Open Access

Kasper Langebeck-Jensen

• To survive and thrive in nature, animals need to adapt their behavior to their environment. Behavioral adaptation is primarily due to changes within the brain and involves changes in the brain proteome (the collection of proteins in the brain). However, thus far very few studies have examined the proteomic changes during behavioral adaptation. Hence, with this work I set out to determine the proteomic changes induced in the brain of zebrafish larvae undergoing behavioral adaptation. Specifically, I examined the changes induced by adaptation to the natural challenge of strong water currents. To this end I took advantage of an assay developed by my collaborators Luis Castillo and Soojin Ryu. In this assay 5 days old zebrafish larvae were exposed to strong water currents. Subsequently they exhibited a reduction in cortisol response and initial locomotion, and increased rheotaxis, as defined by increased swimming directly against the water current when re-exposed to the water current. I employed this assay to investigate the changes to the larval zebrafish brain proteome during behavioral adaptation. Furthermore, I developed a method for extracting larval brains and prepare them for mass-spectrometric analysis. This work not only allowed the comparison of the brain proteome of naïve and behaviorally-adapted larvae, but also resulted in the most comprehensive proteome of the zebrafish brain observed to date and the first proteome of the larval zebrafish brain. In total 4309 proteins were identified in the brain. When the proteome of naïve and behaviorally adapted larvae were compared 41 proteins were found to be more abundant and 16 to be less abundant in the pre-exposed larvae. Of these 57 proteins, 28 have previously been found to have functions in the brain, 17 with functions identified in other tissues, and 12 proteins that have yet to be described. From examining the most relevant function of each protein I propose a speculative model in which the larval brain undergoes behavioral adaptation and becomes less susceptible to stress (reduction in mecp2 and hsp90 protein), form new neuronal connections (regulation of arid1b, fmn2b, ptpra, mycbp2, and pcyt2), modulate existing connections (regulation of asic1b, calsenilin, ptpra, aplp2, dag1, olfm1b, mycbp2, smad3a, and acvr2a abundance), undergo spatial learning in form of navigating the water vortex (increases in calsenilin, ptpra, and pcyt2), show an elevation in protein turnover (increases in lamp2, Ublcp1, larp4b, and ublcp1), have increased and regulated energy production (increases or reduction in rpia, ldhbb, and mitochondrial proteins; nfs1, eci1, MRPS2B, MRPL4, and mrps2), and a decrease in neurogenesis (reduction in smad3a, and ric8a). To further investigate proteomic changes during behavioral adaptation, I investigated the translational response by metabolically labeling the larval forebrain with ANL and visualizing the labeled proteins using the fluorescent non-canonical amino acid tagging (FUNCAT). I detected a general increase in translation within the forebrain as a result of the water vortex adaptation, which correlated well with the range of changes observed in the brain proteome. Specifically, a region within the forebrain correlated with a region in the adult zebrafish that is homologous to the mammalian limbic region. Taken together these results show that during behavioral adaptation, protein synthesis is significantly increased in the larval forebrain, and that throughout the brain regulation of the proteome includes proteins that could support the following functions: changes or modifications in neuronal connectivity, the stress response, spatial learning, changes in energy metabolism and changes in neurogenesis. Lastly, I set out to provide a new tool for zebrafish researchers. Together with Güney Akbalik I introduced metabolic labeling of newly synthesized RNA using 5-ethynyluridine (EU) and subsequent visualization with a copper catalyzed clickreaction to the zebrafish larvae. With 5 hours of EU incubation I was able to visualize newly synthesized RNA and identify pentylenetetrazole-induced transcriptional increases. With this I showed that EU labeling could be implemented to examining transcriptional changes within the brain of zebrafish larvae.
• In der Natur treffen Tiere laufend auf Herausforderungen durch ihre Umwelt. Damit ein Tier diese meistert, muss es ständig sein Verhalten ändern und an diese Herausforderungen anpassen. Diese Verhaltens-Änderungen werden durch änderungen in den neuronalen Netzwerken des tierischen Gehirns bewerkstelligt. Neuronale Plastizität findet auf vielen Ebenen statt - von Änderungen der Rezeptorenmenge in einem postsynaptischen Abschnitt bis hin zur Bildung neuer neuronaler Verbindungen. Allen langanhaltenden Formen von Plastizität gemeinsam ist allerdings, dass diese von Proteinsynthese abhängig sind. Proteine spielen viele Schlüsselrollen in Neuronen - insbesondere an der Synapse - wie z.B. Transporter, Rezeptoren oder als Signalproteine. Während synaptischer Plastizität wird die Menge dieser und anderer Proteine darauf angepasst, den neuen Aktivitäts- und Konnektivitätslevel der Nervenzelle zu unterstützen. Bisher sind die Proteom-Änderungen, die Verhaltens-Anpassungen unterliegen, nicht eingehend untersucht. Die meisten Studien beschränken sich auf wenige Kandidaten-Proteine anstatt die Dynamik des gesamten Gehirn-Proteoms (die Gesamtheit aller Proteine im Gehirn) zu untersuchen. Mit der Untersuchung der Dynamik des Gesamtprotein-Bestandes sollte es möglich sein, neue Proteine mit einer Schlüsselrolle in Verhaltens-Anpassungen zu identifizieren und ein besseres Verständnis für die gesamtheitlichen Proteom-Änderungen bei VerhaltensAnpassungen zu erhalten...