Nitric Oxide and the Regulation of Behavioral State

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Although much has been learned about the transmitter systems regulating behavioral state, we know little about the actual molecular mechanisms causing the brain to sleep and to wake up. This project is directed at defining these mechanisms. Cholinergic neurons of the laterodorsal tegrnental/pedunculopontine tegmental nucleus, which project to the basal forebrain (BF), as well as intrinsic cholinergic neurons of the BF, both contain nitric oxide synthase (NOS), and are active during waking. We found that: 1) NO evokes the release of adenosine from forebrain neurons in culture by a cGMP-independent mechanism; 2) A NOS inhibitor dialyzed into the BF suppressed NREM sleep following sleep deprivation in rats, and a nitric oxide donor dialyzed into the BF increased NREM sleep. These observations are the basis for one of two major hypothesis motivating this project, which is that NO, released during wakefulness in the BF by cholinergic neurons, is a major stimulus to the release of adenosine during waking, and thus is a key factor regulating the extracellular level of this important somnogen. In addition, we found that: 1) The high affinity cGMP degrading enzyme cyclic nucleotide phosphodiesterase (PDE) 9A is expressed in large neurons of the BF; 2) An inhibitor of cGMP degrading PDEs dialyzed into the BF increased NREM sleep. On the basis of these observations, we hypothesize that NO also has important effects relevant to the regulation of behavioral state that are mediated by the NO/cGMP signaling pathway, and are independent of the adenosine-releasing effects of NO. The specific aims of this project are to: 1) Use mierodialysis with behavioral and electroeneephalographie monitoring to test for the role of NO in homeostatic sleep regulation in the rat using NOS inhibitors and NO donors. 2) Determine the anatomic and cellular localization of the components of the NO/cGMP signal transduction system (cGMP hydrolyzing cyclic nucleotide phosphodiesterases, soluble guanylyl cyclase, NO synthase, protein kinase G) in regions of the brain relevant for sleep/wake regulation. 3) Characterize the electrophysiological effects of NO on neurons of the BF and ventrolateral preoptic nucleus (VLPO) using a basal forebrain/preoptic area BF/POA) slice preparation.