Disorders of sleep are estimated to affect 70 million Americans. Sleep is a product of the brain and the cellular mechanisms regulating sleep are incompletely understood. This application is submitted in response to RFA HL-99-001, seeking to stimulate improved molecular, cellular, and systems approaches to investigate sleep in mice. The approach of this application is to use the C57BL/6J mouse (B6) for phenotype characterization of neuronal systems known in other mammals to regulate sleep. The long-term objectives are to advance scientific knowledge by providing data on cholinergic neurotransmission not presently available for mouse. The focus of this application is on muscarinic cholinergic receptors (mAChRs), adenosine A1 receptors, and their associated guanine nucleotide binding proteins (G proteins). Since mammalian sleep is known to be regulated by cholinergic neurotransmission, localizing mAChR subtypes in mouse brain is an essential step for phenotype characterization of the mouse as a model for sleep neurobiology.
Aim 1 will use in vitro autoradiography to localize and quantify mAChR subtypes in nuclei of B6 mouse forebrain and brain stem. These studies will test the hypothesis that mAChRs in mouse brain stem, basal forebrain, and preoptic hypothalamus parallel the distribution of mAChRs in rat and cat. Activation of mAChRs initiates a transmembrane signal transduction cascade mediated by G proteins. Thus, Aim 2 will use autoradiography to quantify G proteins in the brain regions described by Aim 1.
Aim 2 will provide the first phenotype data in B6 mouse localizing cholinergically activated G proteins in brain regions known to regulate sleep and breathing. Concentration-response and antagonist blocking conditions will make it possible to test the hypothesis that G proteins in these nuclei are activated by mAChR stimulation. Cholinergic neuron excitability is modulated by the somnogenic factor adenosine.
Aim 3 will test the hypothesis that sleep deprivation alters muscarinic and adenosinergic activation of G proteins in sleep- and breathing-related brain regions.
Aim 4 will extend characterization of mouse cholinergic phenotype to the cerebral cortex. Cortical electroencephalographic (EEG) activity comprises a defining measure of sleep, and the EEG is cholinergically modulated.
Aim 4 will test the hypothesis that acetylcholine release in frontal association cortex is modulated by muscarinic cholinergic autoreceptors.
Aim 4 will further determine the subtype of cortical muscarinic autoreceptor.
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