An overarching conceptual framework that continues to enrich sleep and respiratory neurobiology is the widely recognized importance of brain stem cholinergic neurotransmission. Within this framework, the long-term objectives of this renewal application are to elucidate signal transduction processes modulating pontine acetylcholine (ACh) release, sleep, and breathing. The four aims are unified conceptually and related to the long-term objectives by focusing on ACh release from laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) neurons and LDT/PPT axon terminals projecting to the medial pontine reticular formation (mPRF).
Specific Aims l and 2 will study the modulatory role of nitric oxide and soluble guanylyl cyclase.
Aims 3 and 4 will determine whether adenosine A1 agonists and antagonists can significantly alter pontine ACh release and cholinergic activation of guanine nucleotide binding proteins (G proteins).
Specific Aim 1 will use microdialysis to simultaneously measure nitric oxide levels and ACh release during wakefulness, NREM sleep, and REM sleep.
Aim 1 will test the hypothesis that nitric oxide levels in the LDT/PPT and mPRF are state-dependent and account for significant variance in ACh release and respiratory rate.
Aim 2 will use microdialysis delivery of a nitric oxide-sensitive soluble guanylyl cyclase inhibitor (ODQ) to the mPRF and the LDT/PPT during wakefulness, NREM sleep, and REM sleep while measuring ACh release, sleep, and breathing.
Aim 2 will test the hypothesis that nitric oxide decreases ACh release via soluble guanylyl cyclase.
Aim 3 will test the hypothesis that mPRF ACh release and sleep are altered by microdialysis delivery of an adenosine A agonist and an antagonist to the mPRF and the LDT/PPT.
Aim 4 will quantify G protein activation in 14 sleep and breathing related nuclei using the [35S]GTPgammaS binding assay.
The Aim 4 in vitro studies will test the hypothesis that cholinergic activation of G proteins is enhanced by an A1 adenosine agonist and decreased by an A1 adenosine antagonist. Together, these four aims will advance scientific knowledge by providing the first data evaluating the roles of soluble guanylyl cyclase, adenosine, and directly measured nitric oxide as modulators of pontine ACh release, sleep, and breathing. These basic studies are potentially health related because the pharmacological management of disrupted sleep and breathing can alter adenosine, nitric oxide, and cholinergic neurotransmission. A better understanding of these molecules will advance sleep and respiratory neurobiology and contribute to rational drug development.
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