Guido Ficarra

Estrogens Modulate the Circadian Rhythm of Hypothalamic Beta-Endorphin Contents in Female Rats

The aim of the present study was to evaluate the changes in the diurnal rhythm of the hypothalamic beta-endorphin (beta-EP) contents in female rats as a function of circulating estrogens. With this purpose we evaluated the diurnal hypothalamic beta-EP changes (1) during the estrous cycle, and (2) in ovariectomized rats with and without acute and chronic estrogen replacement. Ovariectomized rats were treated either acutely with 10 micrograms of estradiol benzoate (EB) or chronically with 2 micrograms/day of EB for 15 days. beta-EP concentrations were measured in acid extracts of medial basal hypothalamus by a specific radioimmunoassay. During the estrous cycle, hypothalamic beta-EP concentrations showed a significant nocturnal increase, with no difference between the 4 days of the cycle. On the day of estrus, beta-EP concentrations between 12.00 and 18.00 h resulted significantly lower than in the other days of the cycle. After ovariectomy, the night-related changes in hypothalamic beta-EP disappeared. The acute administration of EB induced a significant increase in hypothalamic beta-EP after 21 h (18.00 h). On the other hand, the chronic replacement restored the nocturnal peak of hypothalamic beta-EP (18.00, 21.00, 24.00 h). The present data emphasize the role of central beta-EP in regulating the reproductive functions. Moreover, the effect of estrogen in modulating the circadian changes in hypothalamic beta-EP supports the important role of estrogens in brain function.

Effect of chronic intermittent stress on rat pregnancy and postnatal development

The present study evaluates the effect of chronic intermittent cold-swimming stress on body weight gain of pregnant rats and subsequent development of the offsprings after birth, till peripubertal stage. When stress was administered during the first half (1-11 days) of gestation, weight gain of pregnant rats was significantly lower at the 9th and 11th days (P < 0.05 vs. control, respectively). No differences of weight gain in comparison with control rats were found at term gestation in pregnant rats exposed to stress continuously. Similarly, stress administered, starting from 12th day till term gestation, had no effect on weight gain. Even though weight gain of pregnant rats during the second half of pregnancy in group stress 1-11 was restored to normal values, a high mortality rate of neonates 1, 2 and 3 weeks after birth was found in this group (P < 0.02, 0.01 and 0.001 vs. controls). There was no significant difference between stressed and control groups with respect to the number or body weight of litters, as well as weight gain of neonates during the first 21 days of life. In addition, in offsprings from all stressed groups, a high number of small for date animals was found after 14 days of life, and 74.4% of these small for date animals died during the peripubertal period. The present data demonstrate that the exposure to stress in utero may induce damaging effects on postnatal development.

Effect of different chronic intermittent stressors and acetyl-l-carnitine on hypothalamic beta-endorphin and GnRH and on plasma testosterone levels in male rats.

Chronic stress affects the reproductive function by modifying the neuroendocrine homeostasis. The aim of the present study was to clarify the neuroendocrine and the gonadal changes following chronic intermittent stress in male rats and the action of a neuroactive drug, acetyl-l-carnitine (ALC). The effect of two different stressors, cold water swimming or ether, on central beta-endorphin (beta-EP) and GnRH contents, and on plasma testosterone levels was investigated. In addition, the response to an acute stress in chronically stressed rats, treated or untreated with ALC (10 mg/day/rat p.o.), was evaluated. The stressors were applied twice a day for 10 days, and rats were killed before, during and after the last stress session. Mediobasal hypothalamus (MBH) beta-EP and GnRH contents, and plasma testosterone levels were evaluated by radioimmunoassay. The following results were obtained: (1) both chronic swimming and ether stress caused a decrease in hypothalamic beta-EP contents; (2) MBH GnRH contents increased after chronic swimming stress but not after ether stress; (3) chronic swimming stress induced a twofold decrease in plasma testosterone levels, while no changes were observed after ether stress; (4) the treatment with ALC prevented the decrease in plasma testosterone levels after chronic swimming stress, and (5) acute stress in chronically stressed animals caused an increase in MBH-beta-EP. The present data showed that chronic swimming stress reduces the reproductive capacity and impairs the capacity to respond to the acute stress and that ALC modulates the hormonal changes to physical stress and prevents the antireproductive effect of chronic cold swimming.

Melatonin treatment delays reproductive aging of female rat via the opiatergic system.

In female rat age-related reproductive decline is accompanied by progressive impairment of the neuroendocrine mechanisms that regulate LH secretion. The biosynthetic activity of the pineal gland is markedly depressed and the nocturnal secretion of melatonin decreases significantly. The aim of the present study was to evaluate whether the nocturnal administration of melatonin via the drinking water (0.4 micrograms/ml) throughout the course of aging from 14 to 24 months of age could (1) influence the age-related changes that occur in basal serum levels of LH and in the LH response to GnRH or to naloxone stimulation at 16, 18 and 20 months of age, and (2) delay the onset of the postreproductive constant estrous-anovulatory state as evaluated by the daily recording of vaginal smears and by occurrence of polyfollicular ovaries at 24 months of age. Our results demonstrate that melatonin replacement delays the increase in LH serum levels and the decrease in LH response to GnRH that occur in 18-month-old control animals. Furthermore, they show that melatonin treatment prevents the loss of LH response to naloxone manifested in control rats between 16 and 20 months of age. Melatonin also appears to prevent the progressive increase in the monthly occurrence of estrus phases as well as to decrease the number of rats with polyfollicular ovaries at 24 months of age in comparison to control animals. These results suggest that the age-related decrease in circulating melatonin during the night may contribute to the reproductive decline of aging, and that this effect may involve the central opioid system.

Acetyl-L-Carnitine Effect on Pituitary and Plasma β-Endorphin Responsiveness to Different Chronic Intermittent Stressors

The aims of the present study were: 1) to compare the effect of two different chronic intermittent stressors i.e. cold‐swimming versus ether, on the pituitary opioidergic system; 2) to evaluate the response of pituitary and plasma β‐endorphin (βS‐EP) to an acute stress in chronically stressed rats; and 3) to evaluate the effect of acetyl‐l‐carnitine treatment (10 mg/day/rat per os at night) on pituitary and plasma β‐EP changes induced by two different types of chronic stress. The stressors were applied twice a day for 10 days. Rats were killed either before, during or after the last swimming or ether stress session. β‐EP was measured by radioimmunoassay in anterior pituitary and in neurointermediate lobe extracts and in plasma. The following observations were made; 1) Chronic intermittent cold‐swimming stress increased anterior pituitary contents and plasma β‐EP levels; 2) both chronic intermittent cold‐swimming stress and ether stress caused an increase of neurointermediate lobe β‐EP contents; 3) as in control animals, rats exposed to chronic intermittent swimming stress reduced pituitary β‐EP contents and raised plasma β‐EP levels in response to the last acute swimming stress; 4) in contrast to control animals, rats exposed to chronic intermittent ether stress did not show any significant response of the pituitary‐plasma opioidergic system to the last acute ether session; 5) the acetyl‐l‐carnitine treatment counteracted the changes evoked by chronic intermittent cold‐swimming stress on the pituitary and plasma β‐EP levels. The present data show that chronic intermittent ether stress impairs the capacity to respond to the acute stress and that acetyl‐l‐carnitine may modulate the changes of β‐EP levels following chronic cold‐swimming stress exposure.

Acetyl-l-carnitine restores the daily pattern of hypothalamic β-endorphin in rats exposed to continuous light

With the aim of evaluating the effect of acetyl-l-carnitine (ALC) on the daily pattern of hypothalamic beta-endorphin (beta-EP), we studied the effect of chronic treatment with ALC on hypothalamic beta-EP contents after suppression of the dark-phase of the light-dark cycle in female rats. We evaluated the hypothalamic content of beta-EP immunoreactivity every 3 h for 24 h in: (1) female rats treated with ALC for 15 days; (2) female rats treated with ALC for 15 days and exposed to continuous light for 24 h. The concentration of beta-EP immunoreactivity in tissue extracts was measured by radioimmunoassay. The results demonstrate that concentrations of beta-EP immunoreactivity in the medial basal hypothalamus show a circadian rhythm, with beta-EP immunoreactivity levels being higher during the night than during the rest of the day. Exposure to continuous light for 24 h abolished the nocturnal increase in hypothalamic beta-EP immunoreactivity. Rats treated with ALC showed a daily pattern in the beta-EP content of the medial basal hypothalamus similar to that of control rats. These data emphasize the possible role of ALC in restoring or maintaining the endogenous rhythmicity of central beta-EP.

Ontogeny of the circadian rhythm in medial basal hypothalamic ß-endorphin content in female rat

The present study evaluated the possible role of estrogens in generating the circadian rhythm of medial basal hypothalamus content at the time of puberty in female rats. Accordingly, changes in medial basal hypothalamus ß-endorphin (ß-EP) content were investigated in female rats, before and at puberty. Groups of intact or ovariectomized rats were studied after estradiol-benzoate or placebo treatment. The results showed that circadian rhythm of ß-EP content of medial basal hypothalamus is absent in prepubertal rats, while it appears at puberty, associated to a significant increase of ß-EP concentration. The primary involvement of steroids in generating this circadian rhythm was supported by the finding that estradiol-benzoate treatment caused a precocious appearance of ß-EP hypothalamic diurnal changes in prepubertal rats. Moreover, estradiol-benzoate replacement restored the loss of ß- EP nocturnal increase induced by ovariectomy in pubertal animals. Therefore, these data support the significant role of estrogen in inducing the circadian rhythm of ß-EP content in medial basal hypothalamus at the time of puberty in female rats.