The content of olfactory memory in larval Drosophila Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Dushyant Mishra aus Saugor, M.P, Indien Würzburg, 2011 1 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Erster Gutachter: Zweiter Gutachter: Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: 2 Erklärung gemäß § 4 Absatz 3 der Promotionsordnung der Fakultät für Biologie der Bayerischen Julius-Maximilians-Universität zu Würzburg vom 15. März 1999: Die vorgelegte Dissertation besteht aus drei Publikationen und zwei für die Veröffentlichung vorbereiteten Manuskripten, sowie einer zusätzlichen „Allgemeinen Einleitung und Diskussion“. Die Mitwirkung der Co-Autoren jeder Publikation werden auf den folgenden Seiten dargestellt. Ich versichere, dass ich diese Dissertation in allen Teilen selbständig angefertigt habe und dazu keine anderen als die angegebenen Hilfsmittel benutzt habe. Alle aus der Literatur entnommenen Textstellen sind als solche gekennzeichnet. Die vorliegende Arbeit wurde weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegt. Zuvor habe ich keine akademischen Grade erworben oder versucht zu erwerben. Würzburg, den Dushyant Mishra Prof. Dr. Bertram Gerber 3 Structure of the thesis This thesis consists of 2 parts. Part I deals with a behavioural account of how intensity and quality, the two defining features of an odour, are perceived in larval Drosophila. It contains three chapters, corresponding to one manuscript prepared for publication and two publications. The manuscript deals with odour intensity learning, the first publication provides a behaviour based estimate of odour similarity in larval Drosophila, and the second publication shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Part II contains two chapters, corresponding to one manuscript prepared for publication and one publication. The manuscript studies cognitive- enhancement in larval Drosophila where I show that food supplementation with dried roots of Rhodiola rosea dose- dependently improves odour- reward associative learning in larval Drosophila. The publication deals with local loss of function of Synapsin and its consequences for associative plasticity of larval Drosophila. In addition, I present a ‘General Introduction and Discussion’ to give the reader an overview of the background and implications of this thesis. Also included at the end is a brief summary of the studies presented in this thesis. This work would not have been possible without the effort of many people, and the supervision of my mentor Dr. Bertram Gerber. I sincerely acknowledge the co- authors of each manuscript, whose contributions are explicated below. 4 Chapter I.1 Mishra D., Chen Yi., Yarali A., Gerber B.: Olfactory memories can be intensity- specific in larval Drosophila. DM, BG and AY conceived the research and designed the experiments. DM and YC performed the experiments and DM analyzed the data. DM, AY and BG wrote the paper. Chapter I.2 Chen Yi., Mishra D., Schmitt L., Schmuker M., Gerber B. (2010): A behavioural odour- similarity 'space' in larval Drosophila, Chemical Senses, 36(3), 237- 249. YC, DM and BG conceived the research and designed the experiments. YC, LS and DM performed the behaviour experiments. Specially, DM performed the experiments to describe the dose effect curves for different odours which subsequently lead to adjust the odour dilutions for equal learnability. YC, MS and BG analyzed the data. YC and BG wrote the paper. Chapter I.3 Mishra D., Louis M., Gerber B., (2010): Adaptive adjustment of the generalization- discrimination balance in larval Drosophila, J. Neurogenetics, 24(3), 168-175. DM, and BG conceived the research. DM and BG designed the experiments. DM performed the experiments and analyzed the data. DM and BG wrote the paper with inputs from ML. 5 Chapter II.1 Lushchak O.=, Mishra D.=, Haberkern H., Eschbach C., Lushchak V., Niewalda T., Gerber K., Koblowsky M., Gerber B.: 'Cognitive enhancement’ of associative function in larval Drosophila by Rhodiola rosea food supplementation. OL, DM, VL and BG conceived the research. OL, DM and BG designed the experiments. OL, DM, KG, MK performed the learning experiments concerning the larval Drosophila and their controls. KG and TN helped to make the Rhodiola enriched fly food vials. CE and HH performed and analyzed the larval tracking experiments. DM analyzed the data. DM and BG wrote the paper. Chapter II.2 Michels B., Chen Yi., Tanimoto H., Mishra D., Engmann O., Saumweber T., Gerber B., (2011): Site and mode of Synapsin action in associative learning, Learn. Mem. 18(5):332- 44. BM, HT and BG conceived the research and designed the experiments. BM, OE and TS performed the Synapsin rescue experiments. YC performed behavioural experiments with PKA-site mutated transgenes. DM performed behavioural and immunohistochemical analysis with Synapsin- RNAi lines. BM and BG wrote the paper. Wuerzburg, den Dushyant Mishra Prof. Dr. Bertram Gerber 6 Table of Contents General introduction & discussion……………………..................…………................10 Part I Olfactory percepts of larval Drosophila I.1. How is odour intensity integrated into the odour percept? Abstract……………………………………………..………..……………..….....35 Introduction.............................................................................................................36 Materials & Methods..............................................................................................39 Results.....................................................................................................................41 Discussion...............................................................................................................52 Acknowledgements.................................................................................................59 References...............................................................................................................59 Supplementary materials........................................................................................64 I.2. A behavioural odour similarity ‘space’ Abstract....................................................................................................................76 Introduction.............................................................................................................77 Materials & Methods..............................................................................................82 Results.....................................................................................................................86 Discussion...............................................................................................................95 Acknowledgements..................................................................................................99 7 References.............................................................................................................100 Supplementary materials.......................................................................................107 I.3. Odour percepts can be adaptively adjusted Abstract.................................................................................................................115 Introduction...........................................................................................................116 Materials & Methods............................................................................................117 Results...................................................................................................................125 Discussion.............................................................................................................131 Acknowledgements................................................................................................133 References.............................................................................................................134 Supplementary material........................................................................................137 Part II. Mechanisms of learning and memory in larval Drosophila II.1. Cognitive enhancement in larval Drosophila Summary...............................................................................................................143 Results & Discussion............................................................................................144 Experimental procedures......................................................................................160 Acknowledgements...............................................................................................166 References.............................................................................................................166 8 II.2. Loss of function of Synapsin Abstract………….…………………………………………………………….....171 Introduction...........................................................................................................172 Results...................................................................................................................175 Discussion.............................................................................................................193 Materials