| Abstract |
Animal species with strong social interactions and hierarchies exhibit two distinct coping styles, which comprise a group of consistent behavioral and physiological characteristics. Individuals with a proactive coping style are considered dominant, aggressive, bold and with a stereotypic behavior, whereas reactive individuals exhibit a more subordinate, fearful, shy, and flexible behavior. These coping styles are important for survival, homeostasis, social learning, use of the available resources and reproduction. The correlation between aggressive behavior and other behavioral characteristics has been studied in avian species, rodents, and mammals, where aggressive behavior was linked to exploratory behavior and boldness. There are several molecular and neuroendocrinological mechanisms that underlie an animal’s response to negative stimuli and challenges of the natural and social environment. Teleost’s response to stress is regulated through the activation of the Hypothalamic – pituitary – interrenal axis (HPI), leading to the production of cortisol. The effect of cortisol in stress response is regulated through two receptors, the glucocorticoid (GR) and the mineralocorticoid receptor (MR), through genomic and non-genomic effects on physiology and behavior. Zebrafish possesses characteristics that render it an ideal model organism for studying stress. It is a social species, forming schools in nature, yet when kept in pairs, it expresses an aggressive behavior that most of the time leads in the formation of hierarchies. Additionally, under exposure to acute and chronic stress the species has a robust and quantifiable behavioral and physiological response. The overall aim of the present study was to investigate the agonistic behavior and coping styles of zebrafish, as well as the role of the glucocorticoid receptors in the stress response of adult zebrafish and larvae. Initially (Chapter 2), adult male zebrafish characterized as dominants and subordinates through a paired aggression test, were exposed to a battery of behavioral assays, aiming to detect a correlation between the behavioral characteristics of the two coping styles. Results showed that subordinates explored the upper parts of a novel tank more freely than dominants, while individuals with exploratory tendencies exhibited a more anxious phenotype compared to the non – explorers. Given that the behavioral responses observed were not in accordance with the literature, we were not able to detect a consistency in the behavioral response of the two coping styles. The winners and losers of a dyadic fight do not necessary reflect a proactive or reactive coping style, respectively, since the outcome of a fight and the social rank of an animal depend on factors other than the individual’s coping style. In accordance with this, we investigated associated differences in molecular and endocrine regulators of coping styles as well as the role of serotonin in the aggressive behavior of zebrafish. Adult male zebrafish were exposed to a paired aggression test and the resulting dominant and subordinate fish were administered fluoxetine for two hours. After further observing their agonistic behavior and the state of their social hierarchy, fish were euthanized for cortisol determination and gene analysis studies. Acute exposure to fluoxetine had a significant effect on the behavior of the fish, by reducing the aggressive behavior of dominants and the defensive behavior of subordinates. There was no statistically significant difference in cortisol concentration between the two coping styles, however zebrafish exposed to fluoxetine had significantly lower cortisol concentration compared to the fish that were not exposed to the antidepressant. In addition, fluoxetine affected the expression levels of genes related to neural activity, stress and coping styles. The effect of fluoxetine on the HPI axis was further studied in Chapter 4, where after exposing zebrafish larvae to three different doses of fluoxetine for two hours, we subjected them to a behavioral assessment of alternating light and dark conditions. This acute administration of fluoxetine significantly increased the cortisol concentration of larvae and reduced the swimming activity in response to the alternating lighting conditions. The aim of the following experiments was to investigate the role of GR and MR in the neuroendocrine mechanisms that regulate the organism’s response to stress and social challenges of the environment. More specifically, zebrafish mutant lines for gr and mr were used to observe potential differences between the behavioral and endocrine response of these genetic strains under acute and chronic stress conditions (Chapter 5). The results showed a complementary action of the two receptors in regard to the behavioral stress response of adult zebrafish. While there was no difference in the social preference of the two strains compared to the wild type adults, mr -/- adults exhibited a more anxious phenotype compared to gr -/- . Moreover, the higher cortisol concentration under baseline conditions in gr -/- adults, points to the important role of GR in the negative feedback loop of the HPI axis. Along with the higher HPI activity of gr -/- zebrafish, we also observed a more reactive behavioral phenotype of these individuals when exposed to tests that trigger the expression of the species’ agonistic behavior. Finally (Chapter 6), we studied the behavioral and endocrinological response to acute stress in wild type, gr -/- , mr -/- and gr -/- mr -/- zebrafish larvae. Similar to adults, gr -/- larvae exhibited higher cortisol concentrations under baseline and stress conditions. Behavioral tests used in this set of experiments were two light – dark alteration protocols of two different light intensities and a vibrational startle response assay. Larvae knockouts displayed a disturbed response to a series of mechanical stimuli and a significant difference in their habituation levels compared to wild type larvae. This observation suggests an involvement of the glucocorticoid receptors in the activity of the neurons that are responsible for the behavioral response of larvae to these stressors. Furthermore, it was shown that the brightness of the light in assays that expose zebrafish larvae to alternating light and dark conditions, severely affects the intensity of the response as well as the pattern. While the higher light intensity used in these experiments is often chosen for these assays in the literature, our observations suggested that low intensity light could be considered as a mild stressor, while higher intensity could be perceived as an extreme stressor. The differences observed in the response of larvae knockouts under different light conditions could be due to defects in visual processing of stimuli and visual adaptation.
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