J. Gardi

Nuclear factor-κB-like activity increases in murine cerebral cortex after sleep deprivation

Several well-defined sleep regulatory substances, e.g., interleukin-1β, activate the heterodimeric transcription factor nuclear factor-κB (NF-κB). Several substances that inhibit sleep, e.g., interleukin-4, inhibit NF-κB activation. NF-κB activation promotes production of several additional substances thought to be involved in sleep regulation, e.g., nitric oxide. We investigated, therefore, whether there are diurnal rhythms of NF-κB activation in brain and changes in the activation after sleep deprivation. Mice were kept on a 12:12-h light-dark cycle. In one experiment, groups of mice were killed every 3 h across the 24-h cycle. In another experiment, mice were killed at 1500 after 6 h of sleep deprivation, and a group of control mice were killed at the same time. Nuclear proteins were extracted from each brain tissue sample, and NF-κB-like activity was determined with an electrophoretic mobility shift assay. In cerebral cortex, but not other areas of brain, there was a diurnal rhythm in NF-κB-like activation; highest levels were found during the light period. NF-κB-like activation was higher in cerebral cortex after sleep deprivation compared with values obtained from control mice. The results are consistent with the hypothesis that sleep regulation involves multiple gene events, some of which include enhanced production of sleep regulatory substances, the actions of which involve NF-κB activation.

Diurnal variations and sleep deprivation-induced changes in rat hypothalamic GHRH and somatostatin contents

Previous reports indicate that hypothalamic growth hormone-releasing hormone (GHRH) promotes sleep and is involved in sleep regulation. The aim of our experiments was to determine whether the GHRH and somatostatin contents of the rat hypothalamus have diurnal variations and whether they are altered by sleep deprivation (SD). Hypothalamic samples were collected at 10 time points during the 24-h light-dark cycle. SD started at light onset. Hypothalamic samples were obtained after 4 and 8 h of SD and after 1 and 2 h of recovery following 8 h of SD. The peptides were determined by means of radioimmunoassay. GHRH displayed significant diurnal variations with low levels in the morning (a transient rise occurred at 1 h after light onset), gradual increases in the afternoon (peak at the end of the light period and beginning of the dark period), and decreases at night. SD induced significant GHRH depletion, which persisted during recovery. The afternoon rise was delayed, and the nocturnal decline of somatostatin was more rapid than the changes in GHRH. Although the patterns of the diurnal variations in GHRH and somatostatin were similar, there was no significant correlation between them. SD did not alter somatostatin significantly. Comparisons of the present results with previously reported changes in hypothalamic GHRH mRNA suggest that periods of deep nonrapid eye movement sleep (first portion of the light period and recovery sleep after SD) are associated with intense hypothalamic GHRH release.

Central administration of the somatostatin analog octreotide induces captopril-insensitive sleep responses

The effects of intracerebroventricular injections of the long-lasting somatostatin analog octreotide (Oct) were studied on sleep and behavior in rats. Pyrogen-free physiological saline and Oct (0.001, 0.01, 0.1 microgram) or vehicle were administered at light onset, and the electroencephalogram (EEG), motor activity, and cortical brain temperature were recorded during the 12-h light period. Plasma growth hormone (GH) concentrations were measured in samples taken at 30-min intervals after Oct. Oct (0.01 and 0.1 microgram) suppressed non-rapid eye movement sleep (NREMS) for 1-2 h. NREMS intensity (delta EEG activity during NREMS) dose dependently increased in hour 3 postinjection and thereafter (0.1 microgram). Plasma GH concentrations were suppressed after Oct (0.01 and 0.1 microgram), but pulses of GH secretions occurred 90-120 min postinjection in each rat. Oct (0.1 microgram) enhanced behavioral activity, including prompt drinking followed by grooming, scratching, and feeding. Intracerebroventricular injection of the angiotensin-converting enzyme inhibitor captopril (30 microgram, 10 min before Oct), blocked these behavioral responses but not the Oct-induced sleep alterations. The changes in sleep after intracerebroventricular Oct suggest an intracerebral action site and might result from Oct-induced variations in the sleep-promoting activity of GH-releasing hormone.The effects of intracerebroventricular injections of the long-lasting somatostatin analog octreotide (Oct) were studied on sleep and behavior in rats. Pyrogen-free physiological saline and Oct (0.001, 0.01, 0.1 μg) or vehicle were administered at light onset, and the electroencephalogram (EEG), motor activity, and cortical brain temperature were recorded during the 12-h light period. Plasma growth hormone (GH) concentrations were measured in samples taken at 30-min intervals after Oct. Oct (0.01 and 0.1 μg) suppressed non-rapid eye movement sleep (NREMS) for 1-2 h. NREMS intensity (delta EEG activity during NREMS) dose dependently increased in hour 3 postinjection and thereafter (0.1 μg). Plasma GH concentrations were suppressed after Oct (0.01 and 0.1 μg), but pulses of GH secretions occurred 90-120 min postinjection in each rat. Oct (0.1 μg) enhanced behavioral activity, including prompt drinking followed by grooming, scratching, and feeding. Intracerebroventricular injection of the angiotensin-converting enzyme inhibitor captopril (30 μg, 10 min before Oct), blocked these behavioral responses but not the Oct-induced sleep alterations. The changes in sleep after intracerebroventricular Oct suggest an intracerebral action site and might result from Oct-induced variations in the sleep-promoting activity of GH-releasing hormone.

Insulin-like growth factor- 1 (IGF-1)-induced inhibition of growth hormone secretion is associated with sleep suppression

The hypothalamic growth hormone (GH)-releasing hormone (GHRH) promotes non-rapid eye movement sleep (NREMS). Insulin-like growth factor-1 (IGF-1) acts as a negative feedback in the somatotropic axis inhibiting GHRH and stimulating somatostatin. To determine whether this feedback alters sleep, rats and rabbits were injected intracerebroventricularly (i.c.v.) with IGF-1 (5.0 and 0.25 microgram, respectively) and the sleep-wake activity was studied. Compared to baseline (i.c.v. injection of physiological saline), IGF-1 elicited prompt suppressions in both NREMS and rapid eye movement sleep (REMS) in postinjection hour 1 in rats and rabbits. The intensity of NREMS (characterized by the slow wave activity of the EEG by means of fast-Fourier analysis) was significantly enhanced 7 to 11 h postinjection in rats. Plasma GH concentrations were measured in 30-min samples after i.c.v. IGF-1 injection in rats and a significant suppression of GH secretion was observed 30 min postinjection. The simultaneous inhibition of the somatotropic axis and sleep raises the possibility that the sleep alterations also result from an IGF-1-induced suppression of GHRH. The late increases in NREMS intensity are attributed to metabolic actions of IGF-1 or to a release of GHRH from the IGF-1-induced inhibition.

Interleukin-1β stimulates growth hormone-releasing hormone receptor mRNA expression in the rat hypothalamus in vitro and in vivo

Changes in growth hormone‐releasing hormone (GHRH), GHRH‐receptor (R), somatostatin and interleukin (IL)‐1β mRNA levels were determined in fetal rat hypothalamic cultures after administration of IL‐1β (1, 10, 100 ng/ml, 2 h incubation), and in adult rat hypothalamus 5 h after intracerebroventricular injection of IL‐1β (2.5 and 25 ng). IL‐1β stimulated GHRH‐R mRNA expression both in vitro (10 and 100 ng/ml) and in vivo (2.5 and 25 ng). Somatostatin mRNA was significantly stimulated and GHRH mRNA slightly reduced in vitro, while these mRNA species were not altered in vivo in response to IL‐1β. IL‐1β stimulated its own expression both in vitro (10 and 100 ng/ml) and in vivo (25 ng). IL‐1β‐induced mRNA responses occurred 2 h after treatment in vitro (incubation times, 30 min to 6 h). IL‐1β also elicited slight GHRH releases in vitro. Up‐regulation of hypothalamic GHRH‐R by IL‐1β may explain previous findings suggesting that IL‐1β stimulates GHRH activity.

Research reportInsulin-like growth factor-1 (IGF-1)-induced inhibition of growth hormone secretion is associated with sleep suppression1

The hypothalamic growth hormone (GH)-releasing hormone (GHRH) promotes non-rapid eye movement sleep (NREMS). Insulin-like growth factor-1 (IGF-1) acts as a negative feedback in the somatotropic axis inhibiting GHRH and stimulating somatostatin. To determine whether this feedback alters sleep, rats and rabbits were injected intracerebroventricularly (i.c.v.) with IGF-1 (5.0 and 0.25 microgram, respectively) and the sleep-wake activity was studied. Compared to baseline (i.c.v. injection of physiological saline), IGF-1 elicited prompt suppressions in both NREMS and rapid eye movement sleep (REMS) in postinjection hour 1 in rats and rabbits. The intensity of NREMS (characterized by the slow wave activity of the EEG by means of fast-Fourier analysis) was significantly enhanced 7 to 11 h postinjection in rats. Plasma GH concentrations were measured in 30-min samples after i.c.v. IGF-1 injection in rats and a significant suppression of GH secretion was observed 30 min postinjection. The simultaneous inhibition of the somatotropic axis and sleep raises the possibility that the sleep alterations also result from an IGF-1-induced suppression of GHRH. The late increases in NREMS intensity are attributed to metabolic actions of IGF-1 or to a release of GHRH from the IGF-1-induced inhibition.

Sleep loss alters hypothalamic growth hormone-releasing hormone receptors in rats

Previous experiments suggest that sleep deprivation (SD) is associated with growth hormone-releasing hormone (GHRH) release and that GHRH promotes sleep via intrahypothalamic sites of action. Binding of [His(1), (125)I-Tyr(10), Nle(27)]hGHRH(1-32) amide and GHRH receptor (GHRH-R) mRNA levels were determined in the hypothalamus and pituitary of rats subjected to 8 h of SD and of undisturbed control rats. The characteristics of the hypothalamic GHRH binding sites differed from those of the pituitary. High affinity GHRH binding and GHRH-R mRNA levels decreased by 50% in the hypothalamus of SD rats, whereas there were no alterations in the pituitary. The results demonstrate that GHRH-Rs exist in the hypothalamus and they respond differently to SD than the GHRH-Rs in the pituitary. The SD-induced changes are explained by down-regulation of the hypothalamic GHRH-Rs induced by GHRH release during and after SD.

Alterations in GHRH binding and GHRH receptor mRNA in the pituitary of adult dw/dw rats.

Lewis dwarf (dw/dw) rats exhibit growth hormone (GH) deficiency and growth retardation linked to a malfunction of GHRH signaling. In this study, GHRH-receptor (GHRH-R) binding and mRNA in the pituitary of adult male dw/dw and age-matched normal Lewis rats was measured by radioligand binding assay and real-time PCR. Only one of nine pools of dw/dw pituitary membranes revealed detectable binding of [His(1), 125I-Tyr(10), Nle(27)]hGHRH(1-32) amide (B(max); 4.3 fmol/mg protein). In contrast, GHRH-R binding was 22.4 +/- 2.60 fmol/mg protein in normal Lewis rats. mRNA for GHRH-R was detectable in all dw/dw rat pituitaries examined, averaging 21% that of Lewis rats. Low expression of GHRH-R reflects reduced GHRH-R mRNA as well as a possible reduction in translation of the receptor protein.