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Anxiety-like Behavior and Whole-Body Cortisol Responses to Components of Energy Drinks in Zebrafish ( Danio rerio)

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Anxiety-like Behavior and Whole-Body Cortisol Responses to Components of Energy Drinks in Zebrafish ( Danio rerio)

Alia O Alia et al. PeerJ.

Abstract

The current study investigated the independent and combined effects of caffeine and taurine on anxiety-like behavior and neuroendocrine responses in adult zebrafish (Danio rerio). Caffeine (1,3,7-trimethylpurine-2,6-dione), the world's most commonly used psychoactive drug, acts as an adenosine receptor blocker and a mild central nervous system stimulant. However, excessive use of caffeine is associated with heightened anxiety levels. Taurine (2-aminoethanesulfonic acid), a semi-essential amino acid synthesized within the human brain, has been hypothesized to play a role in regulating anxiolytic behavior. Caffeine and taurine are two common additives in energy drinks and are often found in high concentrations in these beverages. However, few studies have investigated the interaction of these two chemicals with regards to anxiety measures. A suitable vertebrate to examine anxiety-like behavior and physiological stress responses is the zebrafish, which has shown promise due to substantial physiological and genetic homology with humans. Anxiety-like behavior in zebrafish can be determined by analyzing habituation to novelty when fish are placed into a novel tank and scototaxis (light avoidance) behavior in the light-dark test. Stress-related neuroendocrine responses can be measured in zebrafish by analyzing whole-body cortisol levels. The goal of this study was to determine if exposure to caffeine, taurine, or a combination of the two compounds altered anxiety-like behavior and whole-body cortisol levels in zebrafish relative to control. Zebrafish were individually exposed to either caffeine (100 mg/L), taurine (400 mg/L), or both for 15 min. Zebrafish in the control group were handled in the same manner but were only exposed to system tank water. After treatment, fish were transferred to the novel tank test or the light-dark test. Behavior was tracked for the first 6 min in the novel tank and 15 min in the light-tark test. Fifteen min after introduction to the behavioral task, fish were euthanized for the analysis of whole-body cortisol levels. The results demonstrate that caffeine treatment decreased the amount of exploration in the top of the novel tank and increased scototaxis behavior in the light-dark test, which supports the established anxiogenic effect of acute exposure to caffeine. Taurine alone did not alter basal levels of anxiety-like behavioral responses nor ameliorated the anxiogenic effects of caffeine on behavior when the two compounds were administered concurrently. None of the drug treatments altered basal levels of whole-body cortisol. The current results of this study suggest that, at least at this dose and time of exposure, taurine does not mitigate the anxiety-producing effects of caffeine when administered in combination, such as with energy drink consumption.

Keywords: Anxiety; Anxiety-like behavior; Caffeine; Cortisol; Energy drink; HPA; HPI; Stress; Taurine; Zebrafish.

Conflict of interest statement

The authors declares that they have no competing interests.

Figures

Figure 1
Figure 1. Measures of zebrafish motor activity in the novel tank test.
Acute exposure to energy drink components (caffeine, taurine, or both) did not alter (A) the total distance traveled and (B) the mean speed while ambulatory in the novel tank test in adult zebrafish. Bars indicate means of each group ±SEM.
Figure 2
Figure 2. Measures of zebrafish freezing behavior in the novel tank test.
Acute exposure to energy drink components (caffeine, taurine, or both) did not alter (A) the total number of immobility bouts and (B) the total time spent immobile in the novel tank test in adult zebrafish. Bars indicate means of each group ±SEM.
Figure 3
Figure 3. Measures of zebrafish exploratory behavior in the novel tank test.
Acute exposure to energy drink components (caffeine, taurine, or both) altered exploratory behavior in the novel tank test in adult zebrafish. Caffeine decreased (A) the distance traveled in the top zone, (B) the number of entries to the top zone, and (C) the time spent in the top zone. Taurine decreased (D) the latency to enter the top zone. Bars indicate means of each group ±SEM.
Figure 4
Figure 4. Measures of zebrafish neuroendocrine function after the novel tank test.
Acute exposure to energy drink components (caffeine, taurine, or both) did not alter whole-body cortisol levels of zebrafish of fish in Experiment 1 (fish were sacrificed after the novel tank test). Bars indicate means of each group ±SEM.
Figure 5
Figure 5. Measures of zebrafish motor activity in the light-dark test.
Acute exposure to energy drink components (caffeine, taurine, or both) affected some aspects of motor activity in adult zebrafish. Acute caffeine decreased (A) the total distance traveled but not (B) the mean speed during ambulation in the light-dark test. Bars indicate means of each group ±SEM and ** indicates significant (p < 0.01) difference from respective non-caffeine treated control (Tukey post hoc).
Figure 6
Figure 6. Measures of zebrafish freezing behavior in the light-dark test.
Acute exposure to energy drink components (caffeine, taurine, or both) altered the total time spent immobile in the novel tank test in adult zebrafish. Caffeine increased the total time immobile in the light-dark test. Bars indicate means of each group ±SEM. *, ** indicates significant (p < 0.05 and p < 0.01, respectively) difference from respective non-caffeine treated control (Tukey post hoc).
Figure 7
Figure 7. Measures of zebrafish exploratory behavior in the light-dark test.
Acute exposure to energy drink components (caffeine, taurine, or both) altered exploratory behavior in the novel tank test in adult zebrafish. Caffeine decreased (A) the distance traveled in the light zone, (B) the number of entries to the light zone, and (C) the number of crossings between compartments. The acute drug treatments did not significantly alter (D) the time spent in the light zone nor (E) the latency to re-enter the light zone after the first visit to the dark zone. Bars indicate means of each group ±SEM. *, ** indicates significant (p < 0.05 and p < 0.01, respectively) difference from respective non-caffeine treated control (Tukey post hoc).
Figure 8
Figure 8. Measures of zebrafish neuroendocrine function after the light-dark test.
Acute exposure to energy drink components (caffeine, taurine, or both) did not alter whole-body cortisol levels of zebrafish of fish in Experiment 2 (fish were sacrificed after the light-dark test). Bars indicate means of each group ±SEM.

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Grant support

This work was supported by the IU Northwest Faculty Grant-in-aid of Research and the Faculty for Undergraduate Neuroscience (FUN) Equipment Loan program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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