed in light of the existence of a bidirectional relationship between this hormone and serotonin as regulators of stress-induced REM sleep rebound.

Neuroendocrine and Peptidergic Regulation of Stress-Induced REM Sleep Rebound.

Sleep homeostasis depends on the length and quality (occurrence of stressful events, for instance) of the preceding waking time. Forced wakefulness (sleep deprivation or sleep restriction) is one of the main tools used for the understanding of mechanisms that play a role in homeostatic processes involved in sleep regulation and their interrelations. Interestingly, forced wakefulness for periods longer than 24 h activates stress response systems, whereas stressful events impact on sleep pattern. Hypothalamic peptides (corticotropin-releasing hormone, prolactin, and the CLIP/ACTH18-39) play an important role in the expression of stress-induced sleep effects, essentially by modulating rapid eye movement sleep, which has been claimed to affect the organism resilience to the deleterious effects of stress. Some of the mechanisms involved in the generation and regulation of sleep and the main peptides/hypothalamic hormones involved in these responses will be discussed in this review.

Role of corticosterone on sleep homeostasis induced by REM sleep deprivation in rats.

Sleep is regulated by humoral and homeostatic processes. If on one hand chronic elevation of stress hormones impair sleep, on the other hand, rapid eye movement (REM) sleep deprivation induces elevation of glucocorticoids and time of REM sleep during the recovery period. In the present study we sought to examine whether manipulations of corticosterone levels during REM sleep deprivation would alter the subsequent sleep rebound. Adult male Wistar rats were fit with electrodes for sleep monitoring and submitted to four days of REM sleep deprivation under repeated corticosterone or metyrapone (an inhibitor of corticosterone synthesis) administration. Sleep parameters were continuously recorded throughout the sleep deprivation period and during 3 days of sleep recovery. Plasma levels of adrenocorticotropic hormone and corticosterone were also evaluated. Metyrapone treatment prevented the elevation of corticosterone plasma levels induced by REM sleep deprivation, whereas corticosterone administration to REM sleep-deprived rats resulted in lower corticosterone levels than in non-sleep deprived rats. Nonetheless, both corticosterone and metyrapone administration led to several alterations on sleep homeostasis, including reductions in the amount of non-REM and REM sleep during the recovery period, although corticosterone increased delta activity (1.0-4.0 Hz) during REM sleep deprivation. Metyrapone treatment of REM sleep-deprived rats reduced the number of REM sleep episodes. In conclusion, reduction of corticosterone levels during REM sleep deprivation resulted in impairment of sleep rebound, suggesting that physiological elevation of corticosterone levels resulting from REM sleep deprivation is necessary for plentiful recovery of sleep after this stressful event.

REM Sleep Rebound as an Adaptive Response to Stressful Situations.

Stress and sleep are related to each other in a bidirectional way. If on one hand poor or inadequate sleep exacerbates emotional, behavioral, and stress-related responses, on the other hand acute stress induces sleep rebound, most likely as a way to cope with the adverse stimuli. Chronic, as opposed to acute, stress impairs sleep and has been claimed to be one of the triggering factors of emotional-related sleep disorders, such as insomnia, depressive- and anxiety-disorders. These outcomes are dependent on individual psychobiological characteristics, conferring even more complexity to the stress-sleep relationship. Its neurobiology has only recently begun to be explored, through animal models, which are also valuable for the development of potential therapeutic agents and preventive actions. This review seeks to present data on the effects of stress on sleep and the different approaches used to study this relationship as well as possible neurobiological underpinnings and mechanisms involved. The results of numerous studies in humans and animals indicate that increased sleep, especially the rapid eye movement phase, following a stressful situation is an important adaptive behavior for recovery. However, this endogenous advantage appears to be impaired in human beings and rodent strains that exhibit high levels of anxiety and anxiety-like behavior.

Modulation of Sleep Homeostasis by Corticotropin Releasing Hormone in REM Sleep-Deprived Rats.

Studies have shown that sleep recovery following different protocols of forced waking varies according to the level of stress inherent to each method. Sleep deprivation activates the hypothalamic-pituitary-adrenal axis and increased corticotropin-releasing hormone (CRH) impairs sleep. The purpose of the present study was to evaluate how manipulations of the CRH system during the sleep deprivation period interferes with subsequent sleep rebound. Throughout 96 hours of sleep deprivation, separate groups of rats were treated i.c.v. with vehicle, CRH or with alphahelical CRH(9-41), a CRH receptor blocker, twice/day, at 07:00 h and 19:00 h. Both treatments impaired sleep homeostasis, especially in regards to length of rapid eye movement sleep (REM) and theta/delta ratio and induced a later decrease in NREM and REM sleep and increased waking bouts. These changes suggest that activation of the CRH system impact negatively on the homeostatic sleep response to prolonged forced waking. These results indicate that indeed, activation of the HPA axis-at least at the hypothalamic level-is capable to reduce the sleep rebound induced by sleep deprivation.

Chronic stress during paradoxical sleep deprivation increases paradoxical sleep rebound: association with prolactin plasma levels and brain serotonin content.

Previous studies suggest that stress associated to sleep deprivation methods can affect the expression of sleep rebound. In order to examine this association and possible mechanisms, rats were exposed to footshock stress during or immediately after a 96-h period of paradoxical sleep deprivation (PSD) and their sleep and heart rate were recorded. Control rats (maintained in individual home cages) and paradoxical sleep-deprived (PS-deprived) rats were distributed in three conditions (1) no footshock--NF; (2) single footshock--SFS: one single footshock session at the end of the PSD period (6-8 shocks per minute; 100 ms; 2 mA; for 40 min); and (3) multiple footshock--MFS: footshock sessions with the same characteristics as described above, twice a day throughout PSD (at 7:00 h and 19:00 h) and one extra session before the recovery period. After PSD, animals were allowed to sleep freely for 72 h. Additional groups were sacrificed at the end of the sleep deprivation period for blood sampling (ACTH, corticosterone, prolactin and catecholamine levels) and brain harvesting (monoamines and metabolites). Neither SFS nor MFS produced significant alterations in the sleep patterns of control rats. All PS-deprived groups exhibited increased heart rate which could be explained by increased dopaminergic activity in the medulla. As expected, PS deprivation induced rebound of paradoxical sleep in the first day of recovery; however, PSD+MFS group showed the highest rebound (327.3% above the baseline). This group also showed intermediate levels of corticosterone and the highest levels of prolactin, which were positively correlated with the length of PS episodes. Moreover, paradoxical sleep deprivation resulted in elevation of the serotonergic turnover in the hypothalamus, which partly explained the hormonal results, and in the hippocampus, which appears to be related to adaptive responses to stress. The data are discussed in the realm of a prospective importance of paradoxical sleep for processing of traumatic events.

[Immune outcomes of sleep disorders: the hypothalamic-pituitary-adrenal axis as a modulatory factor]. / Repercussões imunológicas dos distúrbios do sono: o eixo hipotálamo-pituitária-adrenal como fator modulador.

OBJECTIVE: To review the literature on the interaction between sleep and the immune system. METHOD: A search on Web of Science and Pubmed database including the keywords sleep, sleep deprivation, stress, hypothalamic-pituitary-adrenal axis, immune system, and autoimmune diseases. RESULTS: On Web of Science, 588 publications were retrieved; 61 references, more significant and closer to our objective, were used, including original articles and review papers. CONCLUSION: Sleep deprivation and immune system exert a bidirectional influence on each other. Since sleep deprivation is considered a stressor, inasmuch as it induces elevation of cortisol or corticosterone levels in humans and rodents, respectively, and given the well-known immunosuppressive effect of glucocorticoids, we propose that increased activation of the hypothalamic-pituitary-adrenal axis is a major mediator of the immune alterations observed in patients with insomnia or in sleep deprived subjects.

Repercussões imunológicas dos distúrbios do sono: o eixo hipotálamo-pituitária-adrenal como fator modulador / Immune outcomes of sleep disorders: the hypothalamic-pituitary-adrenal axis as a modulatory factor

OBJETIVO: Revisar a literatura a respeito da interação entre sono e sistema imunológico. MÉTODO: Busca no Web of Science e no PubMed com os descritores: sono, privação de sono, estresse, eixo hipotálamo-pituitária-adrenal, sistema imunológico e doenças auto-imunes. RESULTADOS: Foram encontrados 588 artigos no Web of Science. As 61 referências mais significativas e mais relacionadas aos objetivos do estudo foram utilizadas. Foram incluídos artigos originais e de revisão. CONCLUSÃO: A privação de sono e o sistema imunológico exercem e sofrem influências mútuas. A privação de sono é considerada um estressor, uma vez que induz a elevação do cortisol em seres humanos - ou da corticosterona em roedores. Os glicocorticóides, por sua vez, exercem um efeito imunossupressor. Por essas razões, foi proposto que o aumento da ativação do eixo hipotálamo-pituitária-adrenal seja um importante mediador das alterações imunológicas observadas em pacientes com insônia ou privados de sono.

OBJECTIVE: To review the literature on the interaction between sleep and the immune system. METHOD: A search on Web of Science and Pubmed database including the keywords sleep, sleep deprivation, stress, hypothalamic-pituitary-adrenal axis, immune system, and autoimmune diseases. RESULTS: On Web of Science, 588 publications were retrieved; 61 references, more significant and closer to our objective, were used, including original articles and review papers. CONCLUSION: Sleep deprivation and immune system exert a bidirectional influence on each other. Since sleep deprivation is considered a stressor, inasmuch as it induces elevation of cortisol or corticosterone levels in humans and rodents, respectively, and given the well-known immunosuppressive effect of glucocorticoids, we propose that increased activation of the hypothalamic-pituitary-adrenal axis is a major mediator of the immune alterations observed in patients with insomnia or in sleep deprived subjects.

Comparison of the sleep pattern throughout a protocol of chronic sleep restriction induced by two methods of paradoxical sleep deprivation.

The purpose of the present study was to evaluate the sleep homeostasis of rats submitted to a protocol of chronic sleep restriction by two methods and to evaluate the sleep characteristics during the recovery period. The sleep restriction protocol was accomplished by sleep depriving rats for 18 h everyday for 21 days, using the single platform method (SPM) or the modified multiple platform method (MMPM) of paradoxical sleep (PS) deprivation. Rats were allowed to sleep for 6 h (from 10:00 to 16:00; starting 3 h after lights on) in their individual home-cages, during which their sleep was recorded. At the end of the sleep restriction protocol, rats were recorded in their home-cages for 4 days, where they could sleep freely. Both methods used to induce chronic sleep restriction were effective, in sofar as they resulted in augmented sleep time during the 6h-sleep period, with very few bouts of wakening. Although comparison between the methods did not reveal differences, sleep restriction under MMPM produced a more consistent daily rebound, mainly of paradoxical sleep, with longer episodes. These results showed distinct sleep recovery patterns, suggesting a possible role of the waking experiences (i.e. immobilization stress, social interaction) acting on sleep consolidation.

Sleep homeostasis in rats assessed by a long-term intermittent paradoxical sleep deprivation protocol.

Numerous studies have evaluated the sleep homeostasis of rats after short- or long-periods of sleep deprivation, but none has assessed the effects of prolonged sleep restriction on the rat's sleep pattern. The purpose of the present study, therefore, was to evaluate the sleep homeostasis of rats under a protocol of chronic sleep restriction. Male Wistar rats were implanted with electrodes for EEG and EMG recordings. Using the single platform method, the animals were submitted to 18 h of sleep restriction, beginning at 16:00 h (lights on at 07:00 h), followed by a 6 h sleep window (from 10:00 h to 16:00 h) for 21 days. Immediately after this period, rats were allowed to sleep freely for 4 days (recovery period). The sleep-wake cycle was recorded throughout the entire experiment and the results showed that during the 6h sleep window there was an increase on the percentage of sleep time, reflected by augmented time in high amplitude slow wave sleep and in paradoxical sleep, when compared to baseline sleep, whereas bouts of awakening longer than 1.5 min were greatly reduced, with the animals exhibiting a monophasic-type sleep pattern. During the deprivation period, paradoxical sleep was abolished. High amplitude slow wave sleep was also greatly affected by the protocol. Nonetheless, one day of recovery was sufficient to restore the normal sleep pattern. These findings indicate that this protocol was capable to induce many changes in the rat's sleep patterns, suggesting that during the 6h sleep window there is a sleep adaptive homeostatic process.

Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery.

Vigilance status was continually monitored in socially stable groups of rats exposed to the modified multiple platform (MMP) technique for sleep deprivation. For comparison, sleep parameters were also monitored in socially isolated rats deprived of sleep by the single platform (SP) method. In all cases, sleep was continuously recorded during baseline, during 96 h of sleep deprivation and during 4 days of recovery. Both multiple- and single-platform techniques completely abolished paradoxical sleep (PS) during the deprivation period, but also resulted in significant decreases in slow wave sleep (SWS) (-31% and -37%, respectively). Unexpectedly, animals on large platforms, which are normally intended as controls, also showed significant reductions in PS and SWS, and these effects were more pronounced in rats deprived in groups than in animals deprived in isolation. Another control preparation, rats placed on wire-mesh grids in the deprivation tank, also showed PS reduction (-39%) but no loss of SWS during the 4 test days. Paradoxical sleep rebound was observed in the first 24 h in all groups, except for grid controls. Overall, no significant differences were found between single- and multiple-platform procedures during the 4 days of deprivation. However, sleep rebound was more pronounced in MMP-deprived rats than in SP-deprived rats. Sleep loss in both control groups may reflect residual effect of stress that remain in the platform technique. These findings indicate that the MMP technique is effective in inducing PS deprivation (PSD). However, the fact that SWS is also affected may have implications for conclusions on paradoxical sleep function based upon paradoxical sleep deprivation.

Does paradoxical sleep deprivation and cocaine induce penile erection and ejaculation in old rats?

A recent study has established that paradoxical sleep deprivation (PSD) and cocaine administration elicit genital reflexes (penile erection and ejaculation) in young rats. To discover whether the same effects occurred in old animals submitted to PSD, we administered cocaine (15 mg/kg) to young (3-month) and old (22-month) male rats after a 4-day period of PSD or at the equivalent time-point in control animals. We then evaluated erections and ejaculations. Sixty per cent of the old-PSD group displayed erection, although ejaculation was not observed. Genital reflexes were absent in young and old control groups. We found that PSD reduced testosterone and increased progesterone levels in both young and old PSD groups. In conclusion, our results suggest that although genital reflexes usually decrease with age, testosterone levels alone cannot account for these changes. The interaction of PSD and cocaine probably enhances dopamine transmission in the brain and may elicit penile erection in old rats.