Madhavi Kuchibhotla, Airined Montes, José L. Agosto Rivera, Alfredo Ghezzi.
Department of Biology, University of Puerto Rico, Rio Piedras, San Juan, PR

INTRODUCTION: Alcoholism is the third leading cause of deaths. One of the key components in the development of alcoholism is the gradual increase in resistance to alcohol over repeated exposures. This adaptation is also known as tolerance, which is thought to involve neural adaptations and plasticity within the brain leading to alcohol use disorders (AUD). Recently, the Neuroimmune system has emerged as an important player in the development of AUD. Well Established studies on polymorphisms in macrophages and immune genes were linked to higher risk of alcoholism, supporting the role of immunity and neuroimmune signaling in alcohol addiction. Drosophila studies have identified the RNA binding protein pumilio (pum) as a regulator of ethanol tolerance. Pum has also been linked to innate immunity and alcohol tolerance. Even though it was evident that higher ethanol doses increase the tolerance, the effects of temperature and doses, and their interaction, on tolerance has not been previously studied. We are inclined to hypothesize the tolerance might increase with higher interaction of dose just as doses alone to tolerance, though we aren’t sure if that will be the trend. Therefore, we were motivated to fill this knowledge. Thus, the overall goal of this study is to evaluate the role of pumilio within hemocytes in alcohol induced behaviors as the function of temperature and concentration of ethanol. One of our focuses is to understand how pumilio can alter alcohol responses with temperature.

METHODOLOGY: We employed GAL4/UAS binary system to generate targeted expression of GFP and/or pumRNAi transgenes precisely in hemocyte-specific driver line (hml-GAL4). We performed a 2-day ethanol assay paradigm on F1 age matched female Drosophila melanogaster flies and ethanol sensitivity and tolerances were calculated using recorded sedation scores. All flies were subjected to 50% ethanol at 3 different temperatures (18℃, 22℃ and 27℃) at 3 different instances in regards to temperature. To further reveal the relationship between temperature and ethanol doses and their interaction to ethanol responses, the study was performed at 3 different temperatures (19℃, 25℃, and 28℃) at different instances. At every temperature flies were subjected to 3 different ethanol doses (50%, 75% and 100%) simultaneously. The focus of the study is to reveal the relation between as a factor of ethanol doses and temperature.

RESULTS: Our results indicate that, within the genotype, sensitivity to ethanol increases with temperature and pumilio knockdown increases sensitivity with temperature. Pumilio knockdown demonstrated a significant increase in tolerance index with temperature (p<0.0002) as opposed to its corresponding control flies, which were also statistically significant (p<0.0001). Systematic analysis of temperature and doses revealed that with doses, sensitivity increases irrespective of temperature but at lower temperatures, i.e., at 19℃, pumilio knockdown is less sensitive to ethanol doses than its control, with p-value between the slopes is 0.0026. As temperature increases to 22℃, control flies show decreased sensitivity than lower temperature until both control and pum knock out flies show no significant difference between sensitivities at 28℃ with p value of slopes between them is 0.6044. Irrespective of temperature, control flies shower higher tolerance than pum knockout flies. At lower temperature, ie.,19℃, increased tolerance is observed in terms of tolerance index (T.I) with dose, with p values of slopes between control and pum knockout being 0.0142 but at 25℃, both genotypes have similar slopes with p value being statically non-significant (0.8950). At higher temperature, 28℃, negative slope for control was observed, in contrast to pum knockout showing no changes in tolerances with doses, with p value between them being statistically nonsignificant.

CONCLUSION: Overall, our data suggests that neuronal adaptation mechanisms underlying ethanol tolerance might be different from the perspective of dose only vs interaction of doses and environmental temperature. More specifically, at colder temperatures, one needs high doses of ethanol to be able to develop tolerance, suggesting the nervous system is less excitable and therefore needs more alcohol to be able to trigger plasticity mechanisms. In Contrast, high doses will not develop tolerance as much as at lower, suggesting high temperatures and high ethanol doses too much excitability is observed and plasticity mechanisms are blocked.

ACKNOWLEDGEMENTS: I thank my mentor and co-mentor for their unparallel support, time and intellectual input in shaping this project. I also thank my Colleague Airined in extending her hand. Grateful to every person in my lab who helped me directly or indirectly in this project. Thanks to research organizations for making this project forward with their financial support- NIH Grant 5P20GM103642, NSF Grant 1736026, NSF Grant 1633184, NIH Grant 2RNS080687, NIH Grant P20GM103475, NSF 2131647, 5R25GM061151-19.