The long-range goals of this research program are to define the fundamental cellular mechanisms involved in olfactory perception. A critical and active component of the molecular pathway underlying odorant transduction is the second messenger molecule cAMP, which has been shown to increase rapidly in olfactory sensory neurons upon odor stimulation. The focus of this proposal is on the enzymes that degrade these molecules- - cyclic nucleotide phosphodiesterases (PDEs)-- and are therefore well- situated to act as modulators of the olfactory signals that are ultimately transmitted to the brain. In mammals one of these enzymes is a homolog of the Drosophila dunce gene, which codes for a cAMP-specific PDE. This gene has recently been cloned in the mouse and shown to be abundantly and preferentially expressed in olfactory sensory neurons. A series of studies are proposed to extend this initial discovery. First, the developmental onset of the dunce homolog, called mPDE2, will be established during early formation of the olfactory system. Regulation of this enzyme will then be examined during degeneration and subsequent regeneration of the olfactory mucosa following unilateral removal of the olfactory bulb, which is the target of the sensory neuron axons. Techniques in molecular genetics will then be used to begin addressing basic questions concerning the role of this PDE in olfaction. First, the gene structure of mPDE2 will be studied, including investigations into the protein and/or mRNA heterogeneity in olfactory tissue. Then the mPDE2 gene will be disrupted, or knocked out, using homologous recombination in embryonic stem cells. Once the mice lacking a functional mPDE2 gene are obtained they will be thoroughly analyzed morphologically, physiologically, and behaviorally for any phenotypic abnormalities, particularly with respect to potential deficiencies associated with olfactory function. Finally, to address more completely the issue of PDE function in olfaction, it will be necessary to characterize the other major PDE found in sensory neurons-- a Ca2+ and calmodulin-activatible PDE, or CaM PDE. Therefore, identification of the CaM PDEs expressed in the olfactory system by molecular cloning is proposed. It is expected that these experiments will contribute important information about the role of cyclic nucleotide PDEs in olfaction.
