Leslie B. Vosshall - US grants

The fruit fly, Drosophila melanogaster, has an acute sense of smell that permits it to locate sources of food with great accuracy. Fruit flies strongly prefer fermenting fruit as a food source. The overall goal of this project is to understand how their olfactory system permits these insects to recognize and discriminate the vast number of odorants in the environment, and thereby locate their preferred food source. Proteins called odorant receptors are present in the nerve cells of the antenna, and these are thought to be responsible for relaying olfactory information from the outside world into the fly's brain. Each such nerve cell, or olfactory receptor neuron, expresses a different odorant receptor, permitting it to identify odorants in the environment. Interestingly, a single odorant receptor, called Or83b, is present in all olfactory receptor neurons. As such, its role is at odds with the antenna's function to distinguish among different odorants. This proposal asks what role this unique odorant receptor plays in the olfactory system of the fly. Will flies that lack this protein be unable to respond to olfactory stimuli? Knowledge gained from these experiments may prove to be useful in mitigating the effects of deleterious insects on the environment. Pest insects locate food crops and animal hosts largely through olfactory cues. Understanding the function of Or83b in Drosophila may result in strategies to control the olfactory behavior of deleterious insects that attack agricultural crops and act as human disease vectors. The education component of this CAREER award involves a course entitled Evolutionary Biology of Brain Morphology and Function, which targets pre-graduate students who come to the Rockefeller University campus under the auspices of the University's Science Outreach Program. The course will provide students with hands-on experience in molecular neurobiology, supplemented with outside lectures, and will ask if species-specific behavioral differences have a basis in brain morphology.

The long term goals of this proposal are to understand the molecular and cellular mechanisms that generate the precise connectivity of neurons in the brain. The olfactory system provides an excellent model for axon guidance because a large number of functionally distinct neurons must be wired appropriately for the brain to perceive and discriminate odors. Experiments will be carried out in the fruit fly, Drosphila melanogaster, which has a functionally sophisticated but anatomically simple olfactory system. The availability of the complete genome sequence of this model genetic organism makes it possible to study olfaction at the level of genes, molecules, neurons, and behavior. Approximately 1300 olfactory neurons are wired to 43 olfactory glomeruli in the brain, generating an olfactory sensory map. Odorant recognition is likely to be mediated by a large family of novel odorant receptor genes, which encode 60 different seven transmembrane domain G protein-coupled receptors. In preliminary studies, the P.I. has genetically labeled all neurons expressing a given receptor and demonstrated that axons from these neurons converge with precision to one or two glomeruli in the brain. These studies have led to the hypothesis that functional properties of the olfactory neuron itself are determinants in target selection in the brain. To test this hypothesis, the following specific aims are proposed: (1) Developmental analysis of olfactory axon guidance. (2) Role of the larval antennal nerve as a pioneer fiber in adult olfactory axon guidance. (3) Contribution of the odorant receptor protein to target recognition. (4) Influence of synaptic activity on the formation of the olfactory map. A genetic approach will be used to trace olfactory axon projections in developing animals, in animals whose larval olfactory neurons have been conditionally ablated, and in which neurons expressing a given receptor misexpress a second odorant receptor or a cell- autonomous blocker of synaptic activity. The health relatedness of these studies is that cellular and molecular factors that regulate axonal pathfinding may have direct application to neurological and psychiatric disorders with a developmental basis.

odors; vibration perception; sensory mechanism; olfactions; vibration; olfactory stimulus; aldehydes; biophysics; phenone; clinical research; statistics /biometry; human subject;

DESCRIPTION (provided by applicant): The olfactory system permits animals to perceive a vast number of critical environmental stimuli signaling the presence of food, predators, or mates. Recent advances in the molecular biology of olfaction in diverse species have uncovered these unifying principles: a given olfactory neuron expresses one or a few specific odorant receptor genes; all neurons expressing one odorant receptor converge upon a limited number of targets called glomeruli in the brain; odor stimuli elicit stereotyped patterns of glomerular activation in the brain. However, how the conscious perception of an odor is encoded in the brain is unclear. The long-term goals of this proposal are to understand the molecular and cellular basis for odor coding. Experiments will be carried out in the larval stage of the fruit fly, Drosophila melanogaster, which has an anatomically simple olfactory system amenable to behavior genetic analysis. Preliminary studies have demonstrated that 21 neurons in the dorsal organ at the tip of the larva mediate all olfactory responses to volatile stimuli; that a previously uncharacterized subset of at least 18 Drosophila odorant receptor genes encodes the larval odorant receptors; that a given olfactory neuron expresses one or a few of these odorant receptor genes and targets a dedicated glomerulus in the larval brain; that genetic ablation of a single olfactory neuron leads to selective alterations in olfactory behavior. These results establish the Drosophila larva as the simplest known genetically manipulable model organism that contains all the critical cellular elements found in vertebrate olfactory systems, and set the stage for analyzing odor coding at the level of genes, neurons, circuits, and behavior. These studies have led to the hypothesis that combinatorial activation of olfactory neurons expressing different odorant receptors is required to encode the salient features of a given odorant and activate the appropriate behavioral output. To test this hypothesis, the following specific aims are proposed: (1) Isolating the complete repertoire of larval odorant receptor genes. (2) Determining the peripheral and central organization of larval olfactory neurons. (3) Elucidating the behavioral output of identified larval olfactory neurons. The health relatedness of these studies is that an understanding of circuits underlying sensory perception is relevant for brain disorders with a neurological or psychiatric basis.

Insect vectors of human disease infect hundreds of millions of individuals annually with deadly infectious diseases, including Malaria, Dengue and Yellow Fever, and West Nile Encephalitis. Insects are attracted to human hosts by specific sensory cues, with olfactory cues playing a dominant role. The Broad," Long-Term Objectives of this proposal are to understand the mechanisms by which the olfactory system allows insects to hone in on human hosts. Such knowledge could be translated to a new class of chemical agents that act on the insect olfactory system to interrupt vector insect host-seeking behavior. Responsive to PA-05-154, the overall goal of this specific project is to increase understanding of the molecular structure and function of the odorant receptors (ORs) that mediate odor recognition in insects. In preliminary studies, we have documented a unique chaperoning co-receptor function for OR83b. OR83b is co-expressed and forms OR/ OR83b heterodimers with conventional ORs in most olfactory sensory neurons in vivo and is necessary for the targeting and maintenance of ORs in ciliated dendrites where odor transduction occurs. These preliminary studies have led to the hypothesis that the OR/OR83b complex is essential for insect olfactory function. To test this hypothesis, three Specific Aims are proposed: 1) Identify protein domains that mediate OR83b targeting to olfactory cilia 2) Define protein domains that mediate the formation of heteromeric OR/OR83b complexes 3) Characterize post-translational modifications that modulate the OR/OR83b complex The Research Design involves targeted mutagenesis of OR83b to uncover motifs necessary for trafficking, OR/OR83b heteromerization, and post-translational modifications. The Methods used are phenotypic analysis of Drosophila OR function in vivo and in vitro employing electrophysiological, imaging, cell biological, biochemical, and behavioral techniques. A molecular understanding of insect OR function may aid in the design of novel chemical agents that selectively disrupt insect ORs and thus interrupt host- seeking behaviors of insect disease vectors such as mosquitoes. The Health-Relatedness of this proposal is that such chemical agents may act to reduce infectious disease transmission by vector insects in the future.

DESCRIPTION (provided by applicant): Neuropeptides are important modulators of nervous system function from worms to humans. Produced by specialized neurosecretory cells, their controlled release into local brain circuits and the circulatory system has profound effects on chemosensation, feeding, circadian rhythms, sleep, social behavior, and general physiological homeostasis. In Aedes aegypti mosquitoes, neuropeptides have been implicated in the cyclical behavioral suppression of host attraction that lasts for three days after the female has taken a blood-meal. Although sugar- feeding is sufficient for survival, once female mosquitoes reach reproductive maturity, they require a blood-meal to develop eggs. Attraction to human host cues, including body heat, carbon dioxide, body odor, and taste cues on skin, is only expressed when the female needs blood protein for egg production. For approximately 72 hours after a blood-meal, female attraction to humans is suppressed. This cycle repeats up to 10 times for the approximately one month adult life-span. The spreading of infectious diseases among humans is a by- product of this blood-feeding cycle, so elucidating the mechanisms of host suppression has great health relevance. The broad, long-term objectives of this research are to gain a comprehensive understanding of mosquito neuropeptide and neuropeptide receptor function and to define the mechanisms that mediate the strong suppression of host-seeking behavior after a blood-meal. The proposal has three interlocking specific aims: (1) Annotate neuropeptide and neuropeptide receptor genes in Aedes aegypti and define ligand-receptor interactions. (2) Identify neuropeptide signaling pathways that mediate host-seeking suppression after a blood-meal. (3) Develop a genetic tool-kit for studying neuropeptide signaling in the mosquito. The work is innovative because it uses high-throughput 384-well cell-based screening with a genetically-encoded calcium indicator to deorphanize neuropeptide receptors; pharmacological and neurogenetic tools to probe mechanisms underlying post blood-meal host-suppression; and optimized CRISPR/Cas9 genome-editing to generate a large collection of mutant strains, each lacking a given neuropeptide or neuropeptide receptor. The project will yield significant insights into the ligands and biological function of neuropeptide receptors in this important disease vector insect and will provide new tools to regulate mosquito host-seeking behavior. The strains developed under this project will be a valuable community resource for investigating the physiological functions of neuropeptide signaling.