James Cherry - US grants

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.

DESCRIPTION (provided by applicant): The vomeronasal organ (VNO) projection pathway mediates many of the effects of pheromones on essential psychosexual and neuroendocrine functions in a variety of vertebrate species. We have shown that different populations of neurons in the VNO of male and female mice respond to pheromones derived from male and female conspecifics and that estradiol and testosterone amplify these effects. Experiments are proposed to extend these findings by determining whether sex differences exist in the expression within the basal zone of the VNO of any three different V2R receptors previously cloned in mice, and whether any such differences in mRNA levels for these receptors are modulated by estradiol or testosterone. We will see whether the ability of male pheromones to augment Fos-IR in mitral cells that project to medial amygdaloid nuclei from the rostral as opposed to the caudal AOB differs in gonadectomized, estrogen-treated male and female mice and will use cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH) for the immediate-early-gene (IEG), c-fos, to determine whether mitral cells in the rostral versus caudal zones of the AOB are differentially activated by pheromones from male versus estrous female mice. Finally, we will determine whether the inhibition of male pheromone-induced IEG activation previously seen in the VNO of females two days after mating also occurs in male mice and in response to pheromones from both sexes, and we will assess the possible role of noradrenergic afferents from the superior cervical ganglia on the mating-induced 'silencing' of subsequent odor-induced IEG activation in the VNO. The results obtained in these studies should improve our understanding of the neurobiological mechanisms that underlie the effects of gender, steroid hormones, and previous coital experience on the processing of olfactory signals by the accessory olfactory system.

In humans exposure to body odorants can produce sex-specific neuroendocrine responses, permit the discrimination of scents based on differences in major histocompatibility alleles, and sex-dependently activate regions of the hypothalamus involved in reproduction. However, failure to identify the neural pathways through which such signals access reproductive circuits has impeded our understanding of how human pheromones may affect behavior. Moreover, despite evidence in animals that such biologically relevant odors may act as natural rewards, the routes through which odors are conveyed to the mesolimbic dopamine system that mediates reinforcement have yet to be identified. Using mice, we have identified a direct projection of the main olfactory bulb (MOB) to the medial amygdala (Me), which projects to areas in the hypothalamus that control reproduction. Preservation of this pathway in humans could explain how odors influence sex partner preference and mating behavior. As chemosignals can be detected by both the main and accessory olfactory systems in the mouse, we will compare the involvement of each system in regulating responses to olfactory cues that control sexual motivation and reward. First, we will determine whether the direct pathway from the ventral MOB to the Me is differentially activated by male and female urinary volatiles in the two sexes and whether adult exposure to testosterone modulates the intensity of such responses. Next we will determine whether a subset of olfactory sensory neurons that express the M5 subtype of a TRP cation channel project to MOB glomeruli that are innervated by Me projecting mitral cells and selectively respond to opposite-sex urinary volatiles. Because we have observed that many of the ventral MOB projections to the Me also send branches to the olfactory cortex, we will map the targets of these projections to determine if specific sites in the olfactory cortex are preferentially innervated and differ in males and females. Choi et al (2005) suggested that odors of reproductive significance in mice activate a circuit of neurons that express the transcription factor, Lhx6, in the Me and bed nucleus of the stria terminalis (BNST) and then terminate in the ventromedial hypothalamic nucleus (VMH). We will determine the extent to which main vs accessory olfactory inputs selectively induce Fos (a marker of neuronal activity) in Lhx6- expressing neurons in the Me, BNST and VMH of male mice, and determine whether these neurons send axons to the ventral tegmental area, the location of dopaminergic cell bodies of the mesolimbic system. Finally, we will use a conditioned place preference paradigm to compare the incentive qualities of body odorants detected by the main vs accessory systems in male mice. Our results should provide insight into the olfactory and limbic circuits that process reproductively salient odor cues, and may also help elucidate the mechanism whereby non-olfactory (visual, auditory, or purely cognitive) inputs control sex partner preference and sexual arousal in humans.