Abstracts - Association for Chemoreception Sciences

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P O S T E R S septal epithelium obtained at autopsy (specimens harvested by the National Disease Research Interchange) enables us to identify regions ventral to the cribriform plate that are rich with olfactory neurons. As a consequence, we can obtain olfactory mucosa consistently and orient the tissue properly as required for in-depth analysis of the cell types of the human OE. Immunohistochemistry on sections of post-mortem human olfactory mucosa using a battery of antibodies directed against cytoskeletal proteins, surface antigens, and transcription factors expressed in stem and progenitor cells of rodents, generate staining patterns that closely reflect those observed in the OE of mice and rats. Our results reveal a high degree of similarity between the human and rodent olfactory systems, which was not previously appreciated. Further studies looking for alterations in the cellular composition of human OE obtained via biopsy from patients suffering smell loss and comparing them with patterns of staining in mouse models of injury-induced regeneration will help elucidate the pathophysiology of olfactory disorders in humans. Acknowledgements: R01 DC010242 #P76 POSTER SESSION II: OLFACTORY PHYSIOLOGY & CELL BIOLOGY; TASTE MOLECULAR GENETICS; CHEMESTHESIS & TRIGEMINAL Glomerular targets of olfactory sensory neurons in adult female mice heterozygous for mutated CNGA2 with TRPM5 knockout background David A. Dunston, Christy Thai, Weihong Lin University of Maryland, Baltimore County Baltimore, MD, USA Activation of olfactory sensory neurons (OSNs) influences the proper axonal targeting to the olfactory bulb of adult mice. Odor activation in a majority of OSNs involves the cyclic nucleotide gated channel A2 subunit (CNGA2). We have previously shown that adult female mice heterozygous for the mutated CNGA2 (CNGA2KO-GFP +/-) have more GFP positive glomeruli than CNGA2KO-GFP +/- mice with a knockout of the transient receptor potential channel M5 (TRPM5) (Dunston et al AChemS abstract 2009). Here we further determined the location of the GFP positive glomeruli in these two mouse lines using an olfactory bulb mapping program. Consistent with our previous findings, the density of GFP positive glomeruli in mice that are CNGA2 KO-GFP +/- with TRPM5 knocked out was lower than that of the CNGA2 KO-GFP +/- mice. The overall distribution of these glomeruli was similar in the two lines. We also found that in both lines of mice many glomeruli are targeted by both GFP positive and negative axons in addition to glomeruli that homogenously receive either GFP positive or negative axons. The results imply that OSNs with null CNGA2 may exhibit different levels of activity. Further, we examined the immunolabel intensity for tyrosine hydroxylase (TH), which depends on sensory input. The intensity of the TH immunostaining in glomeruli receiving homogenous GFP positive axons in both lines of mice was decreased but not eliminated. Our data indicate that TRPM5 may contribute to different activity levels of OSNs. Acknowledgements: NIH/NIDCD grant 009269 #P77 POSTER SESSION II: OLFACTORY PHYSIOLOGY & CELL BIOLOGY; TASTE MOLECULAR GENETICS; CHEMESTHESIS & TRIGEMINAL G protein-dependent activation of PLC and PI3K in mammalian olfactory receptor neurons Katharina Klasen 1,3 , Elizabeth A Corey 1 , Daniela Brunert 1 , Kirill Ukhanov 1 , Hanns Hatt 3 , Barry W Ache 1,2 1 Whitney Laboratory, Center for Smell and Taste, and McKnight Brain Institute, University of Florida Gainesville, FL, USA, 2 Department of Biology and Neurobiology, University of Florida Gainesville, FL, USA, 3 Department of Cell Physiology, Ruhr-University Bochum Bochum, Germany Odorants activate both PLC and PI3K in the cilia of olfactory receptor neurons (ORNs) (Klasen et al., 2009). To better understand the potential role of these enzymes in olfactory transduction we asked whether the activation of one or both enzymes was G protein dependent. Isoforms of both PI3K and PLC can be localized to the olfactory cilia by western blot and/or immunohistochemistry. Activation of both enzymes in ORNs can be independently blocked pharmacologically by G proteinspecific inhibitors. The non-hydrolyzable GDP analog GDPbS, as well as suramin, a Gbg inhibitor, independently blocked the activation of PLC and PI3K in vitro as detected by ELISA. The Gbg inhibitor gallein and a closely related compound M119, which is reported to specifically disrupt in vitro G proteindependent activation of PLCb2, PLCb3 and PI3Kg, also suppressed odorant-dependent activation of rodent ORNs in vivo, as measured by calcium imaging. Taken together, these results provide evidence that odorants activate both PI3K and PLC signaling in a G protein-dependent manner in ORNs. Acknowledgements: Supported by grants from the Deutsche Forschungsgemeinschaft and the National Institute on Deafness and Other Communication Disorders. #P78 POSTER SESSION II: OLFACTORY PHYSIOLOGY & CELL BIOLOGY; TASTE MOLECULAR GENETICS; CHEMESTHESIS & TRIGEMINAL Does Olfactory Marker Protein (OMP) function by interacting at calmodulin (CaM) binding sites? Hyun J. Kwon 1 , Kristen Varney 2 , Joyce W. Margolis 3 , David J. Weber 2 , Frank L. Margolis 3 1 Dept. Engineering & Computer Science, Andrews University Berrien Springs, MI, USA, 2 Department of Biochemistry, University of Maryland, School of Medicine Baltimore, MD, USA, 3 Department of Anatomy and Neurobiology, University of Maryland, School of Medicine Baltimore, MD, USA Behavioral, electrophysiological and Ca-imaging analyses have demonstrated the involvement of OMP in olfactory transduction, but the molecular mechanism remains elusive. Our recent studies of protein-protein and protein-peptide interactions have implicated CaM in this process. Bex protein, an OMP binding partner, also binds CaM, suggesting that these three proteins may interact to regulate the availability of CaM. We hypothesized that OMP might interact at CaM binding sites on various proteins in the olfactory transduction cascade. Last year we showed that OMP or CaM can each bind to the auto-inhibitory peptide (XIP) of Na/Ca exchanger 1 (NCX1), a protein involved in Ca2+ 52 | AChemS Abstracts 2010 Abstracts are printed as submitted by the author(s)
extrusion from OSNs. We are characterizing the interaction of OMP with putative CaM binding sites of other proteins involved in olfaction. Peptides from PMCA2, CaM-KII, CNGB1b and PDE1c were analyzed by QCM-D (Quartz Crystal Microbalance with Dissipation Monitoring) for their interactions with CaM or OMP, in the presence or absence of Ca2+. Three of the four peptides (but not the PDE1c peptide) interacted with CaM only in the presence of Ca2+, with low uM Kd values. OMP also interacted with these three peptides, but independent of Ca2+, with 10x higher Kd (lower affinity) values than seen with CaM. These data provide additional evidence that OMP can selectively interact with several peptide sequences that bind Ca2+/CaM. NMR analyses of some of these interactions provide structural evidence for the nature of the complexes. 15N-NMR HSQC titrations of XIP/OMP, XIP/CaM, Bex (50-75)/OMP and Bex (50-75)/CaM demonstrate that OMP and CaM can compete for the same target sites. These data support our hypothesis that OMP can modulate the activities of some CaM binding proteins and thus influence the olfactory signal transduction cascade. Acknowledgements: Supported by Andrews FRG (HJK), NIH DC03112 (FLM) NIH GM58888 (DJW). #P79 POSTER SESSION II: OLFACTORY PHYSIOLOGY & CELL BIOLOGY; TASTE MOLECULAR GENETICS; CHEMESTHESIS & TRIGEMINAL Isolation and characterization of immature olfactory sensory neurons Melissa D. Nickell, Timothy S. McClintock Department of Physiology, University of Kentucky Lexington, KY, USA To detect chemical stimuli, olfactory sensory neurons (OSNs) are necessarily exposed to damage, necessitating life-long neurogenesis to replace damaged OSNs. Mature OSNs (mOSNs) therefore always coexist with immature OSNs (iOSNs). To better understand the respective contributions of the two developmental stages of OSNs, we have simultaneously purified mOSNs and iOSNs from the same mice. We collected three cellular fractions: mOSNs, iOSNs and a residual population of all other cells in the olfactory epithelium. Affymetrix M430 v.2 GeneChip data for each fraction were compared in order to derive mRNA abundance ratios that were statistically evaluated against in situ hybridization data for 396 mRNAs, thereby allowing the assignment of validated probabilities of expression in mOSNs and iOSNs for each transcript. Of the ~10,000 genes that are expressed by OSNs (Sammeta et al., 2007, PMID 17444493) we identified 358 specific to mOSNs and 1,189 specific to iOSNs. The over-represented GeneOntology functions among the mOSN-specific transcripts were ion transport, neurotransmitter transport, synaptic transmission and cilia, consistent with the specializations that define the maturity of these neurons. Transcripts specific to iOSNs represented a more diverse set of biological processes, including axonogenesis, transcription, chromatin modification and nucleic acid metabolism, among others. The iOSN-specific list included 115 transcription factors (~10% of iOSN-specific transcripts) and numerous epigenetic regulators, consistent with the gene expression changes that must accompany neural differentiation. Acknowledgements: Supported by award R01 DC002736 #P80 POSTER SESSION II: OLFACTORY PHYSIOLOGY & CELL BIOLOGY; TASTE MOLECULAR GENETICS; CHEMESTHESIS & TRIGEMINAL Gene Expression Profiling of the Olfactory Neurogenic Lineage Richard C Krolewski, James E Schwob Department of Anatomy & Cellular Biology, Tufts University School of Medicine Boston, MA, USA Olfactory sensory neurons (OSNs) are replaced throughout life during normal maintenance and after injury by the regulated proliferation and differentiation of stem and progenitor cells that persist in the olfactory epithelium (OE) throughout life. The stages in the progression from proliferating progenitor through cell cycle exit to the multistep differentiation of OSNs can be marked via the use of transgenic reporter mice and/or patterns of protein expression. To identify candidate pathways regulating this process we have examined a number of these stages by global transcriptional profiling. Using FACS–purified cells from Ngn1::eGFP (a BAC transgenic line) and DOMP::GFP (gene knock-in to the endogenous locus) mice that are otherwise unmanipulated and others after olfactory bulb ablation, we have obtained the transcriptome of cells at each of four distinct steps in the neuronal lineage: 1) immediate neuronal precursors, 2) activated immediate neuronal precursors, 3) newly differentiating OSNs, and 4) mature OSNs. Functional annotation clustering of highly regulated genes (adjusted p value 2) from these data sets compared to one another and with normal olfactory mucosa has identified candidate pathways and genes for validation and functional studies. Annotation clusters relating to neurogenesis, regulation of transcription, establishment of chromatin architecture, mitotic cell cycle and axonogenesis had high enrichment scores in the globose basal cell and immature neuron–enriched samples, while sensory perception of smell and synaptic transmission were highly enriched terms in the differentiating and mature OSN datasets. These data provide a gateway to examining and understanding the genome–wide events that occur as neurons are born and mature within the OE. Acknowledgements: Supported by R21 DC008568 (J.E.S.) and F30 DC010276 (R.C.K) #P81 POSTER SESSION II: OLFACTORY PHYSIOLOGY & CELL BIOLOGY; TASTE MOLECULAR GENETICS; CHEMESTHESIS & TRIGEMINAL Visualizing the Redistribution of Responses within the Rodent Olfactory Receptor Repertoire : Tracking Chemical, Conformational, and Concentration Changes Zita Peterlin 1 , Yadi Li 2 , Kevin Ryan 2 , Stuart Firestein 1 1 Columbia University : Department of Biological Sciences New York, NY, USA, 2 City College of New York : Chemistry Department New York, NY, USA The combinatorial nature of odor encoding makes the study of the olfactory system fascinating, but at the same time it presents problems for data management. Although tables of probabilities, multidimensional vectors, and other descriptive statistics are the most thorough embodiment of complex data, such modes of presentation are not immediately intuitive to a broad audience. Here, we present a more visual representation that can be used to P O S T E R S Abstracts are printed as submitted by the author(s) Abstracts | 53
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AChemS Association for Chemorecepti
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familiarity and cultural difference
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perception of purine 5’-ribonucle
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(CT), the greater superficial petro
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were significantly different from o
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MSG on NaCl is evidently highlighte
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consisting of motors and IFT comple
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wing. Philip Callahan exposed insec
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seem to indicate that total nasal v
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possibly suggesting non-apoptotic m
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of 7/10, and causing eyes to water
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showed that N1/P2 latencies were lo
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differentiated patients with AD (M=
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pleasantness 3)of mixtures (A,B), A
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Index Aarts, H - P328 Abe, K - P88,
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Haase, L - P3, P4, P29, P355 Haddad
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Murata, Y - P272 Murphy, C - P3, P4
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Veldhuizen, M - P7, P24, P25, P242,
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Registration 7:30 am to 1:00 pm, 6:
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See you next year! AChemS 33rd Annu
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Abstracts - Association for Chemoreception Sciences

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