Karen A. Menuz, PhD - US grants

DESCRIPTION (provided by applicant): Insects that transmit disease often detect humans through olfactory cues, making it imperative to better understand how odors are encoded and elicit behavior in insects. The long term goal of this proposal is to illuminate fundamental processes in insect olfactory signaling, with particular focus on a subset of olfactory neurons, coeloconic neurons, known to detect human odorants. Given the evolutionary conservation of olfactory systems amongst insects, we propose to make use of the genetic and behavioral tools available in Drosophila to understand the molecular basis of attraction to these odorants. In my first aim, I will expand the range of odors known to activate coeloconic neurons by characterizing their responses to additional odorants using electrophysiological recordings. In particular, I will focus on amines and carboxylic acids, attractive odors found in human emanations. In my second aim, I will dissect the behavioral significance of coeloconic neuron signaling by utilizing genetic manipulations and a well-characterized behavioral assay with adult flies. Finally, I will identify additional signaling components enriched in coeloconic ORNs that are necessary for olfactory receptor activity by probing the function and localization of candidate genes obtained from an RNA Sequencing screen. Together, these experiments are designed to elucidate molecular mechanisms of olfactory signaling and the means by which environmental signals are transformed into behavior in insects. PUBLIC HEALTH RELEVANCE: Insect-borne diseases, including malaria, kill more than a million people each year. Insects such as mosquitoes primarily rely upon their olfactory system to detect humans. This project is designed to shed light on how human odorants are encoded and elicit behavior in insects. The results could lead to the development of improved insect repellents and traps.

Olfactory receptor neurons (ORNs) require rapid signal termination mechanisms in order to faithfully report odors that often occur at low and rapidly fluctuating levels. These mechanisms must overcome the obstacle of extremely slow diffusion of odors out of olfactory tissues compared to the rate of environmental fluctuations. In species from insects to mammals, primary olfactory neurons are embedded in non-neuronal auxiliary cells. These cells are thought to mediate odorant clearance, which may contribute to signal termination, but there are few demonstrations of their in vivo effects on neuronal activity. In insects, small stereotyped groups of ORNs and auxiliary cells are encapsulated in hair-like structures known as sensilla. My long term objective is to exploit this organization in the genetic model Drosophila to determine if and how auxiliary cells affect odor encoding and behavior. I recently identified an auxiliary cell transporter that affects responses to ammonia, an odor that attracts many insect vectors of disease. Unexpectedly, loss of this transporter Amt did not simply impair response termination, but instead entirely abolished responses in a particular class of olfactory sensilla. In this R03 proposal, I aim to decipher the relationship between odorant uptake and olfactory receptor sensitivity using ammonia detection as a model system. I will take a multidisciplinary approach combining electrophysiology, molecular biology, genetics, and microscopy. In Aim 1, I will test my hypothesis that ammonia uptake serves to prevent olfactory receptor desensitization. Experiments in Aim 2 are designed to investigate the relationship between olfactory receptors' sensitivity to ammonia and their susceptibility to desensitization. In Aim 3, I will determine whether ammonia responses in other sensilla are supported by Rh50, the only other ammonia transporter in insects, or whether their ammonia responses are independent of ammonia uptake. Based on my preliminary data, I expect that this research will point to odorant uptake as an essential regulator of ORN sensitivity and more firmly establish the integral role of auxiliary cells in olfactory circuit activity. These results will serve as a foundation for an R01 proposal examining how such clearance mechanisms are generalized for the enormous number of odors that olfactory systems can detect.

Complex organisms such as vertebrates and insects have developed specialized chemosensory organs in order to locate food, evaluate mates, and avoid toxins. The sensing of volatile odors is particularly challenging because odors are often present at low levels in turbulent plumes, yet many animal behaviors rely on the faithful coding of odor signals by olfactory receptor neurons. The past few decades have witnessed an explosion of research into olfactory systems, following the cloning of the first odor receptors. Initial research focused on understanding odor receptor function, characterizing their odor response profiles, and understanding their effects on behavior. However, many open questions remain regarding the molecular and cellular mechanisms supporting olfaction in the periphery, particularly in regards to insect olfaction. What signaling mechanisms are downstream of insect odor receptor activation? How are odors removed from the peri-neuronal space? What contribution do non- neuronal support cells make to olfactory neuron activity? How are the support cells of olfactory sensilla similar or different than those in sensilla that mediate other sensory modalities? My lab seeks answers for these questions in the Drosophila antenna, their primary olfactory organ. This versatile model organism is advantageous for such studies because we can exploit the large number of available genetic tools and existing knowledge on its stereotyped receptor and neuronal organization. Here we propose to study the functions of several candidate genes that have arisen from our recent computational screen, and may provide answers to several outstanding questions in field olfaction field as described above. One project centers on several highly conserved antennal-enriched signaling genes and their putative roles in amplification and desensitization downstream of odor receptor activation. Two additional projects focus on the interactions between non-neuronal support cells and the signaling of olfactory neurons. One investigates the role of support cells in odor degradation using candidate metabolic genes that arose from our screen and an RNASeq-based method to identify new candidates. We will also elucidate the potential differences in function between the major support cell classes, which are found not only in olfactory sensilla but also in sensilla that mediate other physiological functions.

Abstract Hematophagous insect vectors of disease locate their human targets in large part through chemosensory- driven behaviors. Development of new insect control measures will require an improved understanding of the molecular and cellular basis underlying their olfactory capabilities. Here, we propose to investigate the mechanisms underlying the ability of insects to detect ammonia, an odor released by humans and attractive to both insect vectors of human disease and non-hematophagous insects. We utilize Drosophila as a genetic model organism due to its wealth of genetic tools that allow us to dissect the fine workings of the system. Using our newly developed genetic tools, we have identified a previously unknown fourth neuron in strongly ammonia sensitive ac1 sensilla. The scientific premise of this proposal is that ammonia responses are mediated by these newly identified Rh50+ neurons and do not depend on the previously implicated IR92a odor receptors or IR92a+ neurons. In Aim 1, we propose to characterize the odor response profile of Rh50+ neurons and determine which neuron mediates electrophysiological and behavioral responses to ammonia. In Aim 2, we will examine whether the ammonia sensitivity of Rh50+ neurons is mediated by a novel type of odor receptor unrelated to the two previously identified odor receptor families in insects. Given the highly conserved nature of this putative receptor and its expression in the antenna of all insect species we have examined, we expect that the proposed findings regarding such ammonia-sensitive neurons and receptors in Drosophila will translate to other insect species, including vectors of human disease.