Studies On Molecular Mechanism Of Ethanol Interfering With Gustatory Plasticity Behavior And Identification Of Its Neuronal Effect By In Vivo Calcium Imaging In C. Elegans

Alcohol is widely used or even abused in modern society. Alcohol can cause serious problems on nervous system and long-term alcohol consumption is correlated with an increased risk of developing tolerance and alcoholism. Although some important results have been achieved in recent years, the molecular mechanisms underlying the behavioral effects of ethanol still need further study.Ethanol can affect the formation of learning and memory in many species. In the model organism Caenorhabditis elegans, gustatory plasticity is a simple learning paradigm in which animals after prolonged pre-exposure to a chemo-attractive salt show chemo-aversion to this salt. Here, our results indicated that ethanol administration during pre-exposure or test stage interfered with gustatory plasticity in well-fed worms, in a dose-independent manner. Genetic analysis revealed that genes play important roles in serotonin signaling such as tph-1, ser-4 and ser-7 were involved in ethanol-mediated gustatory plasticity; serotonin signal also participated in behavior responses affected by ethanol such as locomotion and egg laying. In addition, the gpa-3 mutant animal, carrying mutations in the G-protein a subunit, also showed defects in response to ethanol in modulating gustatory plasticity. Further studies revealed that ethanol could affect the aversive response to diluted octanol, which was another behavioral response dependent on feeding status. These results suggested that serotonin signal play important roles in regulating acute intoxicating effects of ethanol in C. elegans.Another important aspect in this study is that we developed a Y-shaped microfluidic chip for immobilizing and stimulating worms with a one-piece valve for enhanced immobilization of worms. A genetically encoded calcium sensor protein, G-CaMP, was expressed in ASE neurons of C. elegans under the control of specific promoters. Using the well-established interface shifting method, neuronal activities in response to stimuli of immobilized animals could then be monitored by in vivo fluorescence imaging. Results showed that average calcium transients in ASER neurons in response to up-step but not down-step of NaCl concentration were significantly affected by ethanol. ASER and ASEL exhibited different sensitivity in adaptation to NaCl, however ethanol produced significant effect on the adaptation response in ASEL neurons when compared to ASER neurons. Results of calcium imaging indicated that ethanol directly affected the neuronal activity of ASE neuron that plays a dominant role in chemotaxis to salt.Together, our results demonstrated the distinct role of serotonin pathway in modulation of acute response to ethanol in gustatory plasticity in C. elegans. Investigation of the molecular mechanisms underlying ethanol's effect on learning behaviors in C. elegans as well as the direct impact of ethanol on neuronal activity may make a great contribution to the understanding of ethanol's function manner in higher animals.