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To understand the processes underpinning social decision-making, we need to determine how internal states respond to information gathered from the social environment. Brain monoamine neurotransmitters are key in the appraisal of the social environment and can reflect the internal state underlying behavioural responses to social stimuli. Here we determined the effects of conspecific partner cooperativeness during predator inspection on brain monoamine metabolic activity in Trinidadian guppies (Poecilia reticulata). We quantified the concentration of dopamine, serotonin and their metabolites across brain sections sampled immediately after ostensibly expe-riencing cooperation or defection from social partners whilst inspecting a predator model, using a familiar object as a control condition. Our results indicate dopaminergic and serotonergic activity differs with the coopera-tiveness experienced; these different neurotransmission profiles are likely to affect the expression and regulation of downstream behaviours that ultimately contribute to the patterning of cooperative interactions among in-dividuals in a population.

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience.

Introduction; Despite its popularity in research, there is very little scientifically validated knowledge about the best practices on zebrafish (Danio rerio) husbandry, which has led to several facilities having their own husbandry protocols. This study was performed to expand knowledge on the effects of enrichment and fish density on the welfare of zebrafish, with hopes of providing a scientific basis for future recommendations and legislations.

Methods: Zebrafish were reared at three different stocking densities, (1, 3 or 6 fish/L), in tanks with or without environmental enrichment. Agonistic behavior was observed twice a week for 9 weeks directly in the housing tanks. Aspects of welfare is known to be reflected in neuroendocrine stress responses. Thus, cortisol secretion in response to lowering the water level was analyzed for each group. In addition, we assessed cortisol secretion in response to confinement and risk-taking behavior (boldness) using the novel tank diving test for individual fish. At termination of the experiment fish were subjected to stress by transfer to a novel environment and brain tissue was sampled for analysis of brain monoaminergic activity.

Results: Fish kept at the lowest density (1 fish/L) showed a significantly higher level of aggression than fish kept at 3 or 6 fish/L. Moreover, fish kept at this low density showed significantly higher cortisol secretion on a group level than fish kept at the higher stocking densities, when subjected to lowering of the water level. Keeping fish at 1 fish/L also had effects on brain monoamines, these fish showing higher brain dopamine concentrations but lower dopamine turnover than fish kept at higher densities. Neither stocking density or enrichment had any clear effects on the behavior of individual fish in the novel tank diving test. However, fish kept at high densities showed lower and more variable growth rates than fish kept at 1 fish/L.

Discussion: Taken together these results suggest that zebrafish should not be kept at a density of 1 fish/L. The optimal stocking density is likely to be in the range of 3-6 fish/L.

Zebrafish (Danio rerio) are becoming one of the most important model organisms in behavioural neuroscience. It has been shown repeatedly that different zebrafish strains show large behavioural differences. These divergent behavioural profiles may have a genetic basis, but environmental factors and previous experience are also known to greatly affect the behavioural phenotype of zebrafish. It could be expected that behavioural differences at the larval stage should be less affected by environmental factors and experience. In the present study, we screened larvae of zebrafish of the AB strain and offspring of wild-caught zebrafish for boldness, using an open field test. In order to follow the behavioural development, we studied larvae at the age of 5-, 7-, 12- and 30-days post fertilization (dpf). Behaviour, as well as behavioural development, clearly differed between the larvae of the different strains. Wild larvae showed larger total distance moved than AB larvae, both at light and dark conditions. These differences were already present at 12 dpf but became more pronounced with age. Wild larvae had a greater variance compared to AB larvae for most of the variables. We have previously shown that bold and shy adult zebrafish differ in the brain expression of dopamine and opioid receptors. The results of the current study show that wild larvae display significantly higher brain expression of drd2b than AB larvae at 30 dpf, a difference that could be related to differences in activity. We did not detect any differences in the expression of opioid receptors.

Size-selective mortality may evolutionarily alter life-history as well as individual behavioral and physiological traits. Moreover, size-selective mortality can affect group behavioral traits, such as shoaling and collective properties (e.g., shoal cohesion), which are relevant for finding food and reducing risk of predation. Here, we present experimental evidence using selection lines of zebrafish (Danio rerio) that were exposed to positive ( large-harvested), negative (small-harvested), and random (control) size-selective mortality for five generations, followed by eight generations during which harvesting was halted to remove maternal effects and to study evolutionarily fixed outcomes. We investigated changes in shoal cohesion and turnover in monoamines in zebrafish through ontogeny. To that end, we repeatedly measured inter-individual distance in groups of eight fish and the turnovers of dopamine and serotonin in brains of fish from juvenile to the adult stage at 40-day intervals. We, firstly, found that shoal cohesion was overall consistent through ontogeny at group levels suggesting the presence of collective personality. Secondly, we found a decrease in shoal cohesion through ontogeny in the small-harvested and control lines, while the large-harvested line did not show any ontogenetic change. Thirdly, the selection lines did not differ among each other in shoal cohesion at any ontogenetic stage. Fourthly, dopamine turnover increased through ontogeny in a similar way for all lines while the serotonin turnover decreased in the large-harvested and control lines, but not in the small-harvested line. The large-harvested line also had higher serotonin turnover than controls at specific time periods. In conclusion, intensive size-selective mortality left an evolutionary legacy of asymmetric selection responses in the ontogeny of shoal cohesion and the underlying physiological mechanisms in experimentally harvested zebrafish in the laboratory. Significant statement The evolution of animal behavior can be affected by human activities both at behavioral and physiological levels, but causal evidence is scarce and mostly focusing on single life-stages. We studied whether and to what extent size-selective harvesting, a common selection pattern in fisheries, can be an evolutionary driver of the development of shoal cohesion during ontogeny. We used a multi-generation experiment with zebrafish to study cause-and-effects of opposing size-selection patterns. We quantified shoal cohesion, and serotonin and dopamine turnover in the brain. We found that shoal cohesion emerged as a collective personality trait and that behavioral and physiological responses were asymmetrical with respect to the opposing selection patterns.

Stress and aggression have negative effects on fish welfare and productivity in aquaculture. Thus, research to understand aggression and stress in farmed fish is required. The neuropeptides arginine-vasotocin (AVT) and corticotropin-releasing factor (CRF) are involved in the control of stress and aggression. Therefore, we investigated the effect of agonistic interactions on the gene expression of AVT, CRF and their receptors in juvenile rainbow trout (Oncorhynchus mykiss). The social interactions lead to a clear dominant-subordinate relationship with dominant fish feeding more and being more aggressive. Subordinate fish had an upregulation of the AVT receptor (AVT-R), an upregulation of CRF mRNA levels, and higher plasma cortisol levels. The attenuating effect of AVT on aggression in rainbow trout is proposed to be mediated by AVT-R, and the attenuating effect of the CRF system is proposed to be mediated by CRF.

Aggregation of alpha-synuclein (alpha-syn) into Lewy bodies and Lewy neurites is a pathological hallmark in the Parkinson ' s disease (PD) brain. The formation of alpha-syn oligomers is believed to be an early pathogenic event and the A30P mutation in the gene encoding alpha-syn, causing familial PD, has been shown to cause an accelerated oligomerization. Due to the problem of preserving antigen conformation on tissue surfaces, alpha-syn oligomers are difficult to detect ex vivo using conventional immunohistochemistry with oligomer-selective antibodies. Herein, we have instead employed the previously reported alpha-syn oligomer proximity ligation assay (ASO-PLA), along with a wide variety of biochemical assays, to discern the pathological progression of alpha-syn oligomers and their impact on the dopaminergic system in male and female (Thy-1)-h[A30P]alpha-syn transgenic (A30P-tg) mice. Our results reveal a previously undetected abundance of alpha-syn oligomers in midbrain of young mice, whereas phosphorylated (pS129) and proteinase k-resistant alpha-syn species were observed to a larger extent in aged mice. Although we did not detect loss of dopaminergic neurons in A30P-tg mice, a dysregulation in the monoaminergic system was recorded in older mice. Taken together, ASO-PLA should be a useful method for the detection of early changes in alpha-syn aggregation on brain tissue, from experimental mouse models in addition to post mortem PD cases.

Environmental concentrations of the anxiolytic drug oxazepam have been found to disrupt antipredator behaviors of wild fish. Most experiments exposed fish for a week, while evidence from mammals suggests that chronic exposure to therapeutic concentrations of benzodiazepines (such as oxazepam) results in the development of tolerance to the anxiolytic effects. If tolerance can also develop in response to the low concentrations found in the aquatic environment, it could mitigate the negative effects of oxazepam pollution. In the current study, we exposed wild-caught zebrafish to oxazepam (similar to 7 mu g L-1) for 7 or 28 days and evaluated behavioral and physiological parameters at both time points. Females showed reduced diving responses to conspecific alarm pheromone after 7 days, but not after 28 days, indicating that they had developed tolerance to the anxiolytic effects of the drug. Zebrafish males were not affected by this oxazepam concentration, in line with earlier results. Serotonin turnover (ratio 5-HIAA/5-HT) was reduced in exposed females and males after 28 days, indicating that brain neurochemistry had not normalized. Post-confinement cortisol concentrations and gene expression of corticotropin-releasing hormone (CRH) were not affected by oxazepam. We did not find evidence that chronically exposed fish had altered relative expression of GABA A receptor subunits, suggesting that some other still unknown mechanism caused the developed tolerance.

In fish, as well as in other vertebrates, contrasting suites of physiological and behavioral traits, or coping styles, are often shown in response to stressors. However, the magnitude of the response (i.e. stress responsiveness) has been suggested to be independent of stress coping style. One central neurotransmitter that has been associated with both stress responsiveness and differences in stress coping styles is serotonin (5-hydroxytryptamine, 5-HT). In this study, we investigated to what extent stress responsiveness reflects differences in stress coping, and the potential involvement of the 5-HT system in mediating such differences in farmed Gilthead seabream. Initially, fish were classified as proactive or reactive based on their behavioural response to net restraint. Following 1.5 months, fish classified as proactive still showed a higher number of escape attempts and spent longer time escaping than those classified as reactive. These differences were reflected in a generally higher brain stem 5-HT concentration and a lower telencephalic 5-HT activity, i.e. the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT, in proactive fish. Independent of stress coping styles, stress responsiveness was reflected in elevated 5-HIAA concentrations and 5-HIAA/5-HT ratios in telencephalon and brain stem together with increased plasma cortisol concentrations at 0.5 and 2 h following the last net restraint. The current results show that 5-HT signaling can reflect different behavioural output to a challenge which are independent of neuroendocrine responses to stress and lend support to the hypothesis that stress coping styles can be independent of stress responsiveness.

Enriching rearing environment is the strategy suggested for improving the post release survivorship of captive-reared animals. Here, an attempt has been made to evaluate the impact of early rearing enrichment on the hypothalamic-pituitary-interrenal axis (HPI axis), blood glucose, and brain dopaminergic and serotonergic systems of Tor putitora. Fifteen-day-old hatchlings of T. putitora were reared up to advanced fry stage in barren, semi-natural, and physically enriched environments and compared them with regard to pre-stress and post-stress levels of whole-body cortisol, blood glucose, brain serotonergic activity (5HIAA/5HT ratio), dopaminergic activity (DOPAC/DA and HVA/DA ratios) and norepinephrine (NE) levels. Significantly low basal whole-body cortisol, glucose and brain NE levels were observed in a physically enriched group of fish as compared to the other two groups. However, after acute stress, all rearing groups showed elevated levels of cortisol, blood glucose, brain 5HIAA/5HT, DOPAC/DA and HVA/DA ratios and NE levels but the magnitude of response was different among different rearing groups. The barren reared group showed a higher magnitude of response as compared to semi-natural and physically enriched groups. Similarly, the recovery rate of whole-body cortisol, blood glucose, and whole-brain monoamines were long-lasting in barren-reared mahseer. We illustrate that increased structural complexity (physical enrichment) during the early rearing significantly modulates various physiological and stress-coping mechanisms of mahseer.