Christopher J. Potter - US grants

DESCRIPTION (provided by applicant): The sense of smell is caused by odorants activating olfactory sensory neurons in the nose. A pivotal question in the field is how these neuronal activities give rise to odor perceptions. Since the neuronal architecture of the olfactory system i remarkably similar between insects and mammals, studying how the fly brain processes olfactory information could shed light on principles underlying olfaction in other organisms. We previously demonstrated that the olfactory cortex (lateral horn) is divided into two broad domains, one representing food odors and one representing pheromones. Therefore, this olfactory region, which to the naked eye looks homogenous, might be organized into olfactory processing centers that reflect biologically relevant information. These studies were based on simple classifications of odorants as being food or pheromone, but did not take into account the valence of the odorant - that is if it is behaviorally attractive or repulsive. Preliminary data frm our recent work suggests that aversive odors might represent a new processing center in the olfactory cortex. Using a combination of genetics, behavioral analyses, axon tracing, and brain imaging studies, we will test the hypothesis that (1) the olfactory cortex is organized into domains based on three important aspects of a fly's life: food, pheromones, and avoidance. Results from this work will allow us to link repulsive behaviors to their underlying neuronal components and thus model how newly identified and known aversive signals might be represented in the higher olfactory cortex. To understand how attractive signals such as attractive food odors and attractive pheromones are represented in the olfactory cortex, as well as how the olfactory system utilizes them to communicate relevant information regarding an animal's environment, we investigate a novel pheromone signaling system that we have identified. Our preliminary data suggests that this signaling system in Drosophila represents a behavioral link between food and pheromone signaling. Using a combination of genetics, molecular biology, GC-MS, calcium imaging, and electrophysiological recordings, we will test the hypothesis that (2) Drosophila secrete an attractive pheromone when stimulated by attractive food odors to tag sites for positive social behaviors such as egg laying or courtship. Results from the characterization of this new pheromone signaling mechanism will enrich our understanding of how olfactory communications are used to guide behavioral responses to a changing environment. The proposed studies are significant because we will gain insights into how aversive and attractive olfactory information is represented in higher processing centers of an olfactory system that could serve as a model for how olfactory perceptions are encoded in the brains of other animals. !

? DESCRIPTION (provided by applicant): Dopamine (DA) is a critical neurotransmitter, conserved from C. elegans to man, that regulates a wide variety of behaviors, including learning and memory, courtship behaviors, reward-seeking, and sleep/wake rhythms. Dysfunction of DA neural circuits in turn contributes to disorders such as Parkinson's disease, depression, and drug addiction. Yet how DA neural circuits contribute to these behaviors or disorders is not well-understood. The goal of this work is to develop, characterize, and utilize novel genetic reagents for the dissection of the DA neural network in Drosophila melanogaster. Drosophila is an ideal system to carry out these studies, as fruit flies have only ~250 DA neurons to drive many of the same DA-dependent behaviors found in mammals. Moreover, flies are highly tractable to neural circuit analyses, as they have a short generation time, well- developed genetic techniques and resources, and can be easily maintained in large numbers. In previous work, we generated novel transgenic fly lines that allowed us to manipulate distinct subsets of DA neurons. Using these lines, we identified a single pair of DA neurons that promote arousal by projecting to and directly inhibiting a sleep-promoting circuit. In addition, we have recently developed a novel genetic method, CLAMP (Cell Labeling Across Membrane Partners), which allows for identification, morphological characterization, and functional manipulation of neurons based solely on connectivity patterns. Here, we propose to generate novel transgenic DA driver lines, which will be used for the identification, characterization, and connectivity mapping of the DA neural network in the fly brain. First, we will generate new DA transgenic fly lines, based on the genomic enhancers from different genes that express in DA cells. Second, we will screen established Gal4 lines for expression in subsets of DA cells. By using a combinatorial genetic intersectional approach, these fly lines will collectively generate ~22,600 distinct labeling patterns containing small subsets of DA neurons. These lines will be made available to the scientific community to facilitate functional analyses of the DA neural network. Third, we will create a comprehensive database of DA neurons in the fly brain by 1) identifying and naming individual DA cells and 2) by using computer tracing techniques combined with registration to a standard brain model to label projection patterns. Fourth, by using the CLAMP method to systematically map the connectivity of these DA neurons, we will develop a detailed model of the DA neural network in Drosophila. Understanding how DA circuits in Drosophila function to regulate different behaviors would provide insights into related mechanisms in mammals, including humans, and thus set the stage for circuit-based therapeutic interventions for specific neurological and psychiatric diseases.

Project Abstract/Summary The recent Zika crisis once again highlighted the cosmopolitan role the yellow fever mosquito plays pasatahporgoelnifsi,c vector for emerging and resurgent arboviral diseases. To blood-feed and consequently transmit Aedes aegypti and other anthropophilic disease vectors rely on their finely-tuned senses to track a variety ofrfocmhehmuimcaalnasn. dDepshpyisteictahleciuredsirinecclturdoilneginbomdeydoiadtoinr,gcsaurcbhonepdiidoexmidieo,lmogoicisatlulyreim, hpeoartt, aanntdbveihsuavaliocrosn, ttrhaestmeomsqauniattoing Ae. aegypti nmmeoouslreqocunuisltaoar-nbpdoartrnheewc edapiytssoerrasespet hsr.east ednett ekcetyhtuamr gaent s- rteol adtiesdr uspetnms oorsyqsutiitmo ublei hr eamvi oairnalnadr gdeel yveulnopk nnoewwns. tTrhateesgei ecse ltloulcaornatnr odl Here, we propose to develop a genetic method in Aedes aegypti that will facilitate To accomplish this goal, we will use the / system of binary expression to direct the identification, isolation, and transcriptional profiling of mosquito sensory neurons activated by nrdeei rduerwcotilntyhaflatehcxei lpictroaetisnescitoihdneeoni dftetphnretei fsbieci onastcieeonnosfoohrf imCghaoMcsaqPluAciiRtuoIm, saecmnosnoocdreiynfinteredautGri oFonPnssmai nonldpeecvrui oilpel ehttehlraiagtlhcsthe. aAnnspgoperlsyi cfaal upt ioporenensodcfaetgnhecisse (tfearcnohtmennigqnruaeeee,nwtiol l human-related cues. QF2 QUAS mpsceoaannrxtstiiaoclliruanylrianynrgelpyusarewlponen,slposlrrsioynubinrtoeeessducpritosoo,nnlisesdegeastnr,oetwicefyiuxnepsgseesnri)nisamcoclreutyindvtneaasetleulaydrloeabanrysgsyheruetionmspcraroenena-acdrsheienlwaigntiettiohdnttvcaruiaroegclseee.ttlTllsuihtgliaihmsrtau.ccTaltilhicaveisiut:smyp1-)e,dcaneinpefiduecnr2aod)inmepanseltraabicrpietohi:vseearntailsooonrrogisaf ns and 1) To develop a genetic method in Aedes aegypti mosquitoes for labeling sensory neurons activated by human-related We will examine appendages for neuronal CaMPARI labeling using a number of cues, 2) To identify candidate molecular receptors from activated mosquito sensory neurons that may latsaeecsmnteisvproaecrtrayeapdsttutunirmreeeusurmlroie:incp1srr),oewhdsueiesmnswteaiicnnlltg-ipdobeenorr.dfiFovyirenmwdaaloclrylrfm,yawocthsteoe(wrc3yti7ilocl?uCnge)se,snoa(efnwradapht3poe)eltemnrhadonuaisgmscteuarsinrpceotoi(ndortonearlaia,nltlpiainvcregotifhcCiluaaeMmcsiPdiud,AsiaitRnmyIg-=mlRa7oNb0neA%il-alse)aed. nqTndoaenCiusaOrolo2ylna)sts,ee,2sft)otholeloidweendtifbyy detect human-related cues. cnaenudroidnast.eTmhioslsetcuudlayrwreilclegpetnoerrsattheaat nmeawy gmeendeitaictetodoeltetoctdioisnsoecf tthmeosseqtuairtgoestehnusmoraynb-rieollaotgeyd, icdueenstiinfyCpaoMpPuAlaRtiIolnasbeolfed neurons in activated by key human-derived cues, and generate a candidate list of associated receptors that may be targeted for novel mosquito behavioral disruption strategies. Aedes aegypti

Project Abstract/Summary Anopheles gambiae mosquitoes, as the insect vector for the Plasmodium parasite that causes malaria, were responsible for the deaths of >450,000 people last year. Fortunately, mosquitoes have a weakness that our research aims to exploit. Mosquitoes use their sense of smell for most human host-seeking behaviors. This suggests that targeting a mosquito's sense of smell could lead to effective measures that prevent bites and the spread of diseases. Indeed, spatial repellents are volatile odorants that effectively disrupt host-seeking behaviors and keep mosquitoes from approaching. They have widespread use in developed nations as personal protective measures, but global adoption is limited due to high costs, unwanted side-effects (such as skin irritation), or the need to use high-concentrations to remain effective. A major limitation to the identification of new, more tractable, repellents is a lack of understanding of how spatial repellents promote repulsion by impacting the mosquito's sense of smell. This is primarily due to challenging technical hurdles needed to link repellent and other odorants to a variety of olfactory neuron functions. Our introduction of the QF2/QUAS genetic binary system into Anopheles mosquitoes overcomes previous technical barriers, and now enables us to directly visualize the response of olfactory neurons, in living mosquitoes, to repellents and human body odors. Using a combination of calcium imaging, single sensillum electrophysiological recordings, and RNA-seq along with our established and novel genetic techniques, we will test the hypothesis that (1) spatial mosquito repellents function by masking, activating, or scrambling the activity of olfactory neurons. We will examine the activity patterns of olfactory neurons in living Anopheles gambiae mosquitoes when stimulated by 20 commonly used mosquito repellents in the presence and absence of human odorants. In addition, we will identify the odorant receptors expressed by olfactory neurons exhibiting altered activity patterns in response to each repellent. Using a combination of genetic approaches (aimed at modulating the function of targeted olfactory neurons) and behavioral assays, we will test the hypothesis that (2) spatial repellents promote repellent behaviors by altering olfactory system signaling. We will experimentally determine which olfactory neurons and what types of olfactory neuron activity changes are either necessary and/or sufficient to drive repellent behaviors. The proposed studies are significant because we will gain new mechanistic insights into how mosquito repellents target the mosquito's sense of smell and enable the development of rationale biology-based strategies to identify new repellents that are cheaper, safer, and more effective for use on a global scale to prevent the spread of disease.

Project Summary The Neuroscience Training Program (NTP) at Johns Hopkins University was established in 1983 to provide students with advanced instruction and research training in fundamental neuroscience and the basis of neurological diseases. It now includes 73 training faculty in 14 different departments across the university, as well as several associated institutes where neuroscience research is performed and the Janelia Research Campus of the Howard Hughes Medical Institute. The program encompasses a broad array of research areas, including molecular, cellular, developmental, sensory, systems, cognitive and computational neuroscience, as well as neurobiology of disease, providing diverse training options and unique opportunities for collaboration for our students. We typically matriculate 14-16 Ph.D. candidates each year, from a pool of >500 applicants, and 1-4 additional candidates for combined MD/Ph.D. degrees (who are admitted through a separate process). The program is currently supported by eight slots from this T32, as well as institutional funds through the Department of Neuroscience. Students enter the program with diverse backgrounds ranging from computer science to biochemistry. To ensure that they learn the basic tenets of neuroscience, they are required to take a year-long integrative lecture and laboratory course, ?Neuroscience and Cognition, and receive rigorous formal training in quantitative methods, statistics, rigor and reproducibility and neurological diseases. Students learn about research opportunities through a mini-symposium series led by Program Faculty (featuring short chalk talks), the Program Retreat, and Lab Lunches (which feature work-in-progress by NTP faculty). This information is used to help students arrange three 8-12-week laboratory rotations, which are typically completed by the end of the first academic year, and form the basis for selecting a thesis advisor. By the end of the second year, students have completed three elective courses, from 18 small seminar-style courses in different neuroscience specialties or relevant courses offered in other departments. In the spring of Year 2, students write and defend a Thesis Proposal that is written in the form of a Predoctoral NRSA application, and are tested on their understanding of the broader topic area and methods for analysis and reproducibility. Each student is advised by two Pre-thesis Advisors in Years 1-2 (at 3 month intervals) and an individualized Thesis Advisory Committee thereafter (at 6 month intervals). Students complete an Individual Development Plan annually and discuss this with their advisor and the Thesis Advisory Committee. The Graduate Program Steering Committee meets quarterly to carefully track the advancement of each student in the program and establish overall program policy. Currently, 84 students are enrolled in the NTP and the average time to complete the Ph.D. for the past ten years is 6.0 years. Of the students who have graduated from our program, 93% are pursuing careers in a science or medicine-related field. Here, we request funds to support five students during their first two years in the program (10 slots).