Elsevier

Brain Research

Volume 1004, Issues 1–2, 9 April 2004, Pages 91-97
Brain Research

Research report
Sleep deprivation increases the activation of nuclear factor kappa B in lateral hypothalamic cells

https://doi.org/10.1016/j.brainres.2003.11.079Get rights and content

Abstract

Sleep deprivation increases sleep propensity in rats and mice as well as the production of several sleep-regulatory substances. Nuclear factor kappa B (NF-κB) is a transcription factor implicated in the activation of many of these sleep-promoting substances. A unique population of neurons immunoreactive for the p65 subunit of NF-κB was previously localized within the caudal dorsolateral hypothalamus of rats. Therefore, we evaluated the effect of sleep deprivation on NF-κBp65-immunoreactivity (IR) in cells of this region in rats as well as its nuclear translocation in a κB-lacZ transgenic mouse line. In rats after 6 h of sleep deprivation beginning at light onset, the number of neurons with NF-κBp65-IR increased significantly in the caudal lateral hypothalamus, specifically the magnocellular lateral hypothalamus adjacent to the subthalamus. Sleep deprivation also significantly increased the number of cells expressing NF-κB-dependent β-galactosidase in the magnocellular lateral hypothalamus, zona incerta dorsal, as well as the adjacent subthalamus in the transgenic mice. These results suggest that NF-κB expressing cells within the lateral hypothalamus may be important in the maintenance of the sleep–wake cycle.

Introduction

Central autonomic control is tightly linked with the sensory systems monitoring the internal environment as well as the behavioral regulation of the sleep–wake cycle [35], [45]. An integrated network of cells involved in globally regulating behavioral arousal and sleep states includes the basal forebrain and hypothalamus [43]. Physiological processes regulated by the hypothalamus include thermoregulation, food intake, fluid homeostasis and thirst, reproduction, and sleep. Hypothalamic involvement in sleep regulation was first proposed by von Economo [49]. He described patients experiencing sleep disruptions following an epidemic of encephalitis. Patients with lesions in the posterior hypothalamus had increased sleepiness, while patients that had anterior hypothalamic lesions slept less [50]. His results suggested that sleep was an active process, rather than a simple withdrawal of sensory afferents as previously believed. Sixteen years later, Nauta [28] transected the forebrains of rats at the supraoptic region of the hypothalamus and produced large posterior hypothalamic lesions that led to increased sleepiness. Later, Hess [14] found that electrical stimulation of the hypothalamus produced enhancement of motor suppression and parasympathetic activation. A prominent GABAergic projection from the anterior hypothalamus/preoptic area to the posterior lateral hypothalamus is proposed to be involved in the global dampening of arousal activity during sleep [12], [38]. These data might explain why stimulation of the lateral hypothalamus evokes sleep [40], [41], lesions to the anterior hypothalamus can diminish sleep [25] and microinjections into the lateral hypothalamus with the GABA agonist, muscimol, induces sleep in normal cats and anterior hypothalamic lesion-induced insomniac cats [21], [34].

Nuclear factor kappa B (NF-κB) is a transcription factor that influences multiple genes, including several sleep regulatory substances reviewed [19]. An antibody to the p65 subunit of NF-κB strongly labels a population of neurons in the tuberal lateral hypothalamus of rats [15]. Although DNA binding activity of NF-κB in nuclei isolated from the entire hypothalamus was not enhanced by 6 h of sleep deprivation [7], DNA binding activity of NF-κB from the basal forebrain (consisting of horizontal limb of the diagonal band of Broca, the substantia innominata, and the magnocellular preoptic nuclei) was increased following 3 h of sleep deprivation [3], [4]. Since the lateral hypothalamus is included in a larger population of neurons that extends from the reticular formation through to the basal forebrain, it is important to reevaluate whether NF-κB activation occurs in the lateral hypothalamus utilizing the specific antibody to the p65 subunit and a transgenic mouse strain that was created with a lacZ gene containing a nuclear localization signal under a promoter containing NF-κB binding sites [36]. The aim of the current experiments was to determine whether NF-κB is influenced by sleep deprivation in specific cells within the lateral hypothalamic area. To our knowledge, the influence of sleep deprivation on the activation of NF-κB in the lateral hypothalamus has not heretofore been investigated. Therefore, we evaluated the immunoreactivity (IR) of the NF-κBp65 subunit in rats as well as the nuclear translocation of NF-κB subunits in κB-lacZ transgenic mice after 6 h of sleep deprivation and found increases in the magnocellular region of the lateral hypothalamus medial to the subthalamus. This supports our hypothesis that the NF-κB expressing cells within the lateral hypothalamic region may be involved in the maintenance of the sleep–wake cycle.

Section snippets

Materials and methods

All animal experiments were performed in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Washington State University Animal Care and Use Committee.

Results

NF-κBp65 labeled neurons were scattered throughout many areas of the brain although a prominent labeling of large neurons occurred in the caudal lateral hypothalamus (Fig. 1). These cells were intensely stained allowing for visualization of the proximal dendrites as well as a network of fibers interconnecting among the cell bodies. The pattern of labeling was similar between the control and sleep-deprived rats although, the intensity of immunoreactivity was more prominent in the sleep-deprived

Discussion

These results confirm the finding that NF-κBp65 immunoreactivity occurs in the cytoplasm of neurons in the lateral hypothalamus [15]. In other studies, nuclear translocation of NF-κB determined by a gel shift assay on brain tissues homogenates was enhanced, after sleep deprivation, in the cerebral cortex and basal forebrain but not in the hypothalamus [3], [4], [7]. In contrast, results presented here for the caudal lateral hypothalamus show large increases in the NF-κBp65-immunoreactive cells

Acknowledgements

This work was supported by grants from the National Institute of Health (NS25378, NS31453 to J. Krueger). Georgeann Ellis is currently working in the Department of Pathobiology at Auburn University, College of Veterinary Medicine, Auburn, Alabama 36949. Abdur Rehman is currently working in the College of Pharmacy at Washington State University.

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