Browsing by Subject "Axon guidance"

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    Odorantrezeptoren in Axonen olfaktorischer Sinneszellen : in vitro Studien an Explantatkulturen
    (2008) Luxenhofer, Georg; Breer, Heinz
    Olfactory sensory neurons (OSN) expressing a particular odorant receptor (OR) are widely scattered throughout the olfactory epithelium (OE) of the nose and send their axon into a small number of common glomeruli in the olfactory bulb (OB). In a spatially well conserved pattern these axons establish synaptic contacts to second order neurons. The molecular mechanisms underlying the precise wiring are still not well understood. To generate a system which may facilitate the investigation of distinct aspects of this complex process, an in vitro culture with tissue explants from the olfactory system was established in the present work. The use of tissues from transgenic mice which enabled the visualisation of OSN and their processes by intrinsic fluorescence allowed a continuous observation of distinct axonal populations under defined and manipulable conditions. Cells within an explant from the OE harvested at the embryonic stage 14 (E14) extended numerous axonal processes within a few days which grew out radially and without fasciculation. During the initial culture period the explants contained mainly progenitor cells; after several days in culture cells differentiated to OMP-positive, thus mature OSN. Using receptor specific transgenic mouse lines the expression of distinct OR genes in a subpopulation of OSN could be detected. Altogether, the culture conditions thus allowed the differentiation of progenitor cells into OSN with characteristic gene expression. Concerning the key question of how axons of OSN interact with their target tissue, co-culture experiments with OB tissue were performed; they showed that axons were initially repelled by their target. A precultivation of OB tissue, however, resulted in an attraction of axons even from larger distances. Moreover, the bulb tissue exerted a positive effect on the growth rate of OSN axons. During their growth these axons formed bundles which defasciculated in the vicinity of the OB explants. These results showed that characteristic parameters in the generation of OSN, their axonal growth and interactions with the target tissue were recapitulated by the in vitro culture system, thus, providing optimal conditions for the examination of key questions regarding the molecular mechanisms involved in establishing the unique projection pattern. Subsequently, the explant culture system was used to investigate the role of the odorant receptor protein in the process of path finding. Expression of genetically modified receptor variants in the explants revealed novel insights into the subcellular localisation of the odorant receptor mOR256-17. An mOR256-17-EGFP fusion protein could be detected in vesicles transported into the dendrite of OSN, resulting in an accumulation of the OR in the cilia. Using this technique it was possible to observe for the first time OR proteins in vesicles which were transported anterogradely and retrogradely along the entire axon. The OR could be visualised within the growth cones and the attached filopodia. Taking advantage of a novel detection method in which proteins integrated into the plasma membrane were selectively marked, retrogadely transported vesicles containing internalised mOR256-17 protein could be observed. The generation of an OR variant, in which the G-protein binding domain was mutated resulted in a disturbed localisation of the OR protein within OSN. Hence, by developing an improved in vitro explant system, an important tool was generated that allowed novel insights into the function of distinct molecular components and should be valuable for future studies aimed at understanding the complex processes that lead to the precise connection of OSN with their target.