title: A genetically encoded system with high spatio-temporal resolution for in vivo modification of neuronal network activities
creator: Terzi, Firat
description: Despite decades of progress in the field of conditional transgene expression, acute, cell-type specific transgene induction remains difficult to achieve with currently available methods. By combining the inducible Tet system with the conditional, cell-type specific Cre recombinase system, I developed a method that allows genetic manipulation of tissues in vivo at cellular resolution based on adeno-associated viruses (AAV). In addition, the Cre-mediated constitutive expression of a fluorescent reporter highlights cells prior to induction of the transgene via the Tet system. Therefore, cells can be carefully characterized before, during, and after genetic manipulation. The phenotypic consequences of transgene expression can thus be temporally correlated in individual cells in vivo.  The TetOn system is a two-component system of a Tetracycline-dependent transcriptional activator (rtTA) and the Tet-dependent transgene, which I now flanked by Cre recombinase sites. The TetOn system was optimized to increase tightness by additionally introducing the doxycycline-dependent tTR repressor. In vivo, this substantially reduced the notorious leakiness of the TetOn system. The goal of my thesis research was to use this optimized Cre-dependent TetOn system for acute genetic silencing of neurons in mouse cortex by expression of Kir2.1, a potassium channel that cell-autonomously hyperpolarizes membranes. Co-injection of an AAV with the GFP-based calcium indicator GCamP6 allowed in vivo two-photon imaging the spontaneous activity of the same neurons over time in longitudinal experiments. Injection of doxycycline into the brain induced rapid silencing of neurons within hours that lasted for at least 80 h. Using transgenic Parvalbumin-Cre mice, silencing of inhibitory Parvalbumin interneurons significantly increased the spontaneous activity of surrounding neurons in a distance dependent manner as nearby neurons were more affected. No such effect was seen after silencing a random subpopulation of neurons. Finally, I tested if prolonged silencing influences dendritic morphology. Cre-dependent expression of red fluorescent tdTomato was used to monitor spine numbers before and 75 h after Kir2.1 expression. Corroborating previously published in vitro results, I could show for the first time in vivo that silencing individual neurons after synapse formation does not change spine numbers.  This thesis describes the entire journey from the cloning of an optimized TetOn system to its in vivo applications. Together with the establishment of longitudinal in vivo calcium imaging, an analysis pipeline has been created for registration, extracting, and processing of calcium signals for spike estimation. Despite the complexity of the combined tools and the intrinsic variability associated with manipulating and analyzing the same cells over time in vivo, this method yielded exciting data and will provide researchers with the possibility to use this approach in a wide spectrum of experimental settings.
date: 2018
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/24188/1/Thesis_Firat_Terzi_final.pdf
identifier: DOI:10.11588/heidok.00024188
identifier: urn:nbn:de:bsz:16-heidok-241880
identifier:   Terzi, Firat  (2018) A genetically encoded system with high spatio-temporal resolution for in vivo modification of neuronal network activities.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/24188/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng