The broad, long-term objective of this proposal is to elucidate the role of PDE4 in behavioral regulation and in the mediation of the AD effects of PDE4 inhibitors. First, experiments will be carried our to determine which PDE4 subtypes (PDE4A, PDE4B, PDE4D) are involved in the mediation of AD-like behavioral effects, as well as other CNS effects. This will be accomplished by comparing the effects of newly developed PDE4 subtype-selective inhibitors in rats in two behavioral tasks, behavior maintained under a DRL 72-sec schedule and a two-lever rolipram drug discrimination procedure, with their inhibition of recombinant rat PDE4A, PDE4B, PDE4C, and PDE4D. Related experiments will examine the behavioral effects of antisense oligodeoxynucleotides (ODNs) targeted to PDE4A variants (PDE4A1 and PDE4A5) which are hypothesized to be involved in the mediation of AD-like behavioral effects. Second, experiments will be carried out to determine if the behavioral effects are mediated predominantly by inhibitor interaction with the low- or high-affinity rolipram binding sites on PDE4. This will be accomplished by comparing the behavioral effects of PDE4 inhibitors with their affinities for interacting with these sites. Third, experiments will be carried out to determine which PDE4 subtypes are associated with selected receptor-stimulated adenyl cyclase in the brain. This will be accomplished by determining the effects of PDE4 subtype-selective inhibitors, as well as antisense ODNs targeted to PDE4A variants, on the hydrolysis of adenosine-3',5'-cyclic monophosphate (cyclic AMP) formed by stimulation of beta-1 and beta-2 adrenergic or D1 dopaminergic receptor-stimulated adenylyl cyclase in rat brain slices. Fourth, experiments will examine the manner by which presynaptic innervation and cyclic AMP regulate PDE4 in neurons. This will be accomplished by determining the effects of inhibited synaptogenesis, induced with antisense ODNs targeted to synapsin II, or changes in cyclic AMP, induced by an adenylyl cyclase inhibitor or by nonhydrozable cyclic AMP analogs, on the development of PDE4 variants in primary neuronal cultures of rat cerebral cortex. The results of the proposed experiments will show which subtypes of PDE4 are the predominant mediators of the AD and other behavioral effects of PDE4 inhibitors; in addition the relative importance of the low- and high-affinity rolipram binding sites will be established. Finally, the PDE4 variants associated with particular signal transduction systems and the manner by which presynaptic innervation and cyclic AMP regulate these associations will be elucidated. The improved understanding of the function and pharmacology of PDE4 in the brain will begin to clarify its role in the pathophysiology of depression and provide a rational basis for improved pharmacotherapy.
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