| 1. |
- Cocco, Arianna, et al.
(author)
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Characterization of the gamma-aminobutyric acid signaling system in the zebrafish (danio rerio hamilton) central nervous system by reverse transcription-quantitative polymerase chain reaction
- 2017
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In: Neuroscience. - : Elsevier BV. - 0306-4522 .- 1873-7544. ; 343, s. 300-321
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Journal article (peer-reviewed)abstract
- In the vertebrate brain, inhibition is largely mediated by raminobutyric acid (GABA). This neurotransmitter comprises a signaling machinery of GABA(A), GABA(B) receptors, transporters, glutamate decarboxylases (gads) and 4-aminobutyrate aminotransferase (abat), and associated proteins. Chloride is intimately related to GABAA receptor conductance, GABA uptake, and GADs activity. The response of target neurons to GABA stimuli is shaped by chloride-cation co-transporters (CCCs), which strictly control Cl- gradient across plasma membranes. This research profiled the expression of forty genes involved in GABA signaling in the zebrafish (Danio rerio) brain, grouped brain regions and retinas. Primer pairs were developed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The mRNA levels of the zebrafish GABA system share similarities with that of mammals, and confirm previous studies in non-mammalian species. Proposed GABAA receptors are alpha(1)beta(2)gamma(2), alpha(1)beta(2)delta, alpha(2b)beta(3), alpha(2b)beta(3)delta, alpha(4)beta(2)gamma(2), alpha(4)beta(2)gamma, alpha(6b)beta(2)gamma(2) and alpha(6b)beta(2)delta. Regional brain differences were documented. Retinal hetero- or homomeric rho-composed GABAA receptors could exist, accompanying alpha(1)beta(y)gamma(2), alpha(1)beta(y)delta, alpha(6a)beta(y)gamma(2,) alpha(6a)beta(y)delta. Expression patterns of alpha(6a) and alpha(6b) were opposite, with the former being more abundant in retinas, the latter in brains. Given the stoichiometry alpha(6w)beta(y)gamma(z), alpha(6a-) or alpha(6b)-containing receptors likely have different regulatory mechanisms. Different gene isoforms could originate after the rounds of genome duplication during teleost evolution. This research depicts that one isoform is generally more abundantly expressed than the other. Such observations also apply to GABAB receptors, GABA transporters, GABA-related enzymes, CCCs and GABAA receptor associated proteins, whose presence further strengthens the proof of a GABA system in zebrafish.
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| 2. |
- Sundin, Josefin, 1981-, et al.
(author)
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No effect of elevated carbon dioxide on reproductive behaviors in the three-spined stickleback
- 2017
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In: Behavioral Ecology. - : Oxford University Press (OUP). - 1045-2249 .- 1465-7279. ; 28:6, s. 1482-1491
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Journal article (peer-reviewed)abstract
- Ocean acidification, the reduction in ocean pH resulting from anthropogenic emissions of carbon dioxide (CO2), has been predicted to alter the behavior of fishes. During experimental exposure to CO2 concentrations projected for the year 2100 (similar to 1000 mu atm), fish have been reported to display disturbances in activity, learning, behavioral lateralization, and even attraction to predator cues. Reproductive behaviors have received far less attention, despite an intensive research effort on ocean acidification and its ecological importance. Here, we investigate whether elevated levels of CO2 affect reproduction in breeding pairs of the three-spined stickleback, Gasterosteus aculeatus, a model species in behavioral, evolutionary ecology, and environmental toxicology. We found that males under both present day levels (400 mu atm) and future levels (1000 mu atm) of CO2 developed normal sexual ornaments, pursued normal nest building activities, exhibited similar levels of courtship behaviors and displacement fanning, and had the same mating probability. Moreover, fanning behavior during the paternal care period followed what is expected for the species for males from both treatments, and there was no effect of treatment on the numbers of offspring produced. This study is the first to investigate the effect of elevated CO2 on the complete breeding cycle in detail, studying an array of highly fitness-relevant traits. Our study showing surprising resilience of fish reproduction is an important contribution in order to realistically predict the impacts of future ocean acidification.
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| 3. |
- Vossen, Laura E., et al.
(author)
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Chronic Exposure to Oxazepam Pollution Produces Tolerance to Anxiolytic Effects in Zebrafish (Danio rerio)
- 2020
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In: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 54:3, s. 1760-1769
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Journal article (peer-reviewed)abstract
- 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.
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| 4. |
- Vossen, Laura E.
(author)
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Effect of elevated pCO2 and environmental oxazepam on the behavior and physiology of teleost fish
- 2019
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 5. |
- Vossen, Laura E., et al.
(author)
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Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild-caught but not in laboratory zebrafish
- 2020
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In: Science of the Total Environment. - : Elsevier. - 0048-9697 .- 1879-1026. ; 703
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Journal article (peer-reviewed)abstract
- Pollution by psychoactive pharmaceuticals has been found to disrupt anti-predator behaviors of wild fish. The challenge is now to identify which of the many psychoactive drugs pose the greatest threat. One strategy is to screen for behavioral effects of selected pharmaceuticals using a single, widely available fish species such as zebrafish. Here, we show that although such high-throughput behavioral screening might facilitate comparisons between pharmaceuticals, the choice of strain is essential. While wild-caught zebrafish exposed to concentrations of the anxiolytic drug oxazepam as low as 0.57 μg L−1 showed a reduction in the response to conspecific alarm pheromone, laboratory strain AB did not respond to the alarm cue, and consequently, the anxiolytic effect of oxazepam could not be measured. Adaptation to the laboratory environment may have rendered laboratory strains unfit for use in some ecotoxicological and pharmacological studies, since the results might not translate to wild fish populations.
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| 6. |
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| 7. |
- Vossen, Laura E., et al.
(author)
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Sex-Specific Effects of Acute Ethanol Exposure on Locomotory Activity and Exploratory Behavior in Adult Zebrafish (Danio rerio)
- 2022
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In: Frontiers in Pharmacology. - : Frontiers Media S.A.. - 1663-9812. ; 13
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Journal article (peer-reviewed)abstract
- The zebrafish (Danio rerio) is an established model organism in pharmacology and biomedicine, including in research on alcohol use disorders and alcohol-related disease. In the past 2 decades, zebrafish has been used to study the complex effects of ethanol on the vertebrate brain and behavior in both acute, chronic and developmental exposure paradigms. Sex differences in the neurobehavioral response to ethanol are well documented for humans and rodents, yet no consensus has been reached for zebrafish. Here, we show for the first time that male zebrafish of the AB strain display more severe behavioral impairments than females for equal exposure concentrations. Adult zebrafish were immersed in 0, 1 or 2% (v/v) ethanol for 30 min, after which behavior was individually assessed in the zebrafish Multivariate Concentric Square Field (TM) (zMCSF) arena. Males exposed to 2% ethanol showed clear signs of sedation, including reduced activity, increased shelter seeking and reduced exploration of shallow zones. The 1% male group displayed effects in the same direction but of smaller magnitude; this group also explored the shallow areas less, but did not show a general reduction in activity nor an increase in shelter seeking. By contrast, 1 and 2% exposed females showed no alterations in explorative behavior. Females exposed to 2% ethanol did not display a general reduction in activity, rather activity gradually increased from hypoactivity to hyperactivity over the course of the test. This mixed stimulatory/depressant effect was only quantifiable when locomotory variables were analyzed over time and was not apparent from averages of the whole 30-min test, which may explain why previous studies failed to detect sex-specific effects on locomotion. Our results emphasize the importance of explicitly including sex and time as factors in pharmacological studies of zebrafish behavior. We hypothesize that the lower sensitivity of female zebrafish to ethanol may be explained by their greater body weight and associated larger distribution volume for ethanol, which may render lower brain ethanol concentrations in females.
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| 8. |
- Vossen, Laura E., et al.
(author)
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The zebrafish Multivariate Concentric Square Field : A Standardized Test for Behavioral Profiling of Zebrafish (Danio rerio)
- 2022
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In: Frontiers in Behavioral Neuroscience. - : Frontiers Media S.A.. - 1662-5153. ; 16
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Journal article (peer-reviewed)abstract
- The zebrafish (Danio rerio) is an important model organism in the study of the neurobiological basis of human mental disorders. Yet the utility of this species is limited by the quality of the phenotypical characterization tools available. Here, we present a complex testing environment for the quantification of explorative behavior in adult zebrafish, the zebrafish Multivariate Concentric Square Field (TM) (zMCSF), adapted from the rodent equivalent that has been used in > 40 studies. The apparatus consists of a central open area which is surrounded by a dark corner with a roof (DCR), corridors, and an inclined ramp. These areas differ in illumination, water depth, and are sheltered or exposed to different degrees. We quantified behavior of male and female wild-caught and AB strain zebrafish in the zMCSF (day 1) and cross-validated these results using the novel tank diving test (NTDT) (day 2). To assess the effect of repeated testing, AB zebrafish we tested a second time in both tests 1 week later (on days 7 and 8). We detected strong differences between the strains, with wild zebrafish swimming faster and spending more time in the corridors and on the ramp, while they avoided the open area in the center. AB zebrafish were less hesitant to enter the center but avoided the ramp, and often left one or more zones unexplored. No major sex differences in exploratory behavior were detected in either strain, except for a slightly higher velocity of AB males which has been reported before. Importantly, the zMCSF was largely resilient to repeated testing. The diving test revealed only one difference confined to one sex; wild females paid more visits to the top third than AB females. In isolation, this finding could lead to the conclusion that wild zebrafish are more risk-taking, which is incorrect given this strain's avoidance of open areas. To conclude, our results suggest that the zMCSF presents a sophisticated behavioral tool that can distinguish between different magnitudes and types of risk, allowing the user to create an intricate behavioral profile of individual adult zebrafish.
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| 9. |
- Vossen, Laura E., et al.
(author)
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Zebrafish (Danio rerio) behaviour is largely unaffected by elevated pCO2
- 2016
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In: Conservation Physiology. - : Oxford University Press (OUP). - 2051-1434. ; 4:1
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Journal article (peer-reviewed)abstract
- Ocean acidification, the decrease in ocean pH caused by anthropogenic emission of carbon dioxide, can cause behavioural disturbances in marine teleost species. We investigated whether AB-strain zebrafish (Danio rerio) show similar behavioural disturbances in the presence of elevated CO2, because this model species could open up a toolbox to investigate the physiological and neurological mechanisms of CO2 exposure. We found no effect of elevated CO2 (~1600 μatm) on the behaviour of zebrafish in the open field test, indicating that zebrafish are largely insensitive to this elevated CO2 level. In the detour test of lateralization, however, zebrafish exposed to elevated CO2 swam more often to the right, whereas individuals exposed to control CO2 (~400 μatm) had no preference for left or right. This may indicate that some behaviours of some freshwater fishes can be altered by elevated CO2 levels. Given that elevated CO2 levels often occur in recirculating aquaculture and aquarium systems, we recommend that dissolved CO2 levels are measured and, if necessary, the aquarium water should be aerated, in order to exclude CO2 level as a confounding factor in experiments.
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