477 Poster Developmental, Neurogenesis, and ConsumerResearchFORMATION OF THE OLFACTORY PLACODE IN THEZEBRAFISH, DANIO RERIOHarden M.V. 1 , Yang Z. 2 , Lin S. 2 , Whitlock K.E. 1 1 Molecular Biologyand Genetics, Cornell University, Ithaca, NY; 2 Molecular, Cellular andDevelopmental Biology, University of California, Los Angeles, CAIn zebrafish, the olfactory placodes are formed by a convergence oftwo fields of cells located on either side of the developing neural tube(Whitlock and Westerfield, 2000). In order to determine the extent ofcell mixing during formation of the olfactory placodes and cranialneural crest derived structures of the face, we are visualizing cellmovements in the developing embryo. Using a transgenic lineexpressing GFP we are able to visualize the neural crest cells in vivo. Atapproximately 13 hours post fertilization, the neural crest cells migrateanteriorly as a group and separate at the anterior end of the neural tube.Some neural crest cells appear to migrate to the region of the formingolfactory placodes. We are generating a transgenic line that expressesRFP in the olfactory placode fields. By generating animals carryingboth the neural crest GFP and olfactory placode RFP expression we willbe able to visualize the movements of both of these cell types duringdevelopment. Our investigations will provide insight into how theolfactory placode and the neural crest fields mix together duringdevelopment to form the nose. Support: NIH DC0421801 (KEW),Graduate Student Fellowship, Center for Vertebrate Genomics, CornellUniversity (MVH). Whitlock KE and Westerfield M (2000).Development. 127: 3645-3653.478 Poster Developmental, Neurogenesis, and ConsumerResearchMETAMORPHOSIS OF AN OLFACTORY SYSTEM:HORMONAL REGULATION OF GROWTH ANDPATTERNING IN THE ANTENNAL IMAGINAL DISC OF THEMOTH MANDUCA SEXTAFernandez K.A. 1 , Vogt R. 1 1 Biological Sciences, University of SouthCarolina, Columbia, SCPeripheral olfactory systems of insects undergo metamorphosis,transforming from a simple larval antenna to the highly complex adultantenna mediating diverse chemosensory behaviors. Adult antennaederive from imaginal discs which grow during the larval stage, andundergo neurogenesis and morphogenesis during the pupal stage. Weare characterizing patterns of morphogenic activities in the imaginaldisc and early developing antenna to identify hormonally regulatedevents which lead to the patterning of the adult antenna.This study focuses on development the antennal disc in M. sexta.Disc growth occurs throughout most of the fifth larval instar. Theantennal imaginal disc grows inward from an epithelial ringsurrounding the base of the larval antenna. We have quantified DNAcontent during disc growth as an indicator of cell number, observing asharp decline in DNA content just prior to disc eversion. We havesubsequently identifed apoptotic activity in a spatial pattern which isreflected in the spatial organization of the adult antenna. We haveexplored the role of ecdysteroids regulating disc growth. Prior topupation the imaginal discs elongates and everts; we have demonstratedecdysteroid sensitivity of disc eversion, and are currently exploring therole of ecdysteroids in regulating the post eversion apoptotic events.These studies are establishing a foundation for identifying the hormonalregulation of growth and patterning that will give rise to the selection ofspecific chemosensory phenotypes of adult olfactory sensilla.479 Poster Developmental, Neurogenesis, and ConsumerResearchMMP-9 ELEVATION IN THE EARLY RESPONSE TOOLFACTORY NERVE INJURYCostanzo R.M. 1 , Perrino L.A. 1 , Kobayashi M. 1 1 Physiology, VirginiaCommonwealth University, Richmond, VAMatrix metalloproteinases (MMPs) have been implicated inextracellular remodeling that occurs in developmental, reparative andhomeostatic processes. MMP-9 (gelatinase B) has been reported in thecentral nervous system and may be associated with injury processes,including neuronal degeneration and gliosis. We used a welldocumentedmodel of olfactory nerve injury to study the role of MMP-9during degeneration and regeneration processes in the olfactory bulb.By means of Western blot and immunohistochemistry, we studiedMMP-9 and markers for olfactory neuron degeneration and regeneration(Olfactory marker protein, OMP and X-gal staining) and gliosis (Glialfibrillary acidic protein, GFAP) in the olfactory bulbs of P2-tau-lacZmice following bilateral olfactory nerve transection. Data from controlmice and sham surgeries showed almost no MMP-9 in the olfactorybulbs. However, we found that MMP-9 levels rose sharply and abruptly(within hours) following olfactory nerve injury and peaked at 5-7 dayspost-injury. Immunohistochemical analysis showed that MMP-9 waslocalized to the anterior portion of the olfactory bulb, the area thatsustained the greatest injury in our model. After 7 days, MMP-9 levelsdecreased, returning to near control levels at later recovery time points.This is the first report demonstrating an elevation in MMP-9 levels inthe olfactory bulb during the early response to injury, suggesting thatMMP-9 may play a role in neuronal degeneration and gliosis.Supported by NIH-NIDCD R01-DC000165.480 Poster Developmental, Neurogenesis, and ConsumerResearchSUPPORTING CELLS AND OLFACTORY NEURONSEXPRESS DIFFERENT INHIBITORY APOPTOSIS PROTEINSComte I. 1 , Carr V. 1 , Farbman A.I. 1 1 Neurobiology and Physiology,Northwestern University, Evanston, ILIn the olfactory epithelium neurogenesis and neuronal apoptosis arethought to occur continuously throughout life. We believe thatequilibrium between genesis and death of neurons is highly regulated.In this study we used RT-PCR and Northern Blot methods to examinethe expression of three members of the Inhibitory Apoptosis Proteins(IAPs) gene family in rat olfactory epithelium. These IAPs are known toinhibit apoptosis by inhibiting caspase activity and are probablyinvolved in apoptotic regulation. We established that mRNAs ofSurvivin, X-linked IAP (XIAP) and neuronal apoptosis inhibitoryprotein (NAIP) are expressed in ofactory mucosa. Cellular localizationof two proteins for which antibodies were available was studied byimmunohistochemistry whereas localization of mRNA for NAIP wasanalysed using in situ hybridization. All supporting cells are Survivinpositive,but only a subtype with a zonal distribution expresses bothXIAP and Survivin. NAIP is restricted solely to olfactory neurons.Northern blots suggested that the quantities of survivin and XIAPmRNAs did not change significantly after bulbectomy and couldexplain the low turnover of these supporting cells compared to turnoverof neurons. We have also shown by Northern blots that, NAIPexpression is down-regulated 1day after bulbectomy and up-regulated 3to 5 days post-lesion. Our data are consistent with the idea thatapoptosis is regulated in rat olfactory epithelium. Further we suggestthat apoptosis in olfactory sensory neurons and supporting cells areregulated by different molecular mechanisms. Supported by NIH grantnumber: 5R01DC4837120
481 Poster Developmental, Neurogenesis, and ConsumerResearchMICROGLIA IN THE ZEBRAFISH IMMUNE RESPONSE TOINJURYFuller C.L. 1 , Koenig J.J. 1 , Byrd C.A. 1 1 Biological Sciences, WesternMichigan University, Kalamazoo, MIThe zebrafish olfactory system is a good model for studies ofneuronal plasticity and recovery from brain injury or disease. This studyattempts to determine the immune response of the zebrafish brain toinjury, with particular interest in the role of microglia. Microglia arephagocytic cells that respond to neuronal death by removing cellulardebris and they can be identified using a variety of histochemical labelsincluding certain plant lectins. They play an important role in thedefense of the central nervous system and may increase at the site ofinjury as part of an inflammatory response. Here we establish thenormal microglial composition of the adult zebrafish brain and examinethe microglial response to damage. Normal and injured fish wereanalyzed at various time points for the presence of microglia using thelectin marker IsoB4. In normal animals, very few IsoB4 lectin-positiveprofiles were seen in the olfactory bulb and optic tract, although profileswere more prevalent around the ventricles. Injured animals underwenteither olfactory deafferentation or optic nerve crush. In deafferentedolfactory bulbs there were very few lectin-positive cells and noevidence of microglial proliferation. Following optic nerve crush,however, a large number of lectin-positive microglia were visualized inthe optic tract and diencephalon. Thus, we conclude that olfactorydeafferentation elicits a different type of wound response from otherperipheral injury; the olfactory damage response does not involve aproliferation of IsoB4 lectin-positive microglia. We will continue toinvestigate the mechanisms by which the olfactory bulb responds todamage by examining microglia following direct bulb injury. Supportedby NIH DC04262 to CAB482 Poster Developmental, Neurogenesis, and ConsumerResearchCOUMARIN PRODUCES SELECTIVE DEAFFERENTATIONOF THE OLFACTORY BULBSanguino A. 1 1 Psychology, University of South Florida, Tampa, FLCoumarin (1,2-benzopyrone), a compound found in the essential oilsof many plants, had been employed as a fixative and flavoring agent butis now banned in food products because of its potential hepatotoxicity.In rats, doses subthreshold for liver toxicity produce cytotoxicity in theolfactory epithelium, due, in part, to OE-specific P450 bioactivation ofthe toxicant (Gu et al., 1997; Zhuo et al., 1999). We assessed theeffects of ip injections of coumarin on projections from the OE to theolfactory bulb. Anterograde transport of HRP*WGA from OE to theOB was evaluated 7 or 21 days after treatment with 50 or 100 mg/kgcoumarin. Rats appeared normally active 24 hr after treatment. 50mg/kg had little effect on anterograde transport. Seven day 100 mg/kgsurvival cases (n = 5) had dense HRP*WGA reaction product inglomeruli of the AOB but no reaction product in the MOB or very lightreaction product in some glomeruli in the mid lateral and posteriorventral medial bulb. There was considerable recovery of input to all butglomeruli on the dorsal and medial wall of the MOB in 21 day survivalcases (n = 5). Surprisingly, patterns of deafferentation were notbilaterally symmetrical and, in most cases, one bulb had considerablyless input than the other. Higher doses (200–500 mg/kg) were notnecessarily more effective than the 100 mg/kg dose. A study of theeffects of coumarin on odor detection and discrimination is in progress.Supported in part by NIH grant DC04671.483 Poster Developmental, Neurogenesis, and ConsumerResearchMORPHOLOGICAL AND FUNCTIONAL REGENERATION OFTHE OLFACTORY EPITHELIUM DEPENDS UPON THEEXTENT OF THE ABLATIONPlibersek K. 1 , Valentincic T. 1 1 Biology, University of Ljubljana,Ljubljana, SloveniaRegeneration of olfactory lamellae occurs following partial ablationof the olfactory organ of black bullhead catfish (Ameiurus melas).Depending upon the size of the remaining tissue, the olfactoryepithelium regenerated into either small roseta, single lamellae orepithelial tissue. Four months post the ablation, large medial sections ofthe olfactory lamellae (1-3.6 mm x 2 mm x 0.3 mm) regenerated intoeither small rosetae with 14-22 lamellae or fan-like rosetae with 5-11lamellae. The regenerated rosetae contained ciliated and microvillousolfactory receptor neurons (ORNs). Axons of the ciliated ORNsconnected to the anterior area and axons of microvillus ORNsconnected to the lateral area of the ventral olfactory bulb whichindicated full functional regeneration. In a second experiment, smallmedial sections of the olfactory lamellae (0.6-1.4 mm x 1 mm x 0.3mm) regenerated into either flat or fingerlike lamellae or into smalldeformed epithelial tissues. Four months after the ablation there was nobehavioral evidence of olfactory discrimination. The small regeneratedlamellae did not contain ORNs and did not respond to amino acidselectrophysiologically. A year after the ablation, four of the catfish withthe small regenerated lamellae discriminated the conditioned L-norvaline from other amino acids, whereas three catfish with fewregenerated lamellae and four catfish with deformed epithelial tissuesdid not discriminate amino acids. Catfish with functionally regeneratedolfactory organs responded to olfactory stimulation, whereas anosmiccatfish responded to taste stimulation only (Valentincic et al., 1994).Supported by Slovenian Ministry of Higher Education and Sciencegrant P1-0184.484 Poster Developmental, Neurogenesis, and ConsumerResearchHEMOCYTE INFILTRATION OF OLFACTORY RECEPTORNEURON CLUSTERS AFTER AESTHETASC DAMAGE INTHE SPINY LOBSTERSchmidt M. 1 , Derby C. 1 1 Biology, Georgia State University, Atlanta,GAIn the spiny lobster, Panulirus argus, olfactory sensilla (aesthetascs)are comprised of large clusters of olfactory receptor neurons (ORNs)and ensheathing cells. Aesthetascs are continuously generated in adults,and after severe damage they degenerate and subsequently regenerate(Harrison et al. J. Neurobiol. 47:51-66, 2001; Harrison et al. J. Comp.Neurol. 471:72-84, 2004). To study cellular events underlying the localde- and regeneration of aesthetascs, we monitored the tissuecomposition in the olfactory organ with confocal microscopy afterfocally damaging aesthetascs in two ways: shaving off their entire setae,or clipping them at about 50 % of their length. Shortly after the damage(6 h–3 days), infiltration of damaged ORN clusters by granulocytes—aprominent type of circulating hemocytes containing large granules andf-actin—was observed in both treatments. However, this infiltration wasmuch more substantial after clipping the aesthetascs than after shavingthem. Later time points revealed radically different fates of the ORNclusters in both treatments: after clipping, ORN clusters remainedmassively infiltrated by granulocytes for several more days andappeared normal after 3 weeks; after shaving, ORN clusters completelydegenerated within ca. 2 weeks and then started to regenerate by mitoticactivity, without granulocytes being present. These findings indicatethat granulocytes are the main agents of an immune response inducedby physiological relevant damage to aesthetascs and that they contributeto repair mechanisms allowing the damaged ORNs to survive.Acknowledgments: Supported by NIH grant DC00312.121
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