TY  - GEN
ID  - heidok12830
A1  - Dogbevia, Godwin Kofi
AV  - public
Y1  - 2011///
TI  - Tetracycline-controlled inducible gene expression systems to manipulate neuronal circuits in the mammalian brain
KW  - Tetracycline 
KW  -  gene expression
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/12830/
N2  - Regulatable control of gene expression in the mammalian brain is of tremendous importance in understanding the role of neural circuits in the processing, storage and retrieval of information. To understand the fundamental molecular mechanisms underlying these processes, there is the need to develop diverse molecular genetic tools that allow for conditional gene regulation in the central nervous system. To address this issue, we have developed recombinant adeno-associated viruses (rAAVs) equipped with tetracycline (Tet)-controlled genetic switches to inducibly and reversibly manipulate neural circuits, in the mammalian brain. The present work focused on three objectives: In the first part, long-term, Tet-controlled gene regulation in neurons was investigated. In this part, the rAAV system was used to study the kinetics of Tet-controlled gene regulation in vitro and in vivo using the firefly luciferase reporter assay. Furthermore, repeated cycles of gene activation and inactivation in vivo were also demonstrated. In the second part, inducible, subregion- and cell type-restricted gene recombination and gene knockout in the mouse brain was investigated by employing the rAAV system expressing the site-specific Cre recombinase enzyme and a red fluorescent marker protein under Tet control. Conditional and region-specific gene recombination in transgenic reporter mice was carried out by either targeting the viruses to the hippocampus, cortex or both. Gene recombination occurred in these regions only upon Doxycycline (Tet derivative) injection. With the rAAV and Tet-controlled inducible gene expression systems, the N-Methyl-D-aspartate (NMDA) receptors were knocked out in the barrel field. We clearly demonstrated by electrophysiology that loss of NMDA receptors took place after one week of Dox treatment. With the inducible knockout approach, we are currently investigating the function of NMDA receptors in acquisition and maintenance of associative learning and memory.  In the last part, inducible and reversible silencing of synaptic transmission was investigated with the use of tetanus toxin light chain (TeTxLC). By reversible silencing of synaptic transmission with TeTxLC, the role of neurons from different brain regions in memory consolidation processes and behavior will be investigated. With this approach, we have demonstrated that TeTxLC expression results in silencing of synaptic transmission. Moreover, by expressing the TeTxLC in the striatum of the mouse brain, a behavioral phenotype was produced, which was reversed by Dox administration. The original TeTxLC has a long half-life thus, very slow off rate. To reduce the half-life of the TeTxLC, new variants of TeTxLC were generated, named fast one (F1-TeTxLC) and fast two (F2-TeTxLC). With these new variants of TeTxLC, we observed that reversible silencing of synaptic transmission took place within 3 weeks with F1-TeTxLC and less than two weeks with F2-TeTxLC in vivo. We also demonstrated synaptic silencing in organotypic brain slices 24-48 hours after Dox treatment in vitro. A combination of these newly developed genetic tools will be of great advantage to investigate the role of neural circuits in learning, memory consolidation and behavior. 
ER  -