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Introduction Aggression is a competition based survival strategy. The spiegeldanio (spd) strain of zebrafish (Danio rerio), which has a mutation in the fibroblast growth factor receptor 1a, is bolder and more aggressive than the wild type fish [1]. Usually a socially dominant fish has preferential access to food, mate and shelter, and shows very characteristic postures like erection of the fins. It is also aggressive frequently biting, striking and chasing the subordinate fish as well as threatening its own mirror image in mirror tests [2]. However, what happens when an already known bold and dominant fish like spiegeldanio loses a dyadic fight. Spd fish are more aggressive in mirror tests, attacking their mirror image more frequently than wild type conspecifics. However, are they more aggressive in dyadic fights? Do they show an inhibition of aggressive behaviour when losing fights, the typical loser effect? The behavioural inhibition observed in animals losing fights for dominance is at least in part believed to be mediated by an activation of the brain serotonin (5-hydroxytryptamine, 5-HT) system. Do spd fish show a typical increase in brain 5-HT activity in response to social subordination? Dopamine (DA), on the other hand, is associated with aggression and social dominance. What are the effects of winning and losing fights for social dominance in spd fish? In the present study these questions were addressed in an attempt to increase or understanding of the control of agonistic behaviour and social stress. Animals and Methods The Spd strain of zebrafish were raised and reared at 27°C in an Aquaneering Zebrafish system at Uppsala University Biomedical Center. The animals were kept at a 14:10 h of light-dark photoperiod. The water used in the fish tanks was Uppsala municipal tap water (pH 7.2-7.6) of which 10% was exchanged daily. Fish were fed twice daily with Tropical energy food (Aquatic Nature, Belgium) and Artemia (Platinum Grade 0, Argentemia, Argent, Aquaculture, Redmond, USA). The use of animals was approved by the Uppsala Animal Ethical Committee (permit Dnr 55/13) and followed the guidelines of the Swedish Legislation on Animal Experimentation (Animal Welfare Act SFS1998:56), and the European Union Directive on the Protection of Animals Used for Scientific Purposes (Directive 2010/63/EU). The fish were transferred to the individual compartments of dimension 29 x 7.5 x 20 cm (length x breadth x height) in experimental tanks used for dyadic interaction and allowed to recover in isolation overnight. These experimental tanks were made from poly methyl methacrylate plastic and each tank was equipped with a submerged pump with filter (Eheim, typ 2006020, pumping capacity 1/h180, made in China), a heater (Sera aquarium, 25W, made in EU) and an air stone, all of which were placed at the back of the tank separated from the fish by a white perforated PVC screen (Figure 1). The setup of the arena was such that the two fish (1 dyadic pair) had an olfactory but not any visual cue of each other before the dyadic interaction. In the mirror test the fish were made to fight against the mirror image that was displayed in the mirror which was pasted on the wall of the arena. Prior to the beginning of the dyadic contest the mirror was covered with a black plexiglas slide cover. The experiment was carried out in the following sequence: The fishes were netted out and placed in the arena in the compartments A and B (Figure 1) and separated from each other by a partition. The cover of the mirror (opaque black PVC partition, Figure 1) was then removed and fish were made to interact with their own mirror image for 10 minutes. Then the slide covering the mirror was pulled down and the middle separating partition was pulled out and the fish were given an opportunity to fight. Dyadic fight was recorded two times, morning and evening on day one with the help of a video filming camera. Then next day in the morning the dyadic fight was again recorded. During the dyadic interaction the two fishes indulged in mutual display of aggressive behaviour which was followed by chasing and biting attacks performed by the dominant fish over the subordinate fish. Then middle partition was introduced again. Fish were given 6 minutes to habituate and the cover from the mirror was removed and fishes were again allowed to interact with their mirror image. Again the mirror was covered and the fish was allowed to get involved in the dyadic fight. Then each fish was taken out from the compartment at the same time and sacrificed for sampling of brain tissue. The three dimensional model of tank used in the behavioural tests I) Tank used for mirror test and for dyadic fight later on. It consists of two compartments, A and B. The movable partition separating the two compartments would be removed during the dyadic fight test. Compartment C is located at the back and is separated from the compartment A and B with the help of white coloured opaque perforated partition. It contains an air stone (for diffusion of air bubbles), heater (27°C), water pump (for circulation of water) and a drainage tube to exchange the water. II) Diagram of the settings used for dyadic interactions. The mirrors are covered with the help of a black PVC slide and the middle partition is pulled out. This allows the fish to interact. Brain dissection and analysis of monaoamines and monoamine metabolites Brains were divided into forebrain (telencephalon and diencephalon), optic tectum and the rest (here denoted brain stem). The frozen brains were homogenised in 4% (w/v) ice-cold perchloric acid containing 100 ng/ml 3, 4-dihydroxybenzylamine (DHBA, the internal standard) using a Sonifier cell disruptor B-30 (Branson Ultrasonics, Danbury, CT, USA) and were immediately put on dry ice. Subsequently, the homogenised samples were thawed and centrifuged at 15,000 rpm for 10 min at 4o C. The supernatant was used for high performance liquid chromatography with electrochemical detection (HPLC-EC), analysing the monoamines dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) as well as the DA metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), as described by Øverli et al. [3]. In short, the HPLC-EC system consisted of a solvent delivery system model 582 (ESA, Bedford, MA, USA), an autoinjector Midas type 830 (Spark Holland, Emmen, the Netherlands), a reverse phase column (Reprosil-Pur C18-AQ 3 µm, 100 mm × 4 mm column, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) kept at 40° C and an ESA 5200 Coulochem II EC detector (ESA, Bedford, MA, USA) with two electrodes at reducing and oxidizing potentials of -40 mV and +320 mV. A guarding electrode with a potential of +450 mV was employed before the analytical electrodes to oxidize any contaminants. The mobile phase consisted of 75 mM sodium phosphate, 1.4 mM sodium octyl sulphate and 10 µM EDTA in deionised water containing 7 % acetonitrile brought to pH 3.1 with phosphoric acid. The quantification of samples was done by comparing it with standard solutions of known concentrations. DHBA was used as an internal standard to correct for recovery with the help of HPLC software ClarityTM (Data Apex Ltd, Czech Republic). The serotonergic and dopaminergic activity was measured as the ratio of 5-HIAA/5-HT and DOPAC/DA respectively. The brain monoamines were normalized with respect to brain protein weights which were determined with Bicinchoninic acid protein determination kit (Sigma Aldrich, Sweden). The assay was read at a wavelength of 570 nm with the help of a plate reader (Labsystems multiskan 352, Labsystems Thermo Fisher Scientific). Results A clear dominant subordinate hierarchy was established within 30 minutes of dyadic interaction. The number of aggressive acts (bites, strikes and chases) performed by the looser fish decreased significantly from the first dyadic fight to the last (i.e. the fourth) dyadic fight. For the winner fish the number of aggressive acts performed against a mirror during the second mirror test increased or remained same as before after winning a dyadic fight, whereas for the looser fish it decreased significantly. The results from the present study indicate that subordinate fish have higher 5-HIAA/5-HT ratio in the optic tectum as compared to the dominants. More results from this study would be presented at the conference. References 1. Norton W, Bally-Cuif L (2010) Adult zebrafish as a model organism for behavioural genetics. BMC Neurosci. 11:90. 2. Rowland WJ (1999) Studying visual cues in fish behaviour: a review of ethological techniques. Env Biol Fishes. 56:285-305. 3. Øverli Ø, Harris CA, Winberg S (1999) Short-term effects of fights for social dominance and the establishment of dominant-subordinate relationships on brain monoamines and cortisol in rainbow trout. Brain Behav Evol. 54:263-275. |
