1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

5 Symposium Chemosensory Receptors Satellite7 Symposium Chemosensory Receptors SatelliteREPRESENTATION OF NATURAL STIMULI IN THE RODENTMAIN OLFACTORY BULBLin D. 1 , Katz L.C. 1 1 Neurobiology, Duke University, Durham, NCTo understand the organization of natural stimuli in the bulb, werecorded electrophysiological responses of mitral cells and imagedintrinsic signals from glomeruli upon stimulation with volatiles fromnatural stimuli. All natural stimuli including urine are sparselyrepresented, activating less than 10% of mitral cells or glomeruli. Tofurther examine the origins of mitral cell and glomerular responses tonatural stimuli, we fractionated volatiles in natural stimuli using gaschromatography and delivered them sequentially to the animals whilemonitoring neural activities continuously. Among hundreds of volatilespresent in a natural stimulus, mitral cells and glomeruli are frequentlyactivated by a single component whereas individual components onlyevoked activation in a small distinct set of glomeruli. The representationof a complete natural stimulus is largely the simple union ofrepresentations of its individual components. During experiments usingurine volatiles, one cohort of mitral cells were found activatedexclusively by male mouse urine. These cells were determinedresponsive to a previously unknown male specific sulfur containingcompound (methylthio)methanethiol. When added to castrated malemouse urine, synthetic (methylthio)methanethiol significantly enhancedurine attractiveness to female mice. The conclusion from theseexperiments are as followings: (1) natural stimuli and their individualcomponents are sparsely represented in the MOB; (2) mitral cells andglomeruli encode natural odorant stimuli by acting as feature detectors;(3) individual components are processed in the bulb largelyindependently.6 Symposium Chemosensory Receptors SatelliteOLFACTORY RECEPTORS IN THE SEPTAL ORGANGrosmaitre X. 1 , Tian H. 1 , Lee A. 1 , Ma M. 1 1 Neuroscience, University ofPennsylvania, Philadelphia, PAThe septal organ is a distinct chemosensory organ in the mammaliannose. We have identified the odorant receptors expressed in this region.The majority of the septal neurons express only a few odorant receptorsin varying patterns, with the most prevalent receptor (MOR256-3)present in nearly 50% of the neurons and the eight most prevalentreceptors in nearly 95% of the neurons. A single neuron probablyexpresses only one receptor. Using patch clamp recordings, we haveinvestigated how the mouse septal organ neurons in the intactepithelium respond to odorants delivered by pressure ejection (puffing).The majority of the septal organ neurons exhibit both odorant andmechanical responses mediated by adenylyl cyclase. These neurons arerelatively broadly-tuned by responding to multiple distinct odorantswith thresholds at the nanomolar range. In addition, they respond toRinger puffs (mechanical responses) and the response increases linearlywith the pressure of the puff. The septal organ, situated in the air path,may have dual functions in the nose: a sensitive, broadly-tuned odordetector and a mechanical sensor. When the air flows faster in the nose(such as during sniffing), the observed mechanical sensitivity mayincrease the chance for single neurons to fire upon weak stimulation.Furthermore, the mechanical sensitivity may provide a driving force(besides the episodic exposure of odorants during respiration) for theperiodic activities of the olfactory bulb neurons synchronized withbreathing cycles.Supported by NIDCD/NIH, Whitehall Foundation and UPenn IOA(Pilot Grant).8 Symposium Chemosensory Receptors SatelliteFUNCTIONAL AND STRUCTURAL ANALYSIS OFMAMMALIAN ODORANT RECEPTORSLuetje C.W. 1 1 Molecular and Cellular Pharmacology, University ofMiami, Miami, FLMammalian odorant receptors (ORs) are grouped into two broadclasses and numerous subfamilies, which may reflect functionalorganization. We are using Xenopus oocytes to investigate the ligandspecificities of members of OR subfamilies. We find that a wide varietyof Class I and Class II mouse ORs (MORs) can be functionallyexpressed. Co-expression of MORs with Gαolf and the cystic fibrosistransmembrane regulator allows measurement of odorant responsesusing electrophysiological methods. All receptor constructs include theN-terminal 20 amino acid residues of human rhodopsin, and for mostMORs tested this is sufficient for functional expression. Co-expressionof accessory proteins (RTP1, RTP2 and REEP1) allows functionalexpression of additional MORs. Using this assay, we examined theligand specificities of the MOR42 subfamily. MOR42-1 responded todicarboxylic acids (C9-C12). MOR42-2 responded to monocarboxylicacids (C7-C10). MOR42-3 responded to dicarboxylic acids (C8-C10)and monocarboxylic acids (C10-C12). Thus, the receptive range of eachreceptor is unique. However, overlap between the individual receptiveranges suggests that the members of this subfamily are contributing toone contiguous subfamily receptive range, supporting the idea that ORsubfamilies constitute functional units. We are screening a series ofmutant MORs to identify residues that confer differences in ligandspecificity between MOR42-1 and MOR42-3. This analysis, coupledwith computational receptor modeling, provides insight into thestructural basis for odorant recognition by ORs. Support: NIHMH66038, DA08102.RIC-8B, A PUTATIVE GEF FOR G ALPHA OLF, AMPLIFIESSIGNAL TRANSDUCTION THROUGH ODORANTRECEPTORSVon Dannecker L.C. 1 , Mercadante A.F. 1 , Malnic B. 1 1 Department ofBiochemistry, University of Sao Paulo, Sao Paulo, BrazilThe canonical pathway for odorant signal transduction in olfactoryneurons consists of four major olfactory specific components: odorantreceptors, G alpha olf, adenylyl cyclase III and a cyclic nucleotide-gatedchannel. During the last years, a large number of regulatorymechanisms that lead to odorant signal termination have beendescribed, but so far, there was no evidence for regulatory events thatwould result in signal amplification. We identified a protein, Ric-8B,which interacts with G alpha olf. Our results demonstrate that Ric-8B,which is a putative guanylyl nucleotide exchange factor (GEF), shows astriking restricted pattern of gene expression, which co-localizes withthat of G alpha olf: both genes are specifically expressed in maturesensory neurons in the olfactory epithelium and in a few regions in thebrain. In addition, we show that Ric-8B significantly enhances odorantreceptor signaling through G alpha olf in HEK293T cells. Our resultsindicate that Ric-8B can be used to improve high-throughput functionalexpression of odorant receptors in heterologous cells.Supported by grants from FAPESP.2