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Towards a molecular understanding of midbrain-hindbrain neurogenesis and reward in zebrafish [Elektronische Ressource] / Jovica Ninković

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Published 01 January 2006
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Language English
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Technische Universit at Munc hen
GSF-Forschungszentrum fur Umwelt und Gesundheit
Institut fur Entwicklungsgenetik
Abteilung Zebra sc hneurogenetik
Towards a molecular understanding
of midbrain-hindbrain neurogenesis and reward
in zebra sh
Jovica Ninkovic
Vollst andiger Abdruck der von der Fakult at Wissenschaftszentrum Weihstephan fur
Ern ahrung, Landnutzung und Umwelt der Technischen Universit at Munc hen zur Er-
langung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. Erwin Grill
Prufer der Dissertation: 1. Univ.-Prof. Dr. Wolfgang Wurst
2. Univ.-Prof. Dr. Kay H. Schneitz
3. Univ.-Prof. Dr. Alfons Gierl
Die Dissertation wurde am 09.11.2005 bei der Technischen Universit at Munc hen ein-
gereicht und durch die Fakult at Wissenschaftszentrum Weihstephan fur Ern ahrung,
Landnutzung und Umwelt am 01.02.2006 angenommen.Towards a molecular understanding
of midbrain-hindbrain neurogenesis and reward
in zebra sh
Kumulative Arbeit
Jovica NinkovicAbstract
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 identi ed 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 pre gures 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 di ering with respect to their sensitiv-
ity 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 demon-
strated 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 in-
volve 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 di eren t sensitivity thresholds along the medio-lateral axis of the neu-
ral plate. I showed that this di eren tial 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 zebra sh, including a number of crucial
speci cit y controls, such as the assessment of the animal’s stress, vision and memory,
the measure of optimal drug doses, and a veri cation of the dose received into the
animal’s brain. Thanks to this methodology, I demonstrated that more than 95 %
of wild-type zebra sh robustly experience the rewarding e ects of the psychostimu-
lant D-amphetamine. I next focused on the cholinergic system, a known modulator
of dopaminergic transmission in mammals and demonstrated that ache/+ mutant
adult zebra sh, which exhibit higher level of central acetylcholine (ACh) than wild-
type individuals, are strongly resistant to the rewarding e ects of D-amphetamine.
This phenotype cannot be accounted for by alterations in the exploratory activity,
ivision or memory of these mutants. Taken together, my results provide the rst ge-
netic 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 e ect, have been evolutionarily conserved
in vertebrates, and thus validate the zebra sh 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 zebra sh can be used for to produce developmental
mutants and run genetic or chemical screens. I conducted a large-scale screen aimed
to recover dominant modi ers of addiction to D - amphetamine and recovered one
mutation a ecting the response to the rewarding e ects of D - amphetamine. This
m is currently being positionally cloned.
All together, my results set the stage for future forward genetics approaches of
neurogenesis control and reward.
iiList of abbreviations
AC anterior commissure
ACh acetylcholine
AChE acetylcholinesterase
AMPH D - amphetamine
Anf Anterior neural folds homolog
ANP anterior plate
APC Adenomatous Polyposis Coli
Av aversion
Bf1 Brain factor 1
bHLH basic helix-loop-helix
BMP bone morphogenetic protein
BSR brain stimulation reward
Ca caudal reticulospinal neuron
CaP primary motorneuron
ChAT choline acetyltransferase
Ci Cubitus interruptus
CNS central nervous system
CoPA commissural primary ascending interneuron
Cos Costal
CPP conditioned place preference
DAT dopamine transporter
des deadly seven
Di diencephalon
Dll Distal-less
drc dorso-rostral cluster
Dsh disheveled
DVDT dorsoventral diencephalic tract
e epiphysis
iiiE(Spl) Enhancer of split
EAA excitatory amino acid
Eng (En) Engrailed
ENK enkephalin
ENU ethylnitrosourea
ep epiphysial cluster
ERG electroretinography
Esr6 Enhancer of split related epidermal protein-6
Fgf broblast growth factor
GABA gamma-aminobutyric acid
Gbx Gastrulation and brain-speci c homeobox protein
Gli Glioblastoma
GSK3 glycogen synthase kinase-3