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The most successful and widely used animal models in neuroscience are rats and mice, which has been vital for the understanding of the human brain. Another model that has become more prominent during recent years is the zebrafish (Danio rerio). Due to its well-elaborated visual system and learning of aversive events, the zebrafish has become a suitable model for learning and memorizing. In this paper, the relationship between coping styles and learning in different zebrafish strains has been studied by using automatic operant boxes. The strains that were compared are the Tupfel Long Fin (TL). offspring of wild-caught zebrafish (WT) and the Spiegel danio (Spd). The results from the novel tank diving test suggest that there is a difference in coping style depending on the strain of zebrafish. The TL was characterized as a reactive (shy) fish as it had the longest cumulative duration time at the bottom of the tank. The Spd was characterized as a proactive (bold) fish as it had the longest cumulative duration time at the top of the tank. While, the WT characterized as a reactive fish as it exhibited the longest time not moving at all in the tank, indicating a high-stress response profile. An automatic operant box was used to study learning in the different strains. The purpose of the script used was for the fish to associate the feeder mechanism noise and the white light feeder with a food reward. However, no tendency of learning could be observed for any of the strains. 

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.

Scientific investigations, in general, and research in neuroscience, in particular, are becoming ever more complex and require the integration of different techniques. Behavioral assays, which are among the most frequently used methodologies in neuroscience, nowadays rely on advanced, sophisticated technologies that require proficient application. Therefore, behavioral core facilities are becoming essential support units, as they provide the specialized expert research services needed to conduct advanced neuroscience. We here review the lessons learned and insights gathered from managing behavioral core facilities in different academic research institutes. This review addresses several issues, including: the advantages of behavioral core facilities, considerations for establishing a behavioral core facility, and the methodological advances made through calibration and standardization of assay protocols and the development of new assays. Collectively, the review highlights the benefits of both working within and collaborating with behavioral core facility units and emphasizes the potential progress in neuro-phenotyping that such facilities provide.

Stress is known to modulate behavioral responses and rapid decision-making processes, especially under challenging contexts which often occur in social and cooperative interactions. Here, we evaluated the effects of acute stress on cooperative behavior of the Indo-Pacific cleaner wrasse (Labroides dimidiatus) and the implications of pre-treatment with monoaminergic compounds: the selective serotonin reuptake inhibitor - fluoxetine, the 5-HT1A receptor antagonist - WAY-100,635, the D-1 receptor agonist - SKF-38393, and the D-1 receptor antagonist - SCH-23390. We demonstrated that stress decreased the predisposal to interact and increased cortisol levels in cleaners, which are alleviated by fluoxetine and the dopaminergic D-1 antagonist. Overall, our findings highlight the crucial influence of stress on cooperative behavior.

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.

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.

Differences in selection pressure in nature and labs have profound effects on zebrafish strains. The widely used AB strain of zebrafish has been domesticated over several decades. Recently, there has been an upsurge in the availability of genetically modified lines, e.g., the spiegeldanio (spd), which has a mutation in the fibroblast growth factor receptor 1a (fgfr1a) gene. This mutant strain (fgfr1a) has previously been reported to be bolder than fish of the Tubingen strain, from which it was generated. Our knowledge on behavioral differences between different zebrafish strains, relative to wild-caught zebrafish, is limited. In the present study we compare behaviors related to interpretation of boldness in male and female offspring (F1) of wild-caught fish, AB and fgfr1a(-/-) zebrafish. A second aim of the study was to compare the behavior of fish from these strains when tested in different behavioral assays, i.e., shelter seeking, novel tank diving and scototaxis tests. The results demonstrate that behavioral variation exists both within and between the strains, but interpretation of boldness reveals a complex pattern in which behavior differs between strains but is also related to sex and test. Therefore, a careful assessment of various strains of fish using both males and females is warranted in order to strengthen interpretation of results. This is especially important in studies where zebrafish are used as model organisms for human conditions as well as studies evaluating the effects of pharmacological or toxicological substances on behavior.

Zebrafish which carries a mutation in the fibroblast growth factor receptor 1A (fgfr1a), also known as spiegeldanio (spd), has previously been reported to be bolder and more aggressive than wildtype (AB) zebrafish. However, in previous studies aggression has been quantified in mirror tests. In dyadic fights the behavior of the combatants is modified by the behavior of their opponent, and fighting a mirror has been reported to have different effects on brain gene expression and brain monoaminergic systems. In the present study aggression was quantified in fgfr1a mutants and AB zebrafish using a mirror test after which the fish were allowed to interact in pairs, either consisting of two fgfr1a mutants or one AB and one fgfr1a mutant fish. Following dyadic interaction aggressive behavior was again quantified in individual fish in a second mirror test after which the fish were sacrificed and brain tissue analyzed for monoamines and monoamine metabolites. The results confirm that fgfr1a mutants are more aggressive than AB zebrafish in mirror tests. However, fgfr1a mutant fish did not have any advantage in fights for social dominance, and agonistic behavior of fgfr1a mutants did not differ from that of AB fish during dyadic interactions. Moreover, as the AB fish, fgfr1a mutant fish losing dyadic interactions showed a typical loser effect and social subordination resulted in an activation of the brain serotonergic system in fgfr1a mutants as well as in AB fish. Overall the effects of dyadic interaction were similar in fgfr1a mutant fish and zebrafish of the AB strain.

Cleaning interactions are textbook examples of mutualisms. On coral reefs, most fishes engage in cooperative interactions with cleaners fishes, where they benefit from ectoparasite reduction and ultimately stress relief. Furthermore, such interactions elicit beneficial effects on clients' ecophysiology. However, the potential effects of future ocean warming (OW) and acidification (OA) on these charismatic associations are unknown. Here we show that a 45-day acclimation period to OW (+3 degrees C) and OA (980 mu atm pCO(2)) decreased interactions between cleaner wrasses (Labroides dimidiatus) and clients (Naso elegans). Cleaners also invested more in the interactions by providing tactile stimulation under OA. Although this form of investment is typically used by cleaners to prolong interactions and reconcile after cheating, interaction time and client jolt rate (a correlate of dishonesty) were not affected by any stressor. In both partners, the dopaminergic (in all brain regions) and serotoninergic (forebrain) systems were significantly altered by these stressors. On the other hand, in cleaners, the interaction with warming ameliorated dopaminergic and serotonergic responses to OA. Dopamine and serotonin correlated positively with motivation to interact and cleaners interaction investment (tactile stimulation). We advocate that such neurobiological changes associated with cleaning behaviour may affect the maintenance of community structures on coral reefs.

The present study investigated the reproductive toxicity of furan in an Extended One-Generation Reproductive Toxicity Study in rats. Sprague Dawley F0 weaning rats (30 per sex per group) were exposed to furan orally at 0, 1, 2.5, 5, and 10 mg kg(-1) for 10 weeks (males) and 2 weeks (females) and then mated. Results of F0 indicated that in the furan-treated groups (5 mg kg(-1) and 10 mg kg(-1)), body weight (bw) gain decreased during prebreed and gestational period while increased during lactation periods. F0 animals prebreeding exposure resulted in head tilt and foot splay at 10 mg kg(-1). Number of live pups at birth were decreased (p < 0.001) at 10 mg kg(-1). At postnatal day (PND) 70, a significant (p = 0.03) decrease in testosterone levels of male rats and estrogen levels of female rats (p = 0.05) was observed in 10 mg kg(-1) furan-treated group in F1 generation. Luteinizing hormone, follicle-stimulating hormone, and progesterone levels were also reduced, but their reduction was not statistically significant in all groups. In higher dose furan group (10 mg kg(-1)), testicular and ovarian weights were reduced in F1 generation at PND 70, with decreased daily sperm production (p = 0.01) and disturbed estrous cyclicity (p < 0.01). Some histopathological changes were also observed in testis and ovaries in groups whose parents were previously exposed to 10 mg kg(-1) bw of furan group. Based on the above results, it is suggested that exposure to food-based contaminant furan induced remarkable changes in the F0 (parental stage) and F1 (offspring, pubertal, and adult stage) generations of Sprague Dawley rats.

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.

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.

This doctoral thesis investigated the effect of two aquatic pollutants on the behavior and physiology of teleost fish: i) elevated concentrations of carbon dioxide (CO₂) in the context of ocean acidification and ii) low concentrations of the anxiolytic pharmaceutical oxazepam in the context of pharmaceutical pollution.

Anthropogenic emissions of CO₂ are lowering the pH of the oceans. Studies on coral reef fish exposed to CO₂ concentrations projected for the year 2100 (~1000 μatm) reported alarming behavioral effects, of which attraction to predator odor was the most surprising. To explain this behavioral reversal, it was hypothesized that ion-regulatory adjustments to compensate for the decrease in blood pH would result in altered transmembrane gradients of chloride ions, rendering the major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), excitatory. We investigated whether zebrafish (Danio rerio), an often used model species, showed similar behavioral disruptions in elevated CO₂. Zebrafish behavior was however largely unaffected by an approximately month long exposure to ~1600 μatm CO₂. We continued by investigating the reproductive, anxiety-related behavior and aggression in another model species, the three-spined stickleback (Gasterosteus aculeatus). However, also stickleback behavior and responses to social subordination were not affected by CO₂, in contrast to earlier findings. We concluded that CO₂ had no major effect on the behavior of zebrafish and three-spined stickleback.

In the second part of this thesis, I investigated behavioral effects of oxazepam, an anxiolytic pharmaceutical (benzodiazepine) acting on the GABA system. Studies on perch (Perca fluviatilis) have shown that exposure to dilute concentrations of oxazepam (1.8 μg L^-1), close to those found outside the municipal sewage treatment plant in Uppsala (0.58 μg L^-1), can increase activity, decrease sociality and increase feeding rates. I show that similar oxazepam concentrations can also affect zebrafish. Moreover, I show that females are more sensitive to oxazepam showing reduced anti-predator responses at 0.57 μg L^-1 while in males this effect was observed first at 60 μg L^-1. Furthermore, and in contrast to wild-caught zebrafish, laboratory zebrafish did not show any effect of the oxazepam exposure. This finding has implications for the use of laboratory zebrafish in ecotoxicological and pharmacological studies, as results might not translate to wild fish. Finally, I show that zebrafish can develop tolerance to the anxiolytic effects of oxazepam during chronic (28 days) exposure. This is an important discovery that could mitigate the effects of this form of pharmaceutical pollution on wild fish.