The anterolateral projections of the medial basal hypothalamus affect sleep.

The role of the medial basal hypothalamus (MBH) and the anterior hypothalamus/preoptic area (AH/POA) in sleep regulation was investigated using the Halász knife technique to sever MBH anterior and lateral projections in rats. If both lateral and anterior connections of the MBH were cut, rats spent less time in non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS). In contrast, if the lateral connections remained intact, the duration of NREMS and REMS was normal. The diurnal rhythm of NREMS and REMS was altered in all groups except the sham control group. Changes in NREMS or REMS duration were not detected in a group with pituitary stalk lesions. Water consumption was enhanced in three groups of rats, possibly due to the lesion of vasopressin fibers entering the pituitary. EEG delta power during NREMS and brain temperatures (Tbr) were not affected by the cuts during baseline or after sleep deprivation. In response to 4 h of sleep deprivation, only one group, that with the most anterior-to-posterior cuts, failed to increase its NREMS or REMS time during the recovery sleep. After deprivation, Tbr returned to baseline in most of the treatment groups. Collectively, results indicate that the lateral projections of the MBH are important determinants of duration of NREMS and REMS, while more anterior projections are concerned with the diurnal distribution of sleep. Further, the MBH projections involved in sleep regulation are distinct from those involved in EEG delta activity, water intake, and brain temperature.

Cytokine mRNA induction by interleukin-1beta or tumor necrosis factor alpha in vitro and in vivo.

Hypothalamic and cortical mRNA levels for cytokines such as interleukin-1beta (IL1beta), tumor necrosis factor alpha (TNFalpha), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are impacted by systemic treatments of IL1beta and TNFalpha. To investigate the time course of the effects of IL1beta and TNFalpha on hypothalamic and cortical cytokine gene expression, we measured mRNA levels for IL1beta, TNFalpha, interleukin-6 (IL-6), interleukin-10 (IL-10), IL1 receptor 1, BDNF, NGF, and glutamate decarboxylase-67 in vitro using hypothalamic and cortical primary cultures. IL1beta and TNFalpha mRNA levels increased significantly in a dose-dependent fashion after exposure to either IL1beta or TNFalpha. IL1beta increased IL1beta mRNA in both the hypothalamic and cortical cultures after 2-6 h while TNFalpha mRNA increased significantly within 30 min and continued to rise up to 2-6 h. Most of the other mRNAs showed significant changes independent of dose in vitro. In vivo, intracerebroventricular (icv) injection of IL1beta or TNFalpha also significantly increased IL1beta, TNFalpha and IL6 mRNA levels in the hypothalamus and cortex. IL1beta icv, but not TNFalpha, increased NGF mRNA levels in both these areas. Results support the hypothesis that centrally active doses of IL1beta and TNFalpha enhance their own mRNA levels as well as affect mRNA levels for other neuronal growth factors.

Influenza virus-induced glucocorticoid and hypothalamic and lung cytokine mRNA responses in dwarf lit/lit mice.

Influenza virus infection up-regulates cytokines such as interleukin-1beta (IL-1beta) and activates the somatotropic axis and the hypothalamic-pituitary axis. Mice with deficits in growth hormone releasing hormone (GHRH) signaling (lit/lit mice) respond to influenza virus challenge with a progressive decrease in sleep and lower survival rates. Current experiments characterize plasma glucocorticoid responses and hypothalamic and lung mRNA expression of sleep-related genes in lit/lit mice and their heterozygous controls after influenza virus challenge. lit/lit mice had higher basal and post-infection plasma corticosterone levels compared to controls. In contrast, the heterozygous mice increased hypothalamic GHRH-receptor, CRH-type 2 receptor, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) mRNAs after virus treatment while the lit/lit mice failed to up-regulate these substances. In contrast, lung levels of IL-1beta and TNF-alpha mRNAs were greater in the lit/lit mice. These data are consistent with the hypothesis that the sleep response to influenza infection is mediated, in part, by an up-regulation of hypothalamic sleep-related transcripts and they also show that a primary deficit in GHRH signaling is associated with enhanced corticosterone secretion and attenuated hypothalamic cytokine response to infection.

Sleep in spontaneous dwarf rats.

Spontaneous dwarf rats (SDRs) display growth hormone (GH) deficiency due to a mutation in the GH gene. This study investigated sleep in SDRs and their somatotropic axis and compared to Sprague-Dawley rats. SDRs had almost undetectable levels of plasma GH. Hypothalamic GH-releasing hormone (GHRH) mRNA was increased, whereas GHRH-receptor (GHRH-R) and somatostatin mRNAs were decreased in SDRs. Hypothalamic GHRH and somatostatin peptide content decreased in SDRs. Quantitative immunohistochemistry for GHRH and GHRH-R corroborated and extended these findings. In the arcuate nucleus, the number of GHRH-positive cells was significantly higher, whereas GHRH-R-positive perikarya were diminished in SDRs. Cortical GHRH and GHRH-R measurements showed similar expression characteristics as those found in the hypothalamus. SDRs had less rapid eye movement sleep (REMS) and more non-REMS (NREMS) than the control rats during the light period. The electroencephalogram (EEG) delta and theta power decreased during NREMS in the SDRs. After 4-h of sleep deprivation, SDRs had a significantly reduced REMS rebound compared to the controls, whereas NREMS rebound was normal in SDRs. The enhancement in delta power was significantly less than in the control group during recovery sleep. Intracerebroventricular (icv) administration of GHRH promoted NREMS in both strains of rats; however, increased REMS and EEG delta activity was observed only in control rats. Icv injection of insulin-like growth factor 1 increased NREMS in control rats, but not in the SDRs. These results support the ideas that GHRH is involved in NREMS regulation and that GH is involved in the regulation of REMS and in EEG slow wave activity regulation during NREMS.

Rapid eye movement sleep is reduced in prolactin-deficient mice.

Prolactin (PRL) is implicated in the modulation of spontaneous rapid eye movement sleep (REMS). Previous models of hypoprolactinemic animals were characterized by changes in REMS, although associated deficits made it difficult to ascribe changes in REMS to reduced PRL. In the current studies, male PRL knock-out (KO) mice were used; these mice lack functional PRL but have no known additional deficits. Spontaneous REMS was reduced in the PRL KO mice compared with wild-type or heterozygous littermates. Infusion of PRL for 11-12 d into PRL KO mice restored their REMS to that occurring in wild-type or heterozygous controls. Six hours of sleep deprivation induced a non-REMS and a REMS rebound in both PRL KO mice and heterozygous littermates, although the REMS rebound in the KOs was substantially less. Vasoactive intestinal peptide (VIP) induced REMS responses in heterozygous mice but not in KO mice. Similarly, an ether stressor failed to enhance REMS in the PRL KOs but did in heterozygous littermates. Finally, hypothalamic mRNA levels for PRL, VIP, neural nitric oxide synthase (NOS), inducible NOS, and the interferon type I receptor were similar in KO and heterozygous mice. In contrast, tyrosine hydroxylase mRNA was lower in the PRL KO mice than in heterozygous controls and was restored to control values by infusion of PRL, suggesting a functioning short-loop negative feedback regulation in PRL KO mice. Data support the notion that PRL is involved in REMS regulation.

GHRH and sleep.

A significant portion of the total daily growth hormone (GH) secretion is associated with deep non-REM sleep (NREMS). GH secretion is stimulated by the hypothalamic neurohormone, GH-releasing hormone (GHRH). Exogenous GHRH promotes NREMS in various species. Suppression of endogenous GHRH (competitive antagonist, antibodies, somatostatinergic stimulation, high doses of GH or insulin-like growth factor) results in simultaneous inhibition of NREMS. Mutant and transgenic animals with a defect in GHRHergic activity display permanently reduced NREMS which cannot be reversed by means of GH supplementation. GHRH contents and mRNA levels in the hypothalamus correlate with sleep-wake activity during the diurnal cycle and sleep deprivation and recovery sleep. Stimulation of NREMS by GHRH is a hypothalamic action. GABAergic neurons in the anterior hypothalamus/preoptic region are candidates for mediating promotion of NREMS by GHRH. In contrast to NREMS, stimulation of REMS by GHRH is mediated by GH. Simultaneous stimulation of NREMS and GH secretion by GHRH may promote adjustment of tissue anabolism to sleep.

The role of nitric oxide synthases in the sleep responses to tumor necrosis factor-alpha.

It is well established that cytokines such as tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) are involved in physiological sleep regulation, yet their downstream somnogenic mechanisms remain largely uninvestigated. Nitric oxide (NO) is an effector molecule for some TNFalpha actions. Neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) gene knockout (KO) mice sleep differently than their respective controls. In this study, we tested the hypothesis that NO mediates TNFalpha-induced sleep using iNOS and nNOS KO mice and their corresponding wild-type controls. Systemic administration of TNFalpha increased non-rapid eye movement sleep (NREMS) in the two control strains and in the iNOS KO mice during the first 4 h post-injection but failed to increase NREMS in nNOS KO mice. Rapid eye movement sleep (REMS) was suppressed by TNFalpha in nNOS controls but not in the other strains examined. The results suggest that TNFalpha affects sleep, in part, through nNOS.

Different brain structures mediate drinking and sleep suppression elicited by the somatostatin analog, octreotide, in rats.

When injected into the cerebral ventricles, the somatostatin analog, octreotide (OCT) elicits prompt drinking, vasopressin secretion and increases in blood pressure that are attributed to the activation of the intracerebral angiotensinergic system. In addition, OCT induces sleep responses that might be mediated by an inhibition of hypothalamic neurons producing growth hormone-releasing hormone (GHRH). OCT (0.02 microg in 0.2 microl) was microinjected into various brain sites to determine the structures inducing drinking and/or sleep suppression in response to OCT in rats. Drinking (>1 ml water in 10 min) was elicited in 17 rats out of 86 tested. The positive drinking sites resided in or around the subfornical organ (SFO) and the paraventricular nucleus. Both structures are part of the reported angiotensinergic dipsogenic circuit of the brain. These microinjections failed to elicit consistent sleep effects. Sleep suppression (>10% recording time in hour 1) was observed after injection of OCT either into the arcuate nucleus (n=7), where the majority of GHRHergic neurons reside, or into the medial preoptic area/anterior hypothalamus (n=8), where GHRH acts to promote sleep. Administration of OCT into far lateral sites of the lateral preoptic area and lateral hypothalamus stimulated sleep in hour 1 (n=10), perhaps via inhibiting cholinergic neurons previously implicated in arousal. The results are consistent with the hypothesis that somatostatin is involved in the regulation of both water intake and sleep, and suggest that different structures, and therefore different somatostatinergic neuronal pools, mediate these actions.

Biochemical regulation of non-rapid-eye-movement sleep.

The concept, that sleep regulatory substances (sleep factors) exist, stems from classical endocrinology and is supported by positive transfer experiments in which tissue fluids obtained from sleepy or sleeping animals elicited sleep when injected into recipient animals. The transfer experiments concluded with the identification of four sleep factors: delta sleep-inducing peptide (DSIP), uridine, oxidized glutathione, and a muramyl peptide. A physiological sleep regulatory role, however, has not been determined for these substances. In contrast, transfer experiments did not play a part in the development of the strong experimental evidence that implicated the currently known sleep factors in sleep regulation. These substances include adenosine, prostaglandin D2 (PGD2), growth hormone-releasing hormone (GHRH), interleukin-1 (IL1) and tumor necrosis factor (TNF). They promote non-REMS in various species, inhibition of their action or endogenous production results in loss of spontaneous sleep, and their synthesis and/or release display variations correlating with sleep-wake activity. Although the source of these substances vary they all enhance sleep by acting in the basal forebrain/anterior hypothalamus--preoptic region. It is also characteristic of these substances that they interact in multiple ways often resulting in mutual stimulation or potentiation of each other. Finally, there is a third group of substances whose significance in sleep regulation is less clear but for which there are two or more lines of evidence suggesting that they may have a role in modulating non-REM sleep (NREMS). This group includes oleamide, cortistatin, cholecystokinin (CCK), insulin, and nitric oxide (NO). More sleep regulatory substances are likely to be discovered in the future although it is a long and difficult process requiring multiple laboratories to generate sufficient convincing data to implicate any one of them in sleep regulation.

Sleep function.

A theory of sleep function and brain organization positing that sleep serves a neuronal connectivity function and is a fundamental property of highly interconnected groups of neurons (neuronal groups) is presented. Cellular electrical activity within neuronal groups leads to the production of sleep-promoting substances which are also cytokine growth factors. The somnogenic cytokine growth factors (SCGF) in turn, induce molecules necessary for synaptic connectivity. The SCGFs change the synaptic activation patterns within neuronal groups. SCGFs thus induce changes in the input-output relationships of neuronal groups and thereby, cause a neuronal group state shift. Altered input-output relations result in increased efficacy of some synapses. Sleep is thus, targeted to active neuronal groups and serves to incorporate novel stimulus patterns into a synaptic contextual network and also to preserve that network. Coordination of neuronal group state is brought about by sleep regulatory networks. Organism sleep is an emergent property of a population of neuronal groups in the sleep state. After the neuronal group state shift, environmental input is divorced from output. Sleep is thus, useful to keep the animal stationary at a time when its brain is most dysfunctional. Thus, not only is unconsciousness needed because output activity would be out of phase with environmental events, but it is the consequence of the process itself.

Sleep in host defense.

Sleep remains an important enigma in neurobiology; it has a robust adaptive value yet its function remains elusive. Changes in sleep are hallmarks of the acute phase response to infectious challenge. The molecular regulation of these responses involves a cytokine cascade within brain, including interleukin-1 and tumor necrosis factor, and several other substances such as growth hormone releasing hormone, prolactin, nitric oxide and nuclear factor kappaB. These substances are also involved in the regulation of normal spontaneous sleep. Fatigue and sleep disturbances are common in cancer patients and in those receiving cytokine therapy. Regardless, the role of sleep in cancer is relatively uninvestigated.

Alterations in EEG activity and sleep after influenza viral infection in GHRH receptor-deficient mice.

Viral infections induce excess non-rapid eye movement sleep (NREMS) in mice. Growth hormone-releasing hormone receptor (GHRH receptor) was previously identified as a candidate gene responsible for NREMS responses to influenza challenge in mice. The dwarf lit/lit mouse with a nonfunctional GHRH receptor was used to assess the role of the GHRH receptor in viral-induced NREMS. After influenza A virus infection the duration and intensity [electroencephalogram (EEG) delta power] of NREMS increased in heterozygous mice with the normal phenotype, whereas NREMS and EEG delta power decreased in homozygous lit/lit mice. Lit/lit mice developed a pathological state with EEG slow waves and enhanced muscle tone. Other influenza-induced responses (decreases in rapid eye movement sleep, changes in the EEG high-frequency bands during the various stages of vigilance, hypothermia, and decreased motor activity) did not differ between the heterozygous and lit/lit mice. GH replacement failed to normalize the NREMS responses in the lit/lit mice after influenza inoculation. Decreases in NREMS paralleled hypothermia in the lit/lit mice. Lung virus levels were similar in the two mouse strains. Lit/lit mice had a higher death rate after influenza challenge than the heterozygotes. In conclusion, GHRH signaling is involved in the NREMS response to influenza infection.

Sleep in mice with nonfunctional growth hormone-releasing hormone receptors.

The role of the somatotropic axis in sleep regulation was studied by using the lit/lit mouse with nonfunctional growth hormone (GH)-releasing hormone (GHRH) receptors (GHRH-Rs) and control heterozygous C57BL/6J mice, which have a normal phenotype. During the light period, the lit/lit mice displayed significantly less spontaneous rapid eye movement sleep (REMS) and non-REMS (NREMS) than the controls. Intraperitoneal injection of GHRH (50 microg/kg) failed to promote sleep in the lit/lit mice, whereas it enhanced NREMS in the heterozygous mice. Subcutaneous infusion of GH replacement stimulated weight gain, increased the concentration of plasma insulin-like growth factor-1 (IGF-1), and normalized REMS, but failed to restore normal NREMS in the lit/lit mice. The NREMS response to a 4-h sleep deprivation was attenuated in the lit/lit mice. In control mice, intraperitoneal injection of ghrelin (400 microg/kg) elicited GH secretion and promoted NREMS, and intraperitoneal administration of the somatostatin analog octretotide (Oct, 200 microg/kg) inhibited sleep. In contrast, these responses were missing in the lit/lit mice. The results suggest that GH promotes REMS whereas GHRH stimulates NREMS via central GHRH-Rs and that GHRH is involved in the mediation of the sleep effects of ghrelin and somatostatin.

GHRH and IL1beta increase cytoplasmic Ca(2+) levels in cultured hypothalamic GABAergic neurons.

GHRH and IL1beta regulate sleep via the hypothalamus. However, actions of these substances on neurons are poorly understood. In this study, we found both GHRH (100 nM) and IL1beta (1.2 pM) acutely increased cytosolic Ca(2+) in 7.6 and 4.0% of cultured hypothalamic neurons tested, respectively, and 1.2% of neurons responded to both. The neurons that responded were mostly GABAergic (96, 81, and 100% for GHRH, IL1beta, and dual-responsive neurons, respectively).

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.

Humoral Regulation of Sleep.

Cytokines and hormones, including interleukin-1, tumor necrosis factor, growth hormone-releasing hormone, vasoactive intestinal polypeptide and prolactin, are involved in sleep regulation. These substances enhance sleep, inhibition of them inhibits sleep, and their brain levels vary with sleep. This knowledge helps our understanding of the humoral regulation of sleep.