Invariant Natural Killer T cells resilience to paradoxical sleep deprivation-associated stress.

Although several studies demonstrate that stressful situations, such as sleep disturbances, negatively impact the innate and adaptive arms of the immune system, their influence on invariant Natural Killer T (iNKT) cells remains unclear. iNKT cells are CD1d-restricted innate T cells that recognize glycolipid antigens and rapidly produce polarizing cytokines being key players in several immune responses, and a potential target for immunotherapy. iNKT cells differ in several aspects from conventional T lymphocytes, including a unique dependence on CD1d-expressing double-positive (DP) thymocytes for intrathymic maturation. As a consequence of stress, DP thymocytes undergo glucocorticoid-induced apoptosis, which might compromise iNKT developmental pathway. Therefore, we used a paradoxical sleep deprivation (SD) model to determine the impact of sleep disturbance on iNKT cell biology. After 72 h of SD, C57Bl/6 mice exhibited a significant increase in systemic glucocorticoid levels and thymus atrophy. Despite marked decrease in the number of DP thymocytes, the ratio CD1d+/CD1d- was higher in SD mice, and the number of thymic iNKT cells remained unaltered, suggesting that SD did not compromise the iNKT developmental pathway. In contrast, SD reduced hepatic IFN-γ, but not, IL-4-producing iNKT cells, without further effect in the spleen. Despite this fact, SD did not affect stimulation of IFN-γ production by iNKT cells, or cytokine release, in response to α-galactosylceramide, a specific antigen. Furthermore, although SD impaired splenic NK cells activity against tumor cells, it did not affect iNKT cell-specific cytotoxicity. Thus, our study shows that SD-induced stress did not impair the iNKT cells' responses to a cognate antigen.

Effects of lithium and valproate on behavioral parameters and neurotrophic factor levels in an animal model of mania induced by paradoxical sleep deprivation.

The present study aimed to evaluate the effects of treatment with lithium (Li) and valproate (VPA) on behaviors and brain BDNF, NGF, NT-3, NT-4 and GDNF levels in mice submitted to paradoxical sleep deprivation (PSD), which induces an animal model of mania. Male C57BL/6J mice received an intraperitoneal (i.p.) injection of saline solution (NaCl 0.09%, 1 ml/kg), Li (47.3 mg/kg, 1 ml/kg) or VPA (200 mg/kg, 1 ml/kg) once a day for seven days. Animals were randomly distributed into six groups (n = 10 per group): (1) Control + Sal; (2) Control + Li; (3) Control + VPA; (4) PSD + Sal; (5) PSD + Li; or (6) PSD + VPA. Animals were submitted to 36 h of PSD, and then, they were submitted to the open field test. The frontal cortex and hippocampus were dissected from the brain. The manic-like behaviors in the mice were analyzed. Treatment with Li and VPA reversed the behavioral alterations induced by PSD. PSD decreased BDNF, NGF, and GDNF levels in the frontal cortex and hippocampus of mice. The administration of Li and VPA protected the brain against the damage induced by PSD. However, PSD and the administration of Li and VPA did not affect the levels of NT-3 and NT-4 in either brain structure evaluated. In conclusion, the PSD protocol induced manic-like behavior in rats and induced alterations in neurotrophic factor levels. It seems that neurotrophic factors and sleep are essential targets to treat BD.

High Exploratory Phenotype Rats Exposed to Environmental Stressors Present Memory Deficits Accompanied by Immune-Inflammatory/Oxidative Alterations: Relevance to the Relationship Between Temperament and Mood Disorders.

Low-exploratory (LE) and high-exploratory (HE) rodents mimic human depressive and hyperthymic temperaments, respectively. Mood disorders (MD) may be developed by the exposure of these temperaments to environmental stress (ES). Psychiatric symptoms severity in MD patients is related to the magnitude of memory impairment. Thus, we aimed at studying the consequences of the exposure of LE and HE male Wistar rats, during periadolescence, to a combination of ES, namely, paradoxical sleep deprivation (PSD) and unpredictable stress (US), on anxiety-related behavior in the plus maze test, working (WM) and declarative memory (DM) performance. We also evaluated hippocampal immune-inflammatory/oxidative, as consequences of ES, and prevention of ES-induced alterations by the mood-stabilizing drugs, lithium and valproate. Medium exploratory (ME) control rats were used for comparisons with HE- and LE-control rats. We observed that HE-controls presented increased anxiolytic behavior that was significantly increased by ES exposure, whereas LE-controls presented increased anxiety-like behavior relative to ME-controls. Lithium and valproate prevented anxiolytic alterations in HE+ES rats. HE+ES- and LE+ES-rats presented WM and DM deficits. Valproate and lithium prevented WM deficits in LE-PSD+US rats. Lithium prevented DM impairment in HE+ES-rats. Hippocampal levels of reduced glutathione (GSH) increased four-fold in HE+ES-rats, being prevented by valproate and lithium. All groups of LE+ES-rats presented increased levels of GSH in relation to controls. Increments in lipid peroxidation in LE+ES- and HE+ES-rats were prevented by valproate in HE+ES-rats and by both drugs in LE+ES-rats. Nitrite levels were increased in HE+ES- and LE+ES-rats (five-fold increase), which was prevented by both drugs in LE+ES-rats. HE+ES-rats presented a two-fold increase in the inducible nitric oxide synthase (iNOS) expression that was prevented by lithium. HE+ES-rats showed increased hippocampal and plasma levels of interleukin (IL)-1ß and IL-4. Indoleamine 2, 3-dioxygenase 1 (IDO1) was increased in HE+ES- and LE+ES-rats, while tryptophan 2,3-dioxygenase (TDO2) was increased only in HE+ES-rats. Altogether, our results showed that LE- and HE-rats exposed to ES present distinct anxiety-related behavior and similar memory deficits. Furthermore, HE+ES-rats presented more brain and plasma inflammatory alterations that were partially prevented by the mood-stabilizing drugs. These alterations in HE+ES-rats may possibly be related to the development of mood symptoms.

Behavioral, affective, and cognitive alterations induced by individual and combined environmental stressors in rats

Objective: To evaluate whether exposing rats to individual or combined environmental stressors triggers endophenotypes related to mood and anxiety disorders, and whether this effect depends on the nature of the behavior (i.e., innate or learned). Methods: We conducted a three-phase experimental protocol. In phase I (baseline), animals subjected to mixed schedule of reinforcement were trained to press a lever with a fixed interval of 1 minute and a limited hold of 3 seconds. On the last day of phase I, an open-field test was performed and the animals were divided into four experimental groups (n=8/group). In phase II (repeated stress), each group was exposed to either hot air blast (HAB), paradoxical sleep deprivation (PSD) or both (HAB+PSD group) on alternate days over a 10-day period. Control group animals were not exposed to stressors. In phase III (post-stress evaluation), behavior was analyzed on the first (short-term effects), third (mid-term effects), and fifth (long-term effects) days after repeated stress. Results: The PSD group presented operant hyperactivity, the HAB group presented spontaneous hypoactivity and anxiety, and the HAB+PSD group presented spontaneous hyperactivity, operant hypoactivity, impulsivity, loss of interest, and cognitive impairment. Conclusion: A combination of environmental stressors (HAB and PSD) may induce endophenotypes related to bipolar disorder.

Quercetin reduces manic-like behavior and brain oxidative stress induced by paradoxical sleep deprivation in mice.

Quercetin is a known antioxidant and protein kinase C (PKC) inhibitor. Previous studies have shown that mania involves oxidative stress and an increase in PKC activity. We hypothesized that quercetin affects manic symptoms. In the present study, manic-like behavior (hyperlocomotion) and oxidative stress were induced by 24h paradoxical sleep deprivation (PSD) in male Swiss mice. Both 10 and 40mg/kg quercetin prevented PSD-induced hyperlocomotion. Quercetin reversed the PSD-induced decrease in glutathione (GSH) levels in the prefrontal cortex (PFC) and striatum. Quercetin also reversed the PSD-induced increase in lipid peroxidation (LPO) in the PFC, hippocampus, and striatum. Pearson's correlation analysis revealed a negative correlation between locomotor activity and GSH in the PFC in sleep-deprived mice and a positive correlation between locomotor activity and LPO in the PFC and striatum in sleep-deprived mice. These results suggest that quercetin exerts an antimanic-like effect at doses that do not impair spontaneous locomotor activity, and the antioxidant action of quercetin might contribute to its antimanic-like effects.

Effects of different periods of paradoxical sleep deprivation and sleep recovery on lipid and glucose metabolism and appetite hormones in rats.

Sleep has a fundamental role in the regulation of energy balance, and it is an essential and natural process whose precise impacts on health and disease have not yet been fully elucidated. The aim of this study was to assess the consequences of different periods of paradoxical sleep deprivation (PSD) and recovery from PSD on lipid profile, oral glucose tolerance test (OGTT) results, and changes in insulin, corticosterone, ghrelin, and leptin concentrations. Three-month-old male Wistar rats weighing 250-350 g were submitted to 24, 96, or 192 h of PSD or 192 h of PSD with 480 h of recovery. The PSD was induced by the multiple platforms method. Subsequently, the animals were submitted to an OGTT. One day later, the animals were killed and the levels of triglycerides, total cholesterol, lipoproteins (low-density lipoprotein, very-low-density lipoprotein, and high-density lipoprotein), insulin, ghrelin, leptin, and corticosterone in plasma were quantified. There was a progressive decrease in body weight with increasing duration of PSD. The PSD induced basal hypoglycemia over all time periods evaluated. Evaluation of areas under the curve revealed progressive hypoglycemia only after 96 and 192 h of PSD. There was an increase in corticosterone levels after 192 h of PSD. We conclude that PSD induces alterations in metabolism that are reversed after a recovery period of 20 days.

The Pronociceptive Effect of Paradoxical Sleep Deprivation in Rats: Evidence for a Role of Descending Pain Modulation Mechanisms.

The mechanisms underlying the pronociceptive effect of paradoxical sleep deprivation (PSD) are not known. In this study, we asked whether PSD increases tonic nociception in the formalin test, decreases the antinociceptive effect of morphine administered into the periaqueductal gray matter (PAG), and disrupts endogenous descending pain modulation. PSD for either 24 or 48 h significantly increased formalin-induced nociception and decreased mechanical nociceptive paw withdrawal threshold. The maximal antinociceptive effect induced by morphine (0.9-9 nmol, intra-PAG) was significantly decreased by PSD. The administration of a low dose of the GABAA receptor antagonist, bicuculline (30-300 pmol, intra-PAG), decreased nociception in control rats, but not in paradoxical-sleep-deprived ones. Furthermore, the administration of the cholecystokinin (CCK) 2 receptor antagonist, YM022 (0.5-2 pmol) in the rostral ventral medulla (RVM), decreased nociception in paradoxical-sleep-deprived rats but not in control ones. While a dose of the CCK 2 receptor agonist, CCK-8 (8-24 pmol intra-RVM), increased nociception in control rats, but not in paradoxical-sleep-deprived ones. In addition, the injection of lidocaine (QX-314, 2%, intra-RVM) decreased nociception in sleep-deprived rats, but not in control rats, while the lesion of the dorsolateral funiculus prevented the pronociceptive effect of PSD. Finally, PSD significantly increased c-Fos expression in the RVM. Therefore, PSD increases pain independently of its duration or of the characteristic of the nociceptive stimulus and decreases morphine analgesia at the PAG. PSD appears to increase pain by decreasing descending pain inhibitory activity and by increasing descending pain facilitatory activity.

Sleep deprivation alters energy homeostasis through non-compensatory alterations in hypothalamic insulin receptors in Wistar rats.

Studies have shown a gradual reduction of sleep time in the general population, accompanied by increased food intake, representing a risk for developing obesity, type II diabetes and cardiovascular disease. Rats subjected to paradoxical sleep deprivation (PSD) exhibit feeding and metabolic alterations, both of which are regulated by the communication between peripheral signals and the hypothalamus. This study aimed to investigate the daily change of 96 h of PSD-induced food intake, body weight, blood glucose, plasma insulin and leptin concentrations and the expression of their receptors in the hypothalamus of Wistar rats. Food intake was assessed during the light and dark phases and was progressively increased in sleep-deprived animals, during the light phase. PSD produced body weight loss, particularly on the first day, and decreased plasma insulin and leptin levels, without change in blood glucose levels. Reduced leptin levels were compensated by increased expression of leptin receptors in the hypothalamus, whereas no compensations occurred in insulin receptors. The present results on body weight loss and increased food intake replicate previous studies from our group. The fact that reduced insulin levels did not lead to compensatory changes in hypothalamic insulin receptors, suggests that this hormone may be, at least in part, responsible for PSD-induced dysregulation in energy metabolism.

Cardiovascular function alterations induced by acute paradoxical sleep deprivation in rats.

Sleep loss has been implicated in triggering the hypertension. The goal of the present study was investigated the possible mechanisms underlying cardiovascular alterations after acute paradoxical sleep deprivation (PSD). Male Wistar rats were assigned in two experimental groups: (1) control and (2) PSD for 24 h using the modified single platform method. Paradoxical sleep deprived rats exhibited higher blood pressure, heart rate (HR) and impaired baroreceptor sensitivity. After pharmacological autonomic double blockade (propranolol and methylatropine administration), intrinsic heart rate was decreased after PSD. The PSD rats showed a reduction in the vagal tone without affecting sympathetic tone. Isoproterenol administration (0.001, 0.01 and 1 µg/kg) induced an increase in ΔHR responses in PSD group. Electrocardiographic analysis in response to ß-adrenergic stimulation indicated that PSD contributed to ventricular cardiac arrhythmias. Our findings suggest that acute paradoxical sleep loss induce cardiovascular alterations, autonomic imbalance accompanied by impaired baroreflex sensitivity and increased arrhythmia susceptibility.

Acute total sleep deprivation potentiates amphetamine-induced locomotor-stimulant effects and behavioral sensitization in mice.

It has been demonstrated that a prolonged period (48 h) of paradoxical sleep deprivation (PSD) potentiates amphetamine (AMP)-induced behavioral sensitization, an animal model of addiction-related neuroadaptations. In the present study, we examined the effects of an acute short-term deprivation of total sleep (TSD) (6h) on AMP-induced behavioral sensitization in mice and compared them to the effects of short-term PSD (6 h). Three-month-old male C57BL/6J mice underwent TSD (experiment 1-gentle handling method) or PSD (experiment 2-multiple platforms method) for 6 h. Immediately after the sleep deprivation period, mice were tested in the open field for 10 min under the effects of saline or 2.0 mg/kg AMP. Seven days later, to assess behavioral sensitization, all of the mice received a challenge injection of 2.0 mg/kg AMP and were tested in the open field for 10 min. Total, peripheral, and central locomotion, and grooming duration were measured. TSD, but not PSD, potentiated the hyperlocomotion induced by an acute injection of AMP and this effect was due to an increased locomotion in the central squares of the apparatus. Similarly, TSD facilitated the development of AMP-induced sensitization, but only in the central locomotion parameter. The data indicate that an acute period of TSD may exacerbate the behavioral effects of AMP in mice. Because sleep architecture is composed of paradoxical and slow wave sleep, and 6-h PSD had no effects on AMP-induced hyperlocomotion or sensitization, our data suggest that the deprivation of slow wave sleep plays a critical role in the mechanisms that underlie the potentiating effects of TSD on both the acute and sensitized addiction-related responses to AMP.

Anxiety-like effects of meta-chlorophenylpiperazine in paradoxically sleep-deprived mice.

Chlorophenylpiperazines (CPP) are psychotropic drugs used in nightclub parties and are frequently used in a state of sleep deprivation, a condition which can potentiate the effects of psychoactive drugs. This study aimed to investigate the effects of sleep deprivation and sleep rebound (RB) on anxiety-like measures in mCPP-treated mice using the open field test. We first optimized our procedure by performing dose-effect curves and examining different pretreatment times in naïve male Swiss mice. Subsequently, a separate cohort of mice underwent paradoxical sleep deprivation (PSD) for 24 or 48h. In the last experiment, immediately after the 24h-PSD period, mice received an injection of saline or mCPP, but their general activity was quantified in the open field only after the RB period (24 or 48h). The dose of 5mgmL(-1) of mCPP was the most effective at decreasing rearing behavior, with peak effects 15min after injection. PSD decreased locomotion and rearing behaviors, thereby inhibiting a further impairment induced by mCPP. Plasma concentrations of mCPP were significantly higher in PSD 48h animals compared to the non-PSD control group. Twenty-four hours of RB combined with mCPP administration produced a slight reduction in locomotion. Our results show that mCPP was able to significantly change the behavior of naïve, PSD, and RB mice. When combined with sleep deprivation, there was a higher availability of drug in plasma levels. Taken together, our results suggest that sleep loss can enhance the behavioral effects of the potent psychoactive drug, mCPP, even after a period of rebound sleep.

Modafinil ameliorates cognitive deficits induced by maternal separation and sleep deprivation.

Animals exposed to an early adverse event may be more susceptible to a second source of stress later in life, and these stressors may have additive deleterious effects. Sleep deprivation is known to be a stressor, affecting multiple body functions such as the cognition. Modafinil enhances working memory and attention in healthy non-sleep deprived subjects and in animal models of sleep deprivation. The first aim of the present study was to investigate the effects of maternal separation (MS) combined with paradoxical sleep deprivation (PSD) in adulthood on recognition memory in rats. Second, we aimed to evaluate whether the administration of modafinil would be able to ameliorate memory deficits induced by MS and PSD. Wistar rat pups were initially distributed into MS and handling (H) groups, with their litters standardized in 4 females and 4 males. In adulthood, the male rats were submitted to PSD or control condition, being redistributed afterwards in modafinil- or vehicle-treatment immediately after the training session of object recognition task. PSD did not potentiate the cognitive deficit due to MS. However, modafinil was able to recover memory impairments associated to PSD and also to MS in the neonatal period. This study demonstrates for the first time that modafinil ameliorates cognitive deficits associated to MS and to PSD in adulthood, independent from MS in the neonatal period.

Effect of sildenafil (Viagra®) on the genital reflexes of paradoxical sleep-deprived male rats

Since there is evidence that paradoxical sleep deprivation (PSD) elicits penile erection (PE) and ejaculation (EJ), and that the erectile response of rats is mediated by nitric oxide, the present study sought to extend the latter finding by assessing the effects of sildenafil on the genital reflexes of male Wistar rats subjected to PSD. We also determined the influence of sildenafil on hormone concentrations. In the first experiment, sildenafil at doses ranging from 0.08 to 0.32 mg/kg was administered intraperitoneally to rats that had been deprived of sleep for 4 days and to home cage controls (N = 8-10/group). The frequency of PE and EJ was measured for 60 min. PSD alone induced PE in 50 percent of the animals; however, a single injection of sildenafil did not significantly increase the percentage of rats displaying PE compared to PSD-saline or to home cage groups. PSD alone also induced spontaneous EJ, but this response was not potentiated by sildenafil in the dose range tested. Testosterone concentrations were significantly lower in PSD rats (137 ± 22 ng/dL) than in controls (365 ± 38 ng/dL), whereas progesterone (0.9 ± 0.1 vs 5.4 ± 1 ng/mL) and plasma dopamine (103.4 ± 30 vs 262.6 ± 77 pg/mL) increased. These changes did not occur after sildenafil treatment. The data show that although sildenafil did not alter the frequency of genital reflexes, it antagonized hormonal (testosterone and progesterone) and plasma dopamine changes induced by PSD. The stimulation of the genital reflexes by sildenafil did not result in potentiating effects in PSD rats.