The Effects of Caffeine on Learning and Memory in Zebrafish (Danio rerio) Erica Pantelich, Department of Biology, York College INTRODUCTION:  Learning and memory research uses a variety of invertebrate and vertebrate models to investigate the neural basis of cognitive function.  From this research, understanding cognitive function, reasons for cognitive dysfunction, and treatments promoting cognitive improvement have been gained.  Zebrafish are an emerging model for many aspects of neural function.  Researchers like Levin and Chen (2001) observed the effects of nicotine on the memory of zebrafish with a delayed spatial alteration task. Carvan et al. (2004) tested the effects of ethanol on zebrafish memory through a spatial alteration task in a testing apparatus. Darland and Dowling (2001) tested wild type and mutagenzied zebrafish for learning and memory by using a T- maze.  With much focus on the effects of nicotine and ethanol on zebrafish’s learning and memory, little attention has been given to the effects of caffeine.  My research tested the effects of caffeine on learning and memory in zebrafish through the use of a T-maze. QUESTIONS ASKED: 1)Will caffeine enhance the learning and memory (short and/or long term) of the desirable habitat of the T-maze? 2)Will caffeine quicken a decision in a zebrafish to enter either the desirable or undesirable habitat? HYPOTHESIS : Caffeine will enhance the learning and short- term memory, but not the long-term memory of zebrafish. METHODS: Figure 1 Figure 2 Figure 3 FUTURE STUDIES:  Start with many control groups to find the best methodology and sample size.  Label each fish to see each individual zebrafish’s progress.  Use a different testing apparatus such as a spatial alteration task to test learning and memory. ACKNOWLEDGEMENTS: I would like to thank Dr. Boehmler for her instruction with zebrafish and Dr. Rehnberg for his guidance and patience throughout my behavioral study. LITERATURE CITED: Carvan III, M.J., Loucks, E., Weber, D.N., and Williams, F.E Ethanol effects on the developing zebrafish: neurobehavior and skeletal morphogenesis. Neurotoxicology and Teratology 26: Childs, E., and Wit, de H Subjective, behavioral, and physiological effects of acute caffeine in light, nondependent caffeine users. Psychopharmacology. 185: Darland, T., and Dowling J.E Behavioral screening for cocaine sensitivity in mutagenized zebrafish. Proceedings of the National Academy of Science 98: Guo, S Linking genes to brain, behavior and neurological diseases: what can we learn from zebrafish? Genes, Brain and Behavior 3: Levin, E., and Chen, E Nicotinic involvement in memory function in zebrafish. Neurotoxicology and Teratology 26: Levin, E., Limpuangthip, J., Rachakonda, T., and Peterson, M Timing of nicotine effects on learning in zebrafish. Psychopharmacology 184: Young zebrafish were separated equally into four tanks at random (n=13) (Figure 1). 0, 0.06mM, 0.6mM, and 6.0mM of caffeine were prepared for all 10 days of experimentation and stored in 10mL containers. The four tanks were coded to match a corresponding coded 10mL container by an assistant to ensure a blinded study. The fish were exposed to 0, 0.06, 0.6, or 6 mM of caffeine for 3 minutes every day for 10 consecutive days. Exposure took place in a 50 mL beaker. The T-maze (Figure 2) was constructed to assess learning and memory of caffeine-exposed zebrafish and included a desirable habitat and an undesirable one. After exposure, each fish was placed at the starting point of the T-maze and timed until making a decision, either the desirable or undesirable habitat. Until all fish of a tank were tested they were placed into a reserve tank and then placed back into their home tank. Data were analyzed using the binomial equation (Figure 3) and Prism software. RESULTS:  P-values calculated from the binomial equation were plotted for each day for all four groups.  For all four groups, the p-values did not become smaller as the days increased (Figure 4).  The relationship between the number of days that had passed and the amount of time to make a decision whether left or right was plotted and analyzed through a one-way ANOVA (Figure 5).  The time on Day 1 compared to the time on Day 10 is smaller, and there seems to be a decrease in time to a decision as days pass. When statistically analyzed through a one-way ANOVA, there was no significant difference in any of the groups (p>0.05). CONCLUSION:  Due to the probability (p-value) to go to the desirable habitat of the four groups not becoming smaller as days increased, this showed that zebrafish did not choose to go this side more.  Although the graphs display the trend that the zebrafish made a decision faster as time passed, this was not statistically supported (p>0.05).  Overall, because the control group was unable to learn and remember the T-maze, the results of the caffeine groups are inconclusive. Figure 4. P-values represent the probability, calculated from the binomial equation, of choosing the desirable habitat of the T-maze at days 1-10, 13, and 17 for each group (n=13). The dashed line represents a p- value of A=control, B=0.06mM, C=0.6mM, and D=6.0mM. Figure 5. The time it took to make a decision, whether for the desirable or undesirable side, at days 1-10, 13 and 17 of all four groups (n=13). Error bars represent the S.E.M. With the exception of the controls (A), the other groups seem to make a decision faster as time increases. However, after a one-way ANOVA of the days of each group, there was no significant difference between the days (p>0.05). A=control, B=0.06mM, C=0.6mM, and D=6.0mM.