Universität zu Köln

Sukumaran, Sunil Kumar (2005). Development of genetically encoded cell type-specific activity markers for the mouse olfactory system. PhD thesis, Universität zu Köln.

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Abstract

The olfactory system consists of the olfactory epithelium in the nasal cavity, the olfactory bulb, the olfactory cortex and higher brain regions. Olfactory information processing begins at the olfactory sensory neurons that express odorant receptors. These proteins bind odors and transduce a biochemical signal that is converted to action potentials by the neuron. In the olfactory bulb, sensory neurons synapse with projection neurons called the mitral/tufted cells in structures called glomeruli and pass on information to the olfactory cortex. The olfactory bulb is not a simple way station for transmission of odorant information; significant processing of information is carried out by the circuitry in the olfactory bulb. The major neuronal components of the bulb that take part in these interactions are the periglomerular cells, short axon cells and the granule cells. Neural information processing is carried out by ensembles of neurons and it is important to study such populations to reach an understanding of nervous system function. Recent developments in identification of cell type specific promoters and construction of GFP based sensors of Ca2+ influx and voltage changes has made it possible to devise genetic strategies to study them. Another strategy that is frequently employed is studying the expression of Immediate Early Genes, especially C-fos after stimulation. In this study we developed two mice strains, one that can express the short halflife version of EYFP called d2EYFP under the C-fos promoter and another that codes for the calcium sensitive fluorescent protein ratiometric pericam. The expression of both these reporters can be made cell type specific because they are blocked by a floxed stop cassette and can be activated in specific cell types by crossing to mice strains that express the cre recombinase under specific promoters. A Bacterial Artificial Chromosome that carries the Cfos gene was modified by inserting a STOP-IRES-d2EYFP cassette at the C-fos locus after truncating the C-fos-coding region. The loxP site in the BAC's back bone was removed by another round of recombination and circular BAC molecules were used of pronuclear microinjection. One positive founder was identified and is being bred for further analysis. Ratiomeric pericam was cloned under the ubiquitous CAG promoter with a stop cassette in between and transgenic mice were made after testing expression in cell culture. Seven different transgenic lines with different transgene copy numbers were obtained and were subjected to further analysis. No transgene expression could be detected upon breeding to a CMV-Cre mouse strain that should delete in all cells. Analysis of DNA of double transgenic mice showed no deletion of stop cassette. The mice were further crossed to a CamKIIa-cre

Item Type:	Thesis (PhD thesis)
Translated title:	Title Language Entwicklung der genetisch kodierten Art-spezifischen aktivitatstsmarkierungen der Zelle für das Mäuseolfaktorische System German
Translated abstract:	Abstract Language Das Geruchssystem besteht aus dem Riechepithel in der Nasenhöhle, dem Bulbus olfaktorius, dem olfaktorischen Cortex und weiteren Gehirnregionen. Die Informationsverarbeitung im olfaktorischen System beginnt mit den Geruchsrezeptorneuronen, die die Geruchsrezeptorgene exprimieren. Diese Rezeptoren binden Geruchsstoffe und generieren so ein biochemisches Signal, das zu Aktionspotentialen umgewandelt wird. Im Bulbus olfaktorius bilden die sensorischen Neuronen Synapsen mit den Projektionsneuronen in Neuropilknäueln, den sogenannten Glomeruli. Die Projektionsneuronen, Mitral- und Büschelzellen tragen die Information zum Riechcortex. Der Bulbus olfaktorius ist nicht nur eine einfache Relaisstation, hier findet schon Informationsverarbeitung statt durch periglomeruläre Zellen, Short-Axon-Zellen, und Körnerzellen. In allen diesen Interaktionen sind Zellensembles beteiligt, und es ist daher wichtig, die Antworteigenschaften von Populationen von Neuronen zu untersuchen, um das Geruchssystem zu verstehen. Seit kurzem sind genetisch kodierte Calcium-Indikatoren verfügbar geworden, die zusammen mit der Entwicklung zelltypspezifischer Promotoren es erlauben sollten, die neuronale Aktivität auf der Populationsebene zu untersuchen. Solche Promotoren sollten es auch ermöglichen, die Aktivierung von Immediate-Early Genen wie C-fos (welche die neuronale Aktivierung wiederspiegelt) zelltypspezifisch zu charakterisieren. In dieser Dissertation habe ich zwei Mausstämme generiert, in einem wird die destabilisierte Version von EYFP, d2EYFP, unter der Kontrolle des C-fos Promotors exprimiert, im anderen wird der Calciumindikator ratiometrisches Pericam von einem ubiquitären Promotor, CAG, exprimiert. Nichtsdestotrotz kann die Expression in beiden Fällen zelltypspezifisch geführt werden, da eine loxP-STOP-loxP Kassette vorgeschaltet ist, und durch Kreuzung mit einer geeigneten CRE-Rekombinase-exprimierenden Mauslinie entfernt werden kann. German
Creators:	Creators Email ORCID ORCID Put Code Sukumaran, Sunil Kumar tellsparck@yahoo.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-15031
Date:	2005
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Olfactorische system,Pericam, C-fos, German olfactory system, pericam, c-fos English
Date of oral exam:	5 July 2005
Referee:	Name Academic Title Korsching, Sigrun Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/1503