Magda Bignotto

Endocrinological and catecholaminergic alterations during sleep deprivation and recovery in male rats

Since previous data of our group showed increased concentrations in HPA axis hormones in sleep deprived rats, we hypothesized that this augmentation could produce effects in other hormonal systems, particularly in the sexual system. Considering that little is known about how the hormonal system changes during the recovery period after sleep deprivation (SD), our objective was to examine from what point SD alters sexual and stress‐related hormones along with plasma catecholamine concentrations during 4 days. We also sought to verify the time course of their recovery after an equivalent period of recovery sleep. Rats were deprived of sleep by the platform technique for 1–4 days and were allowed to recover for the same period. Plasma catecholamines [dopamine (DA) and noradrenaline (NOR)], testosterone, estrone, progesterone, prolactin, corticosterone and adrenocorticotropic hormone (ACTH) concentrations were measured. Comparisons between groups showed that the SD procedure used in the present study produced marked alterations in almost all studied hormones from 24 h of SD, except for estrone and prolactin (which required 96 h of SD to become altered). Testosterone and estrone decreased, whereas progesterone, prolactin, corticosterone, ACTH, DA and NOR increased. During recovery period, progesterone, prolactin and corticosterone concentrations returned to control levels, whereas testosterone, estrone, NOR and DA did not. In addition, after 48 h of recovery ACTH and NOR decreased below control concentrations, remaining low until 96 h of sleep recovery. Thus, SD showed long lasting, differential effects upon these neurochemicals suggesting that each has its own pattern of responses to SD as well as variable periods of recovery.

Randomized, double‐blind clinical trial, controlled with placebo, of the toxicology of chronic melatonin treatment

The objective of the present study was to assess the toxicology of melatonin (10 mg), administered for 28 days to 40 volunteers randomly assigned to groups receiving either melatonin (N=30) or placebo (N=10) in a double‐blind fashion. The following measurements were performed: polysomnography (PSG), laboratory examinations, including complete blood count, urinalysis, sodium, potassium and calcium levels, total protein levels, albumin, blood glucose, triglycerides, total cholesterol, high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), and very low‐density lipoprotein (VLDL), urea, creatinine, uric acid, glutamic‐oxalacetic transaminase (GOT), glutamic‐pyruvate transaminase (GPT), bilirubin, alkaline phosphatase, gama‐glutamic transaminase (GGT), T3, T4, TSH, LH/FSH, cortisol, and melatonin serum concentrations. In addition, the Epworth Somnolence Scale (ESS) and a sleep diary (SD) were also applied to the volunteers 1 wk before each PSG. In addition, the volunteers were asked about possible side effects (SE) that appeared during the treatment. The study was carried out according to the following timetable: Visit 0, filling out the term of consent and inclusion criteria; Visit 1, PSG, laboratory examinations, ESS, SD, melatonin serum concentrations; Visit 2, SD, melatonin serum concentrations, SE; Visit 3, melatonin serum concentrations, PSG, ESS, SE; Visit 4, laboratory examinations, SE, melatonin serum concentrations, SD; and Visit 5, PSG, ESS, SE. Analysis of the PSG showed a statistically significant reduction of stage 1 of sleep in the melatonin group. No other differences between the placebo and melatonin groups were obtained. In the present study we did not observe, according to the parameters analyzed, any toxicological effect that might compromise the use of melatonin at a dose of 10 mg for the period of time utilized in this study.

Different stress modalities result in distinct steroid hormone responses by male rats

Since both paradoxical sleep deprivation (PSD) and stress alter male reproductive function, the purpose of the present study was to examine the influence of PSD and other stressors (restraint, electrical footshock, cold and forced swimming, N = 10 per group) on steroid hormones in adult Wistar male rats. Rats were submitted to chronic stress for four days. The stressors (footshock, cold and forced swimming) were applied twice a day, for periods of 1 h at 9:00 and 16:00 h. Restrained animals were maintained in plastic cylinders for 22 h/day whereas PSD was continuous. Hormone determination was measured by chemiluminescent enzyme immunoassay (testosterone), competitive immunoassay (progesterone) and by radioimmunoassay (corticosterone, estradiol, estrone). The findings indicate that PSD (13.7 ng/dl), footshock (31.7 ng/dl) and cold (35.2 ng/dl) led to lower testosterone levels compared to the swimming (370.4 ng/dl) and control (371.4 ng/dl) groups. However, progesterone levels were elevated in the footshock (4.5 ng/ml) and PSD (5.4 ng/ml) groups compared to control (1.6 ng/ml), swimming (1.1 ng/ml), cold (2.3 ng/ml), and restrained (1.2 ng/ml) animals. Estrone and estradiol levels were reduced in the PSD, footshock and restraint groups compared to the control, swimming and cold groups. A significant increase in corticosterone levels was found only in the PSD (299.8 ng/ml) and footshock (169.6 ng/ml) groups. These changes may be thought to be the full steroidal response to stress of significant intensity. Thus, the data suggest that different stress modalities result in distinct steroid hormone responses, with PSD and footshock being the most similar.

Effects of paradoxical sleep deprivation on blood parameters associated with cardiovascular risk in aged rats

The effects of 96 h of paradoxical sleep deprivation (PSD) on blood parameters associated with cardiovascular risk were studied in young (3-month old) and aged (22-month old) rats. In general, aging was associated with an overall increase in most measures, irrespective of sleep deprivation condition. The latter manipulation also had significant effects on blood variables, but not in a consistent pattern. Thus, PSD significantly reduced triglyceride levels in both young and aged rats; it reduced blood viscosity in aged but not in young rats, and had no effect on the increased cholesterol levels observed in aged controls. Examinations of cholesterol fractions revealed significant increases in low density lipoprotein and high density lipoprotein in aged PSD rats compared to respective controls, whereas very low density lipoprotein was significant decreased after PSD in both young and aged animals. PSD increased vitamin B(12) levels in aged rats, and significantly decreased homocysteine levels in young but not in aged rats which in turn were already reduced. Folate levels were the only variable that was unaffected by aging and/or PSD. These results indicate that PSD has significant but heterogeneous physiological effects in aged rats and may intensify certain aging-related effects which contribute to cardiovascular disease risk while attenuating others.

Cocaine-induced genital reflexes during paradoxical sleep deprivation and recovery

Paradoxical sleep deprivation (PSD) for 96 h together with cocaine administration elicits genital reflexes (penile erection [PE] and ejaculation [EJ]) in rats. Our objective was to examine genital reflexes after periods of 24, 48, 72, 96, 120, and 144 h of PSD and during a 4-day recovery period in acute cocaine-administered rats. After 24 h of PSD followed by cocaine administration, animals started to display PE and EJ, peaking in the 96th h of PSD, whereas PE and EJ were absent in control animals. The effects of more than 96 h of PSD decrease genital reflexes as observed after 120 and 144 h. Genital reflexes were present in the recovery periods but diminished gradually during the period evaluated. Even short periods of PSD probably cause supersensitivity of dopamine (DA) receptors and exacerbate the effects of cocaine on dopaminergic pathways to induce frequent PE and EJ.

Hormone treatment facilitates penile erection in castrated rats after sleep deprivation and cocaine.

Sleep deprivation is associated with cocaine‐enhanced genital reflexes in male rats, and castration of the male rat causes a decline in sexual behaviour, which can be reversed by hormone administration. We conducted two experiments to determine whether sleep deprivation and cocaine administration could also induce spontaneous penile erection in castrated rats after hormonal treatment (testosterone, progesterone and oestradiol). Different doses of hormones or vehicle were administered to rats during the 4‐day period of sleep deprivation, and in home‐cage control rats. Testosterone did not restore penile erection in castrated sleep‐deprived rats. Progesterone triggered penile erection, and 100 mg/day of progesterone induced the highest proportion of rats displaying penile erection, and restored the frequency of penile erection observed in noncastrated sleep deprived rats. Penile erection was absent in vehicle as well as oestradiol‐treated sleep‐deprived castrated rats. Whereas sleep deprivation increased progesterone concentrations in noncastrated rats, sleep deprivation decreased progesterone concentrations in castrated rats. Corticosterone concentrations were lower in the castrated sleep‐deprived rats than in respective control group. These data show that progesterone treatment facilitates penile erection in sleep deprived‐cocaine castrated rats.

Influence of paradoxical sleep deprivation and cocaine on development of spontaneous penile reflexes in rats of different ages.

Recent studies have established that paradoxical sleep deprivation (PSD) and cocaine administration induce genital reflexes (penile erection and ejaculation) in adult and old rats. To determine whether the same effects would induce spontaneous genital reflexes in rats of different ages (30-90 days old), we administered with cocaine (7 mg/kg) or saline to rats after a 4-day period of PSD, or at the equivalent time-point in control animals, and penile erection and ejaculation were then evaluated. In PSD rats administered cocaine, erection was observed from 30 days old to 90 days old, when both genital reflexes reached a peak. Animals submitted to PSD and saline injection showed erection from 60 to 90 days old. None of the control (saline and cocaine) groups of any age displayed these behaviors. The effects of PSD on steroid hormone levels showed that, although testosterone levels increased with age, PSD caused a marked decrease in testosterone at all ages evaluated. Progesterone and corticosterone levels were higher in PSD groups than in the respective control groups. These findings suggest that the interaction of PSD and cocaine probably enhances dopaminergic transmission in the brain and may accelerate the development of genital reflexes in male rats.

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.

Paradoxical sleep deprivation increases plasma endothelin levels.

The endothelins (ET-1, 2 and 3) constitute a family of 21 amino acid peptides with potent biological activities. ET-1 is one of the most potent endogenous vasoconstrictors so far identified and its increased concentration in plasma appears to be closely related to the pathogenesis of arterial hypertension as well as to obstructive sleep apnea (OSA). OSA patients exhibit repetitive episodes of apnea and hypopnea that result in hypoxia and consecutive arousals. These patients are chronically sleep deprived, which may aggravate the hypertensive features, since literature data show that sleep deprivation results in hypertension both in humans and in animals. Based on the reported relationship between ET-1, hypertension and sleep deprivation consequences, the purpose of the present study was to determine plasma ET concentrations in paradoxical sleep-deprived animals. Male Wistar rats, 3 to 4 months old (N = 10 per group), were deprived of sleep for 24 and 96 h by the platform technique and plasma ET-(1/2) was measured by radioimmunoassay. Analysis of plasma revealed that 96 h of sleep deprivation induced a significant increase in ET-(1/2) release (6.58 fmol/ml) compared to control (5.07 fmol/ml). These data show that sleep deprivation altered plasma ET-(1/2) concentrations, suggesting that such an increase may participate in the genesis of arterial hypertension and cardiorespiratory changes observed after sleep deprivation.

Facilitation of ejaculation after methamphetamine administration in paradoxical sleep deprived rats

This study investigated the effects of methamphetamine (MA) on genital reflexes in paradoxical sleep deprived (PSD) rats. Different doses of MA (0, 4, 16 and 64 mg/kg) were acutely given after PSD or the equivalent time to control animals. We observed enhancement of spontaneous ejaculation in PSD rats with larger doses of MA, the highest of which induced ejaculation in 100% of the PSD rats. This was significantly higher than the 30% in the control. Although testosterone exerts motivational effects on male sexual behavior, our data shows that testosterone levels were lower after the PSD period in saline and in the 64 mg/kg MA groups, which present ejaculation at different rates (20% and 100%, respectively). Progesterone levels were significantly higher in PSD-saline in relation to control group and in the 16 and 64 mg/kg of MA groups compared to the other doses. Since PSD induces dopaminergic alterations and dopamine (DA) has a key role in male sexual behavior, plasma DA was also measured. The DA concentration was enhanced in all PSD groups compared with their control group. The mechanism that activates steroid hormones may represent an important physiological effect through which neurotransmitters can affect behavioral events. These data show that MA facilitates ejaculation in PSD rats, however, further studies need to be carried out in order to clarify the hormonal-neurochemical mechanisms involved.