Production of the glucocorticoid hormone cortisol in response to a stressor is initiated by activation of the hypothalamic-pituitary-interrenal (HPI) axis in fish. Serotonin (5-HT) and negative feedback regulate cortisol production at the whole-animal level; the objective of the present thesis was to investigate their roles in regulating cortisol production by interrenal cells of rainbow trout (Oncorhynchus mykiss). Messenger ribonucleic acid (mRNA) for the 5-HT4 receptor was present in low abundance in interrenal cells. In addition, cortisol production was significantly increased for in vitro head kidney preparations incubated with 5-HT, and this elevated cortisol production was blocked by the 5-HT4 receptor antagonist 5-fluoro-2-methoxy-[1-[2-[(methylsulphonyl) amino] ethyl]-4-piperidinyl]-1h-indole-3-methylcarboxylate sulphamate (GR125487). Thus, 5-HT acts at the head kidney level to regulate cortisol production, probably via the 5-HT4 receptor. Chronic social stress did not appear to regulate the expression of key proteins involved in cortisol biosynthesis or corticosteroid receptors (CR). However, head kidney tissue incubated in vitro with cortisol for 2-8 h showed a reduction in adrenocorticotropic hormone-stimulated cortisol production compared to controls, suggesting the existence of an ultra-short-loop negative feedback mechanism. Thus, the high circulating levels of cortisol in trout experiencing chronic social stress may activate this ultra-short-loop negative feedback mechanism to suppress cortisol production at the head kidney level.

The ability to learn and change future behaviour based on past experiences is crucial for the life and survival of animals. For various behaviours exhibited by animals it is clear that in a seemingly homogeneous population not all individuals behave the same way, even in invertebrates. In crickets (Gryllus bimaculatus), a model system for the mechanisms of intra-specific aggression, agonistic experiences with the underlying impact of neuromodulators have been identified as a cause of inter-individual differences. For mammals and humans, the experience of adversity and stress can have detrimental effects on cognitive abilities and chronic defeat stress is used as a model for depression. In crickets the equivalent, the chronic social defeat stress paradigm, has been established. This thesis first sets out to construct a new model for measuring a conditioned response from multiple behavioural aspects and quantify learning in individual crickets. Video tracking of responses revealed behavioural variables that were included in a binary logistic regression analysis, whereas the resulting multi-variable model proves to be superior to other models constructed and can give the probability of an individual exhibiting a conditioned response. With this, learning indices can be calculated for each individual trained in a differential appetitive olfactory paradigm. With the method at hand, this thesis reveals that the experience of chronic social stress impairs learning in crickets, susceptible and resilient to defeat stress alike. The experience of multiple wins, however, does neither improve nor decrease learning abilities, but a long-term winner effect on aggression could be shown. Although inter-individual differences in learning are present, the aggressive state of crickets is not correlated to the learning indices. The application of serotonergic drugs that block receptors or act as re-uptake inhibitors reveal the influence of serotonin on learning within this paradigm. In addition to maintaining reduced aggressiveness, serotonin promotes the impairment of learning after the experience of chronic social defeat stress.:1 INTRODUCTION.....................................................................................................1 2 MATERIALS AND METHODS............................................................................... 8 2.1 Experimental animals...................................................................................... 8 2.2 Appetitive olfactory conditioning..................................................................... 8 2.2.1 Odour application and rewarding....................................................... 8 2.2.2 Absolute conditioning paradigm........................................................ 10 2.2.3 Differential conditioning paradigm.................................................... 11 2.3 Experimental setup for video-tracking.............................................................. 12 2.4 Binary logistic regression model....................................................................... 13 2.4.1 Binary groups for model building...................................................... 13 2.4.2 Variables of a behavioural response................................................... 14 2.4.3 Calculating a conditioned odour response probability (Presp) ............ 15 2.5 Evaluation of learning with the binary logistic model...................................... 17 2.6 Evaluation of aggression with a standardised fight.......................................... 18 2.7 Multiple agonistic experiences......................................................................... 19 2.7.1 Chronic social defeat stress................................................................ 19 2.7.2 Multiple wins..................................................................................... 20 2.8 Serotonin......................................................................................................... 20 2.8.1 Pharmacological treatments............................................................... 20 2.8.2 Methiothepin and ketanserin.............................................................. 21 2.8.3 Fluoxetine with non-chronic defeat................................................... 21 2.9 Additional data analysis and statistic................................................................ 22 3 RESULTS............................................................................................................ 23 3.1 Binary logistic regression model for quantifying learning............................... 23 3.1.1 Behavioural variables of a conditioned odour response.................... 23 3.1.2 Model building and selection............................................................. 29 3.1.3 Odour response probabilities (Presp)................................................... 31 3.1.4 Application of the regression model to assess the quantification of learning.................................................................................................... 34 3.2 The influence of agonistic experiences on aggression and learning................. 39 3.2.1 Chronic social defeat stress................................................................ 39 3.2.2 Multiple experiences of winning........................................................ 46 3.2.3 Correlation of aggression and learning............................................... 48 3.2.4 Summary of learning capacities – multiple experiences.................... 50 3.3 The influence of serotonergic drugs on learning after chronic defeat.............. 51 3.3.1 Methiothepin and ketanserin.............................................................. 51 3.3.2 Fluoxetine........................................................................................... 57 3.3.3 Summary of learning capacities – chronic defeat and serotonin........ 60 4 DISCUSSION....................................................................................................... 63 4.1 The semi-automated measurement of olfactory learning in individually assayed crickets................................................................................................................... 64 4.2 The influence of multiple agonistic experiences on learning........................... 71 4.3 The role of serotonin in chronic social defeat influenced learning................... 77 4.4 Overall conclusion and outlook........................................................................ 80 5 SUMMARY........................................................................................................... 82 6 ZUSAMMENFASSUNG........................................................................................ 87 7 REFERENCES.................................................................................................... 93 8 APPENDIX................................................................................................... 106 8.1 Figures and tables................................................................................... 106 8.2 Publications and published abstracts....................................................... 108 8.3 Curriculum vitae....................................................................................... 109 8.4 Acknowledgements.................................................................................. 111

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