Neurobiology article review

Chosen Primary Research Article: Acute Fluoxetine Differently Affects Aggressive Display in Zebrafish Phenotypes (2019). Entire reference available in the reference page.

Introduction/Background

            The biological understanding of the factors behind aggression is limited though there are disorders that state aggression is a symptom. In numerous animal models, when serotonergic transmission is acutely increased, it inhibits aggressive behavior, and even though this observation is true in most studies, there are some gaps in the literature, especially when it comes to basal vertebrates such as fish. Even within Zebrafish, there are phenotypic differences between leopard zebrafish and longfin zebrafish. The study aimed to find differences in aggressive display with longfin and leopard zebrafish, and the effect of acute fluoxetine in both populations.

Hypothesis

The authors hypothesized that the hyper-serotonergic phenotype of leopard zebrafish would produce increased aggressive behavior, and that fluoxetine would inhibit this display. 

Methods Used to Test Hypothesis

            The scientists used outbred populations because of their increased genetic variability which decreased the effects of a random genetic drift that could lead to the development of other heritable traits that are unique. Therefore, the animals used in the experiments represented the natural populations in the wild more accurately. They came to the laboratory at an approximate age of three months, were quarantined for two weeks, and the experiment began when they were approximately four months. Both genders were kept in the same tank before the experiments. They were kept in 40L tanks with a maximum of 25 fish per tank, for at least two weeks before the commencement of the experiments. The tanks were filled with non-chlorinated water at room temperature (28 °C) and a pH of 7.0-8.0, the provision of lighting was through fluorescent lamps in a cycle of 14-10 hr (LD), and the water quality parameters were: pH 7.0-8.0. Hardness 100-150 mg/L CaCO3, dissolved oxygen 7.5-8.0 mg/L, and ammonia and nitrite <0.001 ppm.

            For MIA, the zebrafish were transferred individually to a tank containing 1L of water, with the tank being lit from above with white light. The zebrafish were allowed to acclimate to their new surroundings for five minutes, and after that a mirror was positioned on the outside of the tank, and the behavior of the zebrafish was recorded through a video camera that was positioned on the wider side of the tank, and was analyzed by observers who were blind to the treatment by use of the event recording software X‐Plo‐Rat. The endpoints analyzed were time in the square closest to the mirror (s), frequency (N), and duration(s) of aggressive display, and the total number of squares that were crossed. Aggressive display meant swimming posture with erect caudal, dorsal, anal, and pectoral fins.

            There were a total of two experiments. For the first experiment, the zebrafish were allocated to each group based on their phenotype. Fifteen zebrafish were used in each group, and displayed either the longfin (Group LOF) or leopard (Group LEO) phenotypes. They were randomly drawn from the tank right before tested and the order in which the phenotypes were tested was randomized through the generation of random numbers using a number randomizer website. They were immediately individually transported to the experiment room and left for thirty minutes without any disturbance, and were then exposed to the MIA test explained in the previous paragraph. The differences between the groups were analyzed by using the Approximative Two-Sample Fisher-Pitman Permutation Tests 10,000 Monte Carlo re-samplings.  The data analyst was blinded to phenotype through the use of coding to reflect treatments in the resulting datasets, and after the execution of analysis, the data unblinded. 

            For the second experiment, ten zebrafish were used in each group, and displayed either the leopard (Group LEO) or longfin (Group LOF) phenotypes. Just as with experiment one, the zebrafish were randomly taken out from the tank right before testing, and the order in which the phenotype were tested was randomized through a randomizer website. The zebrafish from each phenotype were also randomly allocated to treatment (vehicle or either fluoxetine dose) also via the randomizer website. The zebrafish were injected with drug or vehicle and left undisturbed for thirty minutes and then exposed to the MIA test. The differences between the groups were then analyzed by using two-way analyses of variance on Huber’s M-estimators. The data analyst in this experiment was also blinded to phenotype through coding to represent treatments in the resulting databases, and after analysis, this data was unblinded..

Results

            In experiment one LEO zebrafish showed longer latencies to display than LOF animals (Z=3.3925, p=0.0005, d=1.5779), shorter display durations (Z= -2/5659, p=<2.2e-16, d- -1/0605), time spent near the mirror (Z= -3/2284, p=<2.2e-16, d= -1.4593), and frequency (Z= -2.7073, p= 0.003, d= -1.1372). There were no differences that were found in complete locomotion (Z= -0.69887, p=0.4965, d= -.2573). For experiment two, no main effects of phenotype (p=0.5736 and partial ϵ2 = 0.0326) or FLX dose (p = 0.5044; partial ϵ2 = 0.0482) were found for latency, but there was an important interaction that was found (p = 0.0412; partial ϵ2 = 0.1622). The main effects of phenotype (p = 0.0132; partial ϵ2 = 0.2429) and FLX dose (p = 0.0062; partial ϵ2 = 0.2297), and also an interaction effect (p = 0.009; partial ϵ2 = 0.1986), were found for display duration. There were no main effects of phenotype (p = 0.2692; partial ϵ2 = 0.3704) that were found for time near mirror, but a main effect of FLX dose (p = 0.0004; partial ϵ2 = 0.0164) and an interaction effect (p < 0.0001; partial ϵ2 = 0.5760). There were also no main or interaction effects found for total locomotion.

Discussion

            The results showed how zebrafish with the leopard skin phenotype show less aggressive readiness and less aggression than longfin zebrafish. Also, a different pattern of fluoxetine was observed, where fluoxetine decreased aggressive display but not readiness in longfin zebrafish, but increased it in leopard zebrafish. The different dose-response profile between the phenotypes led to different results. With the low dose (2.5 mg/kg) aggression was decreased in longfin zebrafish, and with the high dose (5.0 mg/kg) increased it in leopard zebrafish. These results suggest that either an ‘optimal’ serotonergic tone is needed in order to maintain aggression levels (more likely), or it suggests that serotonin transporters are either downregulated or desensitized in the leopard zebrafish population. 

Conclusion/Critical Evaluation of Study

            In conclusion, the experiments in this study showed the differences in aggressive behavior when it comes to longfin and leopard zebrafish, and the effect of fluoxetine on both of the aforementioned populations. These results are salient and relevant in order to comprehend the role of phasic and tonic serotonin neurotransmission on aggressive behavior in preclinical models, and therefore may contribute to a more informed appreciation of the intricate and complex roles of this monoamine in the controlling of vertebrate aggression. However, there are limitations in this study because the sample size is not as large as is needed to establish authority on the aggression of these phenotypes, and that is why other researchers have to undertake further research and have even large sample sizes, for which they will have to secure exorbitant amounts of funding.

References

Barbosa, Hellen P., Maximino, Monica G. Lima, Caio Maximino, and Monica G Lima-Maximino. (2019). Acute fluoxetine differently affects aggressive display in zebrafish phenotypes. Aggressive Behavior, 45(1), 62–69.