The Role of Adenosine in Wake/Sleep Transition

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DESCRIPTION (provided by applicant): Adenosine (AD) activation of A1 adenosine receptors (A1R) can inhibit neurons of the brainstem and basal forebrain cholinergic arousal centers and facilitate slow wave sleep as demonstrated by the somnogenic effect of local application of exogenous AD to these centers. This suggests the hypothesis that local increases in AD, correlated with neuronal activity, are sufficient to facilitate slow wave sleep (SWS). This hypothesis may be tested by induction of temporally and anatomically restricted A1R gene deletion using transgenes with selective promotion of the recombinase, Cre, and with an adeno-associated viral vector that expresses the recombinase, Cre. The Cre recombinase will catalyze the gene deletion in transgenic mice with loxP sequences flanking the target, the functionally essential A1R exon. The deletion is predicted to increase waking under baseline conditions and to dampen the rebound sleep response to sleep deprivation. A physiologically relevant means of increasing A1R inhibition is by activation of NMDA receptors. This will be characterized in LDT using in vitro slice and whole cell voltage clamp recording techniques. AD cytoplasmic levels are in equilibrium with extracellular levels, and are, accordingly, of potential importance to extracellular, AD mediated inhibition and sleep/wake state modulation. The role of signal transduction processes in regulating the AD cytoplasmic levels will be investigated. The neuronal form of the major adenosine metabolizing enzyme, adenosine kinase (AK), will be characterized with regard to expression levels and localization at the RNA and protein level. The potential regulation of intracellular adenosine by protein phosphorylation/dephosphorylation of AK will be studied using a combination of molecular biological, protein biochemical, and neuropharmacological approaches. The state of phosphorylation of adenosine kinase across behavioral states and in response to sleep debt and rebound sleep will be assessed in whole animals (both rats and mice) and in brain slices. This may provide the requisite target cellular mechanism(s) for NMDA receptors and other potential modulators of extracellular AD.