This research program was initiated in 1999 in response to RFA HL99001. The goal of the RFA was to stimulate improved molecular, cellular, and systems approaches to investigate sleep in mice. Every human gene has a mouse homologue. This remarkable homology means that the mouse genome may provide unique insights into human disease. Advances in sequencing the mouse genome now require complimentary data regarding normal and abnormal phenotypes. In accord with consensus statements published by The Jackson Laboratory, this application focuses on the C57BL/6J (B6) mouse strain. The long-term objectives are to advance scientific knowledge by providing data not presently available concerning molecules that regulate ACh release and alter electroencephalographic (EEG) excitability, sleep, and breathing.
Aim 1 will test the hypothesis that microinjecting neostigmine into the pontine reticular formation of B6 mouse causes a REM sleep-like state and changes in breathing that are concentration-dependent, site-specific within the pons, and blocked by atropine.
Aim 2 will use in vivo microdialysis and high performance liquid chromatography (HPLC) to test the hypothesis that dialysis delivery of an adenosine A1 receptor agonist into the prefrontal cortex of B6 mouse will decrease cortical ACh release and EEG power, and delay wake-up time from anesthesia.
Aim 3 will use combined microdialysis and microinjection to test the hypothesis that ACh release in the pontine reticular formation of B6 mouse is altered by nitric oxide donors and by inhibitors of nitric oxide synthase.
Aim 4 will use a quantitative Western assay to test the hypothesis that brain expression of M2 muscarinic receptor protein varies significantly as a function of mouse strain and brain region.
These aims will take this research program in new directions by developing a pharmacological model of rapid eye movement sleep in mouse (Aim 1), quantifying the effects of endogenous neuromodulators on ACh release (Aims 2 and 3), and initiating strain comparisons of muscarinic receptor protein expression (Aim 4). The unifying conceptual scheme of this proposal is that higher level phenotypes such as sleep and breathing (Aim 1) emerge from the expression of lower level phenotypes such as ACh (Aims 2 and 3) and muscarinic cholinergic receptors (Aim 4).
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