Abstracts - Association for Chemoreception Sciences

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P O S T E R S RA affects neurogenesis in postnatal mammals and the reduction in ORNs in VAD OE results from local depletion of RA. To test this hypothesis, we are developing a defined mouse embryonic stem cell (mESC) model to investigate effects of RA signaling on mESC differentiation. Addition of RA to mESCs induces neurogenesis presumably by complexing with nuclear RA receptors (RAR) that mediate gene expression. The goal of this study was to localize RARa and RARg expression in mESCs. Two different mESC strains were grown on microscope slides. One strain (R1ES) was grown in undefinded media (supplemented with 15% FBS) and the other (Bl6 ES) was grown in chemically defined media. Fixed cells were reacted with RARa and RARg antibodies using immunofluorescent and electron microscopy (EM) protocols. Negative controls showed no reactivity. MCF-7 cells, a positive control for RARa and RARg, showed positive nuclear staining with both antibodies. The RARa and the RARg antibodies positively stained the nuclei of R1ES and Bl6 mESCs. Staining was variable in intensity throughout mESC colonies, suggesting there is variable expression of the protein in different cells. Unexpectedly, incubation of mESCs with either 0.5 mM RA or 0.5 mM VA for 24h did not increase or diminish staining by the antibodies. The results further confirm the authenticity of the antibodies. Future work will be directed at monitoring RAR expression as cells differentiate into neurons and in identifying cell types in the olfactory organ and bulb of rodents. Acknowledgements: NIH/NIGMS/MBRS/SCORE S06 GM 008092 #P300 POSTER SESSION VI: PERIPHERAL AND CENTRAL TASTE; PERIPHERAL OLFACTION Genetic Manipulation of Sox2 in the Adult Olfactory Epithelium During Lesion-Induced Regeneration Adam I. Packard 1 , James E. Schwob 1 1 Tufts University School of Medicine Boston, MA, USA The transcription factors Sox2 and Pax6 are co-expressed in multiple cell types of the adult OE, including HBCs, “upstream” GBCs, and Sus cells, but not in “downstream” GBCs nor olfactory sensory neurons (OSNs). We used transduction by retroviral vector (RVV) to over-express them individually and together during recovery from MeBr lesion to test the hypothesis that the two factors suppress neuronal differentiation. As described last year, Sox2-IRES-eGFP-encoding RVV significantly suppresses OSN formation compared to empty vector (EV), though many OSN-containing clones remain; half of the GFP (+) OSNs (and only the OSNs among the cells in the clones) lack detectable Sox2 protein. Is co-expression of Pax6 and Sox2 required for complete OSN suppression and to maintain Sox2 protein in OSNs? A Sox2-Pax6-eGFP-encoding RVV (SEP) also produced only partial suppression of OSNs, although Sus cells were increased relative to the Sox2 RVV. As before, both Sox2 and Pax6 proteins were undetectable in many SEP-transduced OSNs. Since Sox2 over-expression suggests that it may enhance the proliferation within upstream neural progenitor cells, we eliminated Sox2 expression by infecting Sox2 loxP/loxP mice with a Cre-encoding RVV. Elimination of Sox2 caused a significant reduction in clone size, probably by preventing the expansion of neural progenitor cells, but both OSNs and Sus cells are still produced, suggesting that Sox2 is not required for either cell type. In summary, Sox2 is neither necessary nor sufficient to drive the differentiation of OSNs or Sus cells. However, Sox2 regulates progenitor cell proliferation and acting with Pax6 biases in favor of Sus cell formation. Lastly, OSNs tightly regulate their constellation of transcription factors to achieve and maintain their differentiated state. Acknowledgements: NIH grant R01 DC002167 #P301 POSTER SESSION VI: PERIPHERAL AND CENTRAL TASTE; PERIPHERAL OLFACTION The Transcription Factor p63 is Required for the Differentiation of Horizontal Basal Cells During Development Nikolai Schnittke, Adam Packard, James E Schwob Department of Anatomy & Cellular Biology, Tufts University School of Medicine Boston, MA, USA The capacity of the adult olfactory epithelium (OE) to replace neurons lost through senescence, axotomy, or trophic factor depletion and to reconstitute itself after severe injury is incumbent on the retention of multipotent, neurocompetent stem cells. Olfactory stem cells reside amongst the basal cells of the epithelium, of which at least two types exhibit a capacity for multipotency – horizontal basal cells (HBCs) and globose basal cells (GBCs). GBCs, defined on the basis of transcription factor expression, are present early in embryonic development, repopulate the tissue throughout adult life, and contribute to tissue regeneration after severe injury. In contrast, the population of HBCs, defined by adherence to the basal lamina and by the expression of HBC-specific proteins (including cytokeratins (CK) 5 and 14), is not established until after birth. HBCs remain dormant during normal tissue maintenance, but contribute to tissue regeneration after severe injury. Here we report that p63, a member of the p53 transcription factor family, is necessary for the emergence of HBCs during the development of the OE. We show that p63 precedes both expression of CK5 and 14 and the migration toward the basal lamina, and that p63-mutant mice generate all cell types of the OE except for HBCs. Finally, we demonstrate that p63 expression anticipates HBC reappearance in the ventral OE of adult rats after methyl bromide lesion, and that HBC activation to multipotency during regeneration is accompanied by a transient loss of p63. Together these data suggest a model in which p63 acts to set aside a reserve stem cell population that can be activated by severe lesion. Moreover, loss of p63 expression appears to be a hallmark feature of such activation. Acknowledgements: R01 DC002167 #P302 POSTER SESSION VI: PERIPHERAL AND CENTRAL TASTE; PERIPHERAL OLFACTION IFT88 Regulates Olfactory Cilia Maintenance and Function Jeremy C. McIntyre, Paul M. Jenkins, Dyke P. McEwen, Jeffrey R. Martens University of Michigan Ann Arbor, MI, USA Odorant detection begins in the cilia of olfactory sensory neurons (OSNs), where the binding of odorants to receptors initiates the canonical G-protein signaling pathway. The components of this pathway are compartmentalized in cilia, yet none are translated there. In order to reach the ciliary membrane, proteins are transported from the basal bodies into the cilia by the process of intraflagellar transport (IFT). While olfactory cilia are critical for the detection of odorants, relatively little is known about their development, maintenance and protein trafficking. IFT particles, 126 | AChemS Abstracts 2010 Abstracts are printed as submitted by the author(s)
consisting of motors and IFT complex proteins such as IFT88, are responsible for the transport of cargo into and out of cilia. Mutations in IFT88 have been shown to affect ciliogenesis in several other cell types. Here, we used the ORPK mouse, an IFT88 hypomorphic mutant, to investigate IFT in the development and maintenance of OSN cilia. Disruption of IFT88 affects olfactory cilia organization and olfactory function, but does not result in the complete loss of cilia. Although electroolfactogram recordings show that ORPK mice are largely anosmic, scanning electron microscopy shows the OSNs from ORPK mice do contain cilia, although ORPK olfactory cilia are fewer and shorter compared to wild-type littermates. Immunohistochemistry reveals a decrease in gamma- and acetylated-tubulin staining consistent with a decrease in cilia in ORPK mice as well as a concomitant loss of the signaling proteins, ACIII, CNGA2, and Gg13 from the ciliary layer. Staining for MOR28 shows odorant receptor protein remains localized to the dendritic knobs and remaining cilia. These data indicate that IFT88 is not required for olfactory ciliogenesis, but rather IFT88 is important for the maintenance of cilia and hence olfactory function. Acknowledgements: This work was support by National Institutes of Health Grants DC009606 (J.R.M.), DC00011 (J.C.M., P.M.J., and D.P.M.) and National Research Service Award Fellowship DC009524 (P.M.J.) #P303 POSTER SESSION VI: PERIPHERAL AND CENTRAL TASTE; PERIPHERAL OLFACTION Fasciculation of Molecularly Defined Subsets of Axons in the Developing Olfactory Nerve Pathway Lydia R. Maurer 1 , Alexandra M. Miller 1, 3, 4 , Charles A. Greer 1,2,3 1 Department of Neurosurgery, Yale University School of Medicine New Haven, CT, USA, 2 Department of Neurobiology, Yale University School of Medicine New Haven, CT, USA, 3 Interdepartmental Neuroscience Program (INP) New Haven, CT, USA, 4 Medical Scientist Training Program New Haven, CT, USA Olfactory sensory neuron (OSN) axons exit the olfactory epithelium (OE) and grow toward the olfactory bulb (OB) where they coalesce into glomeruli. Each OSN expresses 1 of ~1,200 odor receptors (ORs) and the axons sort into 2-3 glomeruli in a reproducible manner based on OR expression. Deletion or substitution of the OR results in convergence failure, or convergence in an ectopic site. While ORs appear necessary for appropriate convergence of axons, they are likely not sufficient to fully account for the behavior of OSN axons. The OSNs expressing any 1 OR are distributed roughly in 1 of 4 zones of the OE, but within a zone are not contiguous, as their distribution appears stochastic. Recent work has implicated that pre-target axon sorting first occurs in the olfactory nerve (Bozza et al., 2009; Imai et al., 2009). We recently established that in mice OSNs expressed ORs as early as embryonic day (E) 9 and that axons first emerge from the OE, crossing the basal lamina into the undifferentiated mesenchyme, at E10. Using regional and OR specific markers, we have now asked when homotypic fasciculation first occurs and assessed the degree to which subpopulations of axons may remain segregated as they extend toward the nascent OB. Here, we show that immediately upon crossing the basal lamina axons uniformly turn sharply, at ~90 degrees toward the OB. Molecularly defined sub-populations of axons begin to segregate by E12, forty-eight hours after the OSN axons have crossed the basal lamina but prior to the axons first reaching the nascent OB. Homophilic axon fasciculation appears to be a hierarchical process. While regional segregation occurs in the mesenchyme, ultimate convergence of OR-specific subpopulations does not occur until the axons reach the inner nerve layer of the OB. Acknowledgements: Generously supported by the NIH/NIDCD/NIA. #P304 POSTER SESSION VI: PERIPHERAL AND CENTRAL TASTE; PERIPHERAL OLFACTION Bridging Multiple Time-Scales in the Signal Transduction of the Mouse Olfactory Receptor Neuron Daniel P. Dougherty Michigan State University East Lansing, MI, USA Our goal is to identify the salient or essential dynamic components of the olfactory signal transduction cascade in mouse. We use an integrative approach which takes advantage of electrophysiological data from the olfactory receptor neuron (ORN) of several mutant mouse lines. This data is used concurrently to develop physiologically-based computational models of the slow (transduction) and fast (action potential) currents in ORN. A key question for our group is, “Can a single general model be developed which predicts the ORN electrophysiology of all the observed mutant lines?” In this poster, we present application of a novel Bayesian model-fitting algorithm called Wedge to the electrophysiological data from several mutant mouse lines. We illustrate how a diversity of responses across a population of ORN can be addressed through a fully Bayesian dynamical systems approach using this Wedge algorithm. We conclude our analysis by summarizing which components of the ORN cascade appear to be most responsible for the diversity or variability observed in the current response of mouse. Acknowledgements: This work was supported by Award Number R01DC009946 (NIDCD/NIH). #P305 POSTER SESSION VI: PERIPHERAL AND CENTRAL TASTE; PERIPHERAL OLFACTION A neural code for binary odorant mixture interactions in the nose Ginny E Cruz, Graeme Lowe Monell Chemical Senses Center Philadelphia, PA, USA As a step towards elucidating the neural mechanisms of odorant mixture interactions, we examined binary mixture interactions at the level of olfactory receptor neurons (ORN) in an intact mouse. We used transgenic mice that expressed synaptopHluorin (spH) selectively in ORNs. We measured odor-evoked neurotransmitter release, reported as an increase in spH fluorescence signal, by distinct populations of ORNs converging onto defined glomeruli in the olfactory bulb. We obtained maps of activity across the dorsal surface of the bulb in response to eugenol (EG), methyl iso-eugenol (MIEG) or their binary mixture. EG and MIEG, which have highly similar chemical structures but different smells, activated overlapping sets of receptors; however, the apparent odorant binding affinity and sensitivity varied with the different receptors. Both EG and MIEG evoked dose-response relations that showed wide dynamic range, saturation, and only weak cooperativity (Hill coefficient ~ 1). Below response saturation, glomerular responses to binary P O S T E R S Abstracts are printed as submitted by the author(s) Abstracts | 127
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AChemS Association for Chemorecepti
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AChemS Association for Chemorecepti
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We are pleased to announce that sev
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#1 GIVAUDAN LECTURE Normal and Canc
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and against delta ENaC is being pur
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#13 SYMPOSIUM - GENETICS OF HUMAN O
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#19 SYMPOSIUM - CILIA, SENSORY DYSF
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#26 PLATFORM PRESENTATIONS - POLAK
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esponse selectivity for both OSNs a
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elicit glucagon-like peptide-1 (GLP
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contrast, changing the concentratio
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of OBPs, we have systematically sup
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whereas mouse and joystick reaction
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POSTER PRESENTATIONS #P1 POSTER SES
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PROP strips and PROP solutions. PAV
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#P11 POSTER SESSION I: TASTE IMAGIN
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TGI. The present study investigated
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delivered in the scanner intra-oral
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a universal odor descriptor map. Ho
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acetate or ethyl butyrate v/v in mi
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#P54 POSTER SESSION II: OLFACTORY P
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hours later, the levels of prolifer
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extrusion from OSNs. We are charact
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anchor of ‘strongest sensation of
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cells within gustatory papillae, we
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excite menthol- and/or CA-sensitive
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gene, HMBS was used. All primers we
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oom, without time keeping devices,
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#P113 POSTER SESSION III: OLFACTORY
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decreasing of endogenous volatiles
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Abstracts - Association for Chemoreception Sciences

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