Muramyl peptides. Variation of somnogenic activity with structure.

Sleep-promoting activities of muramyl dipeptide (MDP) (NAc-Mur-L-ala-D-isogln) and the naturally occurring muramyl peptide(s), factor S, have recently been demonstrated. We now have amplified our understanding of structural requirements for somnogenic activity. The effects of several analogs of MDP on rabbit slow-wave sleep are presented and these results are compared to the dose-response relationship for MDP. Some tentative conclusions as to structural requirements for somnogenic activity are presented; most notably, amidation of the free gamma-carboxyl of MDP and several of its analogs resulted in the loss of somnogenic activity. MDP also can induce febrile and immunostimulatory responses. In the present paper, we show that some analogs possess immunostimulatory and pyrogenic activity but not somnogenic activity, thus suggesting that these biological activities of muramyl peptides may, in part, be mediated by separate mechanisms.

Tumor necrosis factor alpha: activity dependent expression and promotion of cortical column sleep in rats.

Cortical surface evoked potentials (SEPs) are larger during sleep and characterize a sleep-like state in cortical columns. Since tumor necrosis factor alpha (TNF) may be involved in sleep regulation and is produced as a consequence of waking activity, we tested the hypothesis that direct application of TNF to the cortex will induce a sleep-like state within cortical columns and enhance SEP amplitudes. We found that microinjection of TNF onto the surface of the rat somatosensory cortex enhanced whisker stimulation-induced SEP amplitude relative to a control heat-inactivated TNF microinjection. We also determined if whisker stimulation enhanced endogenous TNF expression. TNF immunoreactivity (IR) was visualized after 2 h of deflection of a single whisker on each side. The number of TNF-IR cells increased in layers II-IV of the activated somatosensory barrel column. In two separate studies, unilateral deflection of multiple whiskers for 2 h increased the number of TNF-IR cells in layers II-V in columns that also exhibited enhanced cellular ongogene (Fos-IR). TNF-IR also colocalized with NeuN-IR suggesting that TNF expression was in neurons. Collectively these data are consistent with the hypotheses that TNF is produced in response to neural activity and in turn enhances the probability of a local sleep-like state as determined by increases in SEP amplitudes.

Sleep and Microbes.

Sleep is profoundly altered during the course of infectious diseases. The typical response to infection includes an initial increase in nonrapid eye movement sleep (NREMS) followed by an inhibition in NREMS. REMS is inhibited during infections. Bacterial cell wall components, such as peptidoglycan and lipopolysaccharide, macrophage digests of these components, such as muramyl peptides, and viral products, such as viral double-stranded RNA, trigger sleep responses. They do so via pathogen-associated molecular pattern recognition receptors that, in turn, enhance cytokine production. Altered sleep and associated sleep-facilitated fever responses are likely adaptive responses to infection. Normal sleep in physiological conditions may also be influenced by gut microbes because the microbiota is affected by circadian rhythms, stressors, diet, and exercise. Furthermore, sleep loss enhances translocation of viable bacteria from the intestine, which provides another means by which sleep-microbe interactions impact neurobiology.

Deficiency of growth hormone-releasing hormone signaling is associated with sleep alterations in the dwarf rat.

The somatotropic axis, and particularly growth hormone-releasing hormone (GHRH), is implicated in the regulation of sleep-wake activity. To evaluate sleep in chronic somatotropic deficiency, sleep-wake activity was studied in dwarf (dw/dw) rats that are known to have a defective GHRH signaling mechanism in the pituitary and in normal Lewis rats, the parental strain of the dw/dw rats. In addition, expression of GHRH receptor (GHRH-R) mRNA in the hypothalamus/preoptic region and in the pituitary was also determined by means of reverse transcription-PCR, and GHRH content of the hypothalamus was measured. Hypothalamic/preoptic and pituitary GHRH-R mRNA levels were decreased in the dw/dw rats, indicating deficits in the central GHRHergic transmission. Hypothalamic GHRH content in dw/dw rats was also less than that found in Lewis rats. The dw/dw rats had less spontaneous nonrapid eye movement sleep (NREMS) (light and dark period) and rapid eye movement sleep (REMS) (light period) than did the control Lewis rats. After 4 hr of sleep deprivation, rebound increases in NREMS and REMS were normal in the dw/dw rat. As determined by fast Fourier analysis of the electroencephalogram (EEG), the sleep deprivation-induced enhancements in EEG slow-wave activity in the dw/dw rats were only one-half of the response in the Lewis rats. The results are compared with sleep findings previously obtained in GHRH-deficient transgenic mice. The alterations in NREMS are attributed to the defect in GHRH signaling, whereas the decreases in REMS might result from the growth hormone deficiency in the dw/dw rat.

Somnogenic activity of immune response modifiers.

Sleepiness is a presenting symptom in nearly all infectious diseases. James Krueger describes how microbial products, such as muramyl peptides, lipid A and double-stranded RNA, as well as endogenous products elicited by these substances, such as interleukin 1, modulate sleep. The altered sleep during infection seems to result from an exaggerated activation of physiological sleep mechanisms, since normal sleep is controlled by a wide range of substances including many of these immune response modifiers.

Microbial products and cytokines in sleep and fever regulation.

Excessive sleepiness and fever are constitutional symptoms associated with systemic infection. Although fevers have been investigated for many years, sleep responses to infectious challenge have only recently been investigated. Inoculation of animals with bacterial, viral, protozoan and fungal organisms result in complex sleep responses dependent upon the microbial agent and route of administration. The general pattern is characterized by an initial robust increase in non-rapid eye movement sleep (NREMS) followed by a period of NREMS inhibition. REMS is inhibited after infectious challenge. The sleep responses are accompanied by fever but the two responses are, in part, independent from each other. Sleep responses, like fevers, may be beneficial to host defense although this area is relatively uninvestigated. Microbial products likely responsible for sleep and fever responses include bacterial muramyl peptides and endotoxin, and viral double stranded RNA. These microbial products induce sleep and fever responses in animal models. The exact mechanism of how these structurally diverse microbial products elicit sleep and fever remain unknown; however these substances share the ability to induce cytokine production. Cytokines such as interleukin-1 (IL-1), tumor necrosis factor, acidic fibroblast growth factor (FGF), and interferon-alpha (IFN-alpha) are somnogenic whether given directly into brain or intravenously. Other cytokines lack somnogenic activity, e.g., IL-2, IL-6, IFN beta and basic FGF. The somnogenic actions of cytokines probably involve growth hormone-releasing hormone (GHRH) and nitric oxide. Anti-GHRH or inhibition of NO production inhibits normal sleep and inhibits IL-1-induced sleep. In conclusion, cytokines are likely key mediators of fever and sleep responses to infection. The microbial-cytokine altered sleep likely results from an amplification of physiological sleep mechanisms which include cytokines, several neuropeptides and neurotransmitters such as nitric oxide.

Interactions of cytokines with the hypothalamus-pituitary axis.

Major humoral mechanisms include the endocrine and immune systems, and there is substantial literature describing interactions between these systems during infection and inflammatory processes. Within the brain, such interactions are less well known. One major brain function altered during infection and inflammation and by several endocrine hormones is sleep. These changes in sleep provide a useful illustration of the interactions between cytokines and the hypothalamus-pituitary axis (HPA). Experimental evidence is reviewed that illustrates the interaction of cytokines, especially interleukin-1 (IL-1), with the HPA in regard to their effect on sleep. The evidence linking IL-1, growth hormone-releasing hormone/growth hormone, and corticotropin-releasing hormone to sleep regulation is reviewed. There is also evidence that shows that these two major sleep-regulatory systems are linked to each other.

Interleukin-10 inhibits spontaneous sleep in rabbits.

Proinflammatory cytokines, including interleukin-1beta(IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are involved in sleep regulation. IL-10 is an anti-inflammatory cytokine that inhibits proinflammatory cytokine production. We hypothesized that IL-10 could attenuate sleep. Thirty-one male rabbits were used. Three doses of IL-10 (5 ng, 50 ng, and 250 ng) were injected intracerebroventricularly during the rest (light) period. One dose of IL-10 (250 ng) was injected during the active (dark) cycle. Appropriate time-matched control injections of saline were given to the same rabbits on different days. The two highest doses of IL-10 significantly inhibited spontaneous nonrapid eye movement sleep if IL-10 was given during the light cycle. The highest dose of IL-10 (250 ng) also significantly decreased rapid eye movement sleep. IL-10 administered at dark onset had no effect on sleep. The sleep inhibitory properties of IL-10 provide additional evidence for the hypothesis that a brain cytokine network is involved in regulation of physiologic sleep.

Sleep-associated changes in interleukin-1beta mRNA in the brain.

Much evidence implicates interleukin-1beta (IL-1beta) in sleep regulation. Two previous studies indicated that levels of IL-1beta in mRNA were affected by sleep. In the current study, levels of IL-1beta mRNA and IL-1 receptor assessory protein (IL-1RAP) mRNA were determined 1 h after the beginning of light and dark periods and after sleep deprivation, using the reverse transcriptase-polymerase chain reaction (RT-PCR) and mutated internal standards. Daytime samples contained relatively more IL-1beta mRNA than nighttime samples, and levels of IL-1beta mRNA were higher after sleep deprivation. These changes occurred in the hypothalamus, hippocampus, cerebral cortex, and mesencephalon/pons. In contrast, the IL-1 RAP mRNA level did not seem to be affected by sleep.

Modulation of human leukocyte antigenDR expression in glioblastoma cells by interferon gamma and other cytokines.

To study the regulation of major histocompatibility complex class II antigen by central nervous system cells, the expression of one of these antigens, human leukocyte antigenDR (HLADR) in three human glioblastoma cell lines (HTB14, 16 and 17) and a neuroblastoma cell line (HTB11) was determined. Interferon-gamma (IFN gamma) induced HTB16 and HTB17 cells to express HLADR, and enhanced the antigen expression in HTB14 cells, but it failed to induce HLADR expression in HTB11 cells. Tumor necrosis factor-alpha amplified and accelerated the expression of HLADR induced by IFN gamma in HTB16 cells. Interleukin-1 beta, prostaglandin E2 and transforming growth factor-beta suppressed IFN gamma-induced HLADR expression in HTB16 cells. Several other substances tested did not affect HLADR expression or IFN gamma-induced HLADR. These findings confirm that IFN gamma plays a role in the regulation of HLADR expression in cells derived from the brain and that some other cytokines modify IFN gamma-HLADR interactions.

Tumor necrosis factor receptor fragment attenuates interferon-gamma-induced non-REM sleep in rabbits.

Although the somnogenic actions of interferon-alpha (IFNalpha) and IFNbeta have been reported, the sleep effects of IFNgamma remained unknown. Thus, we investigated the effects of intracerebroventricular injection of human IFNgamma on sleep in rabbits. IFNgamma dose-dependently increased nonrapid eye movement sleep (NREMS), electroencephalographic slow wave activity and brain temperature (Tbr). These effects were markedly attenuated after the heat treatment of IFNgamma. IFNgamma suppressed rapid eye movement sleep if given during the light period, but not if given at dark onset. Although a tumor necrosis factor receptor fragment did not affect any sleep parameters when given at dark onset, it significantly attenuated IFNgamma-induced NREMS and Tbr. These data suggest that IFNgamma may be involved in the sleep responses during infection. Further, IFNgamma may have a synergistic interaction with intrinsic TNFalpha in the brain.

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1 beta or an IL-1 beta fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor.

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1 beta, tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. Cells were also tested under identical conditions with an IL-1 beta synthetic peptide fragment (IL-1 beta 208-240) previously shown to possess biological activity. IL-1 beta, TNF alpha and IFN gamma potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1 beta 208-240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1 beta, rabbit IL-1 beta 208-240 and human IL-1 beta 208-240 was blocked by the IL-1ra, while TNF alpha- and IFN gamma-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1 beta and human IL-1 beta 208-240 was also blocked by the sIL-1R. Our findings suggest that IL1 beta 208-240 acts as an IL-1 beta agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.

Diurnal variations of interleukin-1 beta mRNA and beta-actin mRNA in rat brain.

Interleukin-1 beta (IL-1 beta) is posited to play an important physiological role in brain functions in addition to its better defined role in pathology. The experiments described herein were performed to determine if IL-1 beta mRNA and beta-actin display diurnal rhythms in various areas of brain. Rats were sacrificed at 4 h intervals across a 12:12 h light/dark cycle. Hypothalamic, hippocampal and cortical IL-1 beta mRNA peaked just after lights were turned on, declined slightly during the remaining light period and stayed low in the dark. There were no significant changes in IL-1 beta mRNA in brain stem or cerebellum samples. beta-actin mRNA levels were relatively constant across the day in the hypothalamus, brain stem and cerebellum. However, beta-actin mRNA levels were lower during the day than during the night in the hippocampus and cortex.

Fos-immunoreactivity in the hypothalamus: dependency on the diurnal rhythm, sleep, gender, and estrogen.

Diurnal variations and sleep deprivation-induced changes in the number of Fos-immunoreactive (Fos-IR) neurons in various hypothalamic/preoptic nuclei were studied in the rat. The nuclei implicated in sleep regulation, the ventrolateral preoptic (VLPO), median preoptic (MnPO), and suprachiasmatic (SCN, dorsomedial subdivision) nuclei, displayed maximum c-fos expression in the rest (light) period. Sleep deprivation (S.D.) suppressed Fos-IR in the dorsomedial subdivision of SCN but failed to alter Fos in the VLPO. Fos-IR increased in the VLPO during recovery after S.D. A nocturnal rise in Fos expression was detected in the arcuate (ARC), anterodorsal preoptic (ADP) and anteroventral periventricular (AVPV) nuclei whereas the lateroanterior hypothalamic nucleus (LA) and the ventrolateral subdivision of SCN did not display diurnal variations. S.D. stimulated Fos expression in the ARC, ADP, and LA. Statistically significant, albeit modest, differences were noted in the number of Fos-IR cells between males and cycling female (estrus/diestrus) in the VLPO, MnPO, ARC, LA, and AVPV, and the female ADP did not display diurnal variations. Ovariectomy (OVX) was followed by marked reduction in Fos expression in the VLPO, SCN, and AVPV, and the diurnal rhythm decreased in the VLPO, and vanished in the dorsomedial SCN, and AVP. Estrogen administration to OVX female rats stimulated Fos expression in most nuclei, and the lost diurnal variations reoccurred. In contrast, castration of male rats had little effect on Fos expression (slight rises in diurnal Fos in the ARC and ventrolateral SCN). The results suggest that Fos expression is highly estrogen-dependent in many hypothalamic nuclei including those that have been implicated in sleep regulation.

Gadolinium chloride pretreatment prevents cafeteria diet-induced sleep in rats.

The liver Kupffer cells constitute the largest population of fixed macrophages in the body and reside at a strategic position in liver sinusoids to interact with mediators from the gut. Previously, we showed that cafeteria feeding increases sleep by a subdiaphragmatic mechanism and increases interleukin-1beta (IL-1beta) mRNA expression in rat liver and brain. Thus, the aim of the present experiment was to test the hypothesis that macrophages, in particular liver Kupffer cells, contribute to the excess sleep observed in cafeteria diet fed rats. Sleep-wake activity and brain temperature (T(br)) were examined in rats injected with gadolinium chloride (GdCl3) alone and in rats fed a cafeteria diet with or without prior pretreatment with GdCl3. The intravenous administration of GdCl3 alone, using a dose that blocks phagocytosis and eliminates large Kupffer cells (7.5 mg/kg), increased sleep in the dark period of the light-dark cycle and decreased sleep in the light period. Sleep-wake activity returned to baseline levels 24 h after the injection. In control rats, cafeteria feeding increased non-rapid eye movement sleep (NREMS) and T(br), and decreased rapid eye movement sleep (REMS) and electroencephalographic slow-wave activity (SWA) during NREMS. GdCl3 pretreatment prevented the increase in NREMS, but did not significantly affect REMS, T(br), or SWA during NREMS compared with the control rats. These results suggest that liver Kupffer cells contribute to the excess NREMS that accompanies increased feeding possibly via their capacity to produce IL-1beta.

Effect of sleep deprivation on serum influenza-specific IgG.

Intra-pulmonary protection against influenza virus in immune mice, largely dependent upon serum lgG, is reported to be suppressed by 7 hours of sleep deprivation following viral challenge. This implies that sleep deprivation may accelerate the catabolism of influenza-specific antibodies. To determine the effects of sleep deprivation on the catabolism of serum antibodies, BALB/c mice were passively immunized intravenously with lgG anti-influenza monoclonalantibodies and catabolism kinetics monitored for 6 days. Mice were then sleep-deprived for either 9 hours (one episode) or for 9 hours followed by 6 hours on the consecutive day (two episodes) and the serum titer of influenza-specific monoclonal antibodies monitored for an additional 8 days via ELISA. One episode of sleep-deprivation had only minor effects on lgG catabolism; however, two episodes of sleep-deprivation caused significant changes in the kinetics of lgG catabolism, resulting in elevated lgG levels (p = 0.02) for 2 days post-sleep deprivation. Elevation of serum influenza-specific lgG (p = 0.005) was also seen in actively immune mice following two episodes of sleep-deprivation. Serum chemistries ruled out dehydration as a cause of the increased antibody levels; however, some anomalies were noted: total protein and albumin were elevated, although not significantly, and P and Ca were decreased. Thus, our data do not support the hypothesis that sleep-deprivation lowers existing serum antibody titers by accelerating antibody catabolism.

Effects of short-term sleep deprivation on murine immunity to influenza virus in young adult and senescent mice.

Sleep has been proposed as an innate host defense, exerting effects on both specific and nonspecific immunity. In one of the more striking papers dealing with the effects of sleep on specific immunity, Brown et al (Reg. Immunol. 1989; 2: 321-325) reported that depriving influenza virus-immune mice of sleep for 7 hours following total respiratory tract viral challenge abrogated anti-viral immunity within the lungs and lowered the level of anti-influenza antibody in lung homogenates. In the solidly-immune convalescent mouse, nasobronchial immunity to influenza virus has been shown to be due to secretory IgA (S-IgA) within the mucosal mucocilliary blanket, while serum IgG has been shown to mediate protection within the lung parenchyma. In this study we attempted to duplicate the work of Brown et al in solidly immune mice. We were unable to abrogate mucosal anti-influenza viral immunity with a single post-viral-challenge sleep-deprivation episode, nor were we able to depress this immunity with one pre- and two post-challenge sleep-deprivation episodes in young adult or old mice, or with two pre-challenge sleep-deprivation episodes in old mice. Sleep deprivation did not depress the level of serum influenza-specific IgG antibodies, and resulted in increased influenza-specific serum IgG compared with normally sleeping mice in aged immune mice boosted 3 weeks before challenge and sleep deprived once before and twice after challenge (p = 0.005). No differences in anti-viral respiratory immunity were apparent between young and old mice. We conclude that short-term sleep deprivation has minimal effects on pre-existing mucosal and humoral immunity in either the young adult or the senescent mouse.

Differential effects of total and upper airway influenza viral infection on sleep in mice.

Sleepiness is a common perception during most infectious diseases, including viral infections. Previously, we observed that a lethal strain of influenza virus (H1N1) causes a greater increase in non-rapid eye movement sleep (NREMS) than a nonlethal strain of influenza virus (H3N2), suggesting that the magnitude of sleep responses after viral inoculation depends on the severity of the infection. The aim of the present experiment was to further test this possibility. The effects of total airway infection versus upper airway infection on sleep were tested in two groups of mice using the same strain of virus (H1N1). After 2-3 days of baseline sleep recordings. Swiss-Webster mice were infected intranasally with H1N1 influenza virus. Sleep was determined again for an additional 3 days and 6 hours. Total airway infection significantly increased NREMS beginning about 24 hours after the viral inoculation and significantly suppressed rapid eye movement sleep (REMS) with a longer latency. Both the increase in NREMS and the decrease in REMS lasted until the end of the experiment. Total airway infection also significantly decreased the body weight of the mice. In contrast, upper airway infection did not induce clear changes in sleep and body weight.

Sleep modifies glutamate decarboxylase mRNA within the barrel cortex of rats after a mystacial whisker trim.

STUDY OBJECTIVES: Determine the effects of time of day and sleep deprivation on glutamate decarboxylase 67,000 MW (GAD67) mRNA during cortical synaptic reorganization induced by a unilateral trimming of the mystacial vibrissae in rats. DESIGN: Two experiments were conducted--One measured GAD67 mRNA in the barrel cortex at 3 or 6 h after a unilateral whisker cut at either light or dark onset; the other measured GAD67 mRNA after a unilateral whisker cut at light onset with or without sleep deprivation. SETTING: University-based Research Laboratory. PARTICIPANTS: Sprague-Dawley rats (250-350 g). INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: The reverse transcriptase polymerase chain reaction was used to measure the time of day changes in GAD67 mRNA after a unilateral whisker cut. GAD67 mRNA decreased in the barrel cortex at 3 and 6 h after a whisker trim at dark onset when the rats were mainly awake. No changes were observed in GAD67 mRNA levels after a whisker cut at light onset, a time when the rats mainly sleep. In situ hybridization for GAD67 mRNA supported these findings; no changes in GAD mRNA occurred in layer 4 of the barrel cortex that received input from the mystacial whiskers that were trimmed at light onset. However, when the rats were sleep-deprived, GAD67 mRNA increased in the barrel cortex receiving input from the lip hairs surrounding the trimmed mystacial whiskers. CONCLUSIONS: These data imply that sleep modifies GAD67 mRNA expression and that this effect is dependent upon the nature of the ongoing synaptic reorganization. They support the notion that sleep serves a synaptic function.

Prolactin and rapid eye movement sleep regulation.

During the past few years data have accumulated suggesting the involvement of prolactin (PRL) in rapid eye movement sleep (REMS) regulation. Pituitary PRL secretion seems to be, at least in part, sleep-dependent. PRL is also found in the central nervous system. PRL-containing neurons in the hypothalamus project to various structures in the brain. Systemic injection of PRL promotes REMS in rats, cats and rabbits. Intracerebroventricular injection of PRL enhances REMS in rats. Stimulation of endogenous PRL secretion by vasoactive intestinal peptide (VIP) also promotes REMS. Immunoneutralization of blood-borne PRL slightly reduces REMS. Various observations (hypoprolactinemic and hyperprolactinemic rats) indicate that PRL may act on REMS via modulating the diurnal rhythms of REMS. It is likely that hypothalamic PRL is more important for sleep regulation than circulating PRL. Hypothalamic PRL is likely involved in the mediation of the REMS-promoting activity of VIP. We conclude that PRL has a role in REMS regulation.