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Ninkovic, J.

Towards a molecular understanding of midbrain-hindbrain neurogenesis and reward in zebrafish.

München, Technische Universität, Fakultät Wissenschaftszentrum Weihenstephan, Diss., 2006, 211 S.
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The developmental functionality of neural networks involved in complex diseases, such as addiction, is an important determinant of adult behavior. Thus, understanding the principles of embryonic neurogenesis is of prime importance. To approach this issue, I focused on neurogenesis control in the midbrain-hindbrain domain, which contains a long-lasting progenitor pool, the Intervening zone (IZ). I identified the Hairy/E(Spl) factors Him and Her5 as the crucial determinants of IZ formation. The expression of these two factors at the end of gastrulation prefigures and later during development precisely delineates in space the IZ. The IZ is formed as a two-partite area (lateral (LIZ) and medial (MIZ)), these two domains differing with respect to their sensitivity to the “Him + Her5” inhibitory activity. Using single and double knockdowns of him and her5, as well as a him + her5 deletion mutant background (b404), I demonstrated that Him and Her5 are equally necessary for MIZ formation, and that they act redundantly in LIZ formation in vivo. I showed that these processes do not involve cross-regulation between Him and Her5 expression or activities. Increasing the function of one factor when the other is depleted, I further showed that Him and Her5 are functionally interchangeable. My results are in agreement with a model where the global “Him + Her5” activity inhibits ngn1 expression in a dose-dependent manner and through different sensitivity thresholds along the medio-lateral axis of the neural plate. I showed that this differential sensitivity of the MIZ and LIZ were based on graded Gli signaling along the medio-lateral neural plate axis at the level of the IZ, and that Gli1 activity in this process was regulated by the PKA/ GSK3 phosphorylation tandem. According to my results, Gli1 increases the threshold level for “Him + Her5” inhibitory activity in the MIZ and loss of Gli1 function render the MIZ into the LIZ in respect to “Him + Her5” inhibitory activity. In parallel, to approach brain functionality, I developed a reliable conditioned place preference methodology to score addiction in zebrafish, including a number of crucial specificity controls, such as the assessment of the animal’s stress, vision and memory, the measure of optimal drug doses, and a verification of the dose received into the animal’s brain. Thanks to this methodology, I demonstrated that more than 95 % of wild-type zebrafish robustly experience the rewarding effects of the psychostimulant D-amphetamine. I next focused on the cholinergic system, a known modulator of dopaminergic transmission in mammals and demonstrated that ache/+ mutant adult zebrafish, which exhibit higher level of central acetylcholine (ACh) than wildtype individuals, are strongly resistant to the rewarding effects of D-amphetamine. This phenotype cannot be accounted for by alterations in the exploratory activity, i vision or memory of these mutants. Taken together, my results provide the first genetic arguments supporting manipulations of acetylcholinesterase (AChE) activity as a promising avenue towards limiting addiction behavior to psychostimulants. Second, they show that the rewarding potential of amphetamine, as well as the importance of the cholinergic system in modulating this effect, have been evolutionarily conserved in vertebrates, and thus validate the zebrafish as a reliable model to give insight into the molecular neurobiology of drug-induced reward in vertebrates. This is of crucial interest given the ease with which zebrafish can be used for to produce developmental mutants and run genetic or chemical screens. I conducted a large-scale screen aimed to recover dominant modifiers of addiction to D - amphetamine and recovered one mutation affecting the response to the rewarding effects of D - amphetamine. This mutation is currently being positionally cloned. All together, my results set the stage for future forward genetics approaches of neurogenesis control and reward.  
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Publication type Other: Thesis
Thesis type Doctoral thesis
Corresponding Author
Keywords Zebrabärbling; Neurogenese; Molekulargenetik
Quellenangaben Volume: , Issue: , Pages: 211 S. Article Number: , Supplement: ,
University Technische Universität
University place München
Faculty Fakultät Wissenschaftszentrum Weihenstephan
Non-patent literature Publications
Institute(s) Institute of Developmental Genetics (IDG)