Sleep research has historically been neuron-centric; much effort has focused on determining neuronal circuits and/or actions of transmitter substances produced by neurons. Historically, glia were thought to be merely passive brain residents, yet new research demonstrates that glial cells, particularly astrocytes and microglia, are active participants in sleep regulation and in sleep-immune interactions. For example, recent studies demonstrate that inhibiting vesicular release from astrocytes reduces EEG slow wave activity and attenuates sleep deprivation-induced increases in non-rapid eye movement (NREM) sleep. However, much remains to be learned about the relative contribution of astrocytes to mechanisms underlying alterations in sleep during immune challenge. The cytokines interleukin-1? (IL-1) and tumor necrosis factor-? (TNF) are involved in the regulation of sleep and in the alterations in sleep that occur during immune challenge. Importantly, IL-1 and TNF are produced by neurons and glia, their receptors are present on neurons and glia, and they constitute an important mechanistic link in neuronal-glial communication. Our knowledge about astrocyte contributions to sleep-immune interactions will be advanced if actions of this cell type can be specifically, independently, and reversibly modulated in vivo. Thus, the overall objective of this R21 Exploratory / Developmental Research Grant Award is to selectively stimulate G protein-coupled signaling cascades in astrocytes to elucidate their contributions to the regulation of sleep during immune challenge. To accomplish this task we will use designer receptors exclusively activated by designer drugs (DREADDs). DREADDs are engineered human muscarinic G protein-coupled receptors that are unable to bind their endogenous ligand, acetylcholine. Instead, they bind a chemically inert synthetic ligand, clozapine-N-oxide (CNO), which then stimulates intracellular signaling. To date, DREADDs have almost exclusively been used to modulate activity of neurons. The central hypothesis to be tested by experiments proposed in this application is that stimulating G protein-coupled signaling cascades in astrocytes will attenuate changes in sleep that are induced by immune challenge. We will use the Cre-loxP system, commercially-available GFAP:Cre mice, and adeno-associated virus vectors to selectively express DREADDs on astrocytes. We will then test our overall hypothesis by conducting experiments within the context of two Aims.
In Aim 1 we will stimulate Gq or Gi G protein-coupled signaling in the absence of immune challenge and quantify regional changes in mouse brain cytokine protein.
In Aim 2 we will activate these same signaling cascades and determine the impact of this manipulation on lipopolysaccharide (LPS)-induced alterations in mouse sleep patterns and brain cytokine protein. Upon completing these studies we will not only possess a new toolkit, but we will have novel data that will contribute to our understanding of mechanisms and functions for sleep-immune interactions. These achievements will have a prolonged and sustained impact on the field by filling a gap in our knowledge of sleep and gliotransmission during immune challenge.
Sleep-wake behavior is altered during immune challenge. Mechanisms responsible for altering sleep during immune challenge include the actions in brain of immune-active substances called cytokines. The cellular source of cytokines in brain during immune challenge and interactions between different cell types (neurons, glia) are not well known or understood. We hypothesize that glia, specifically astrocytes, are critical mediators of changes in sleep during immune challenge. We will test this hypothesis using novel mice in which astrocytes can be selectively modulated.
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