In vivo imaging of temperature-response in Drosophila neurons and genetic screening of the genes affecting temperature-preference & circadian behavior

Abstract

Temperature is the essential factor for the survival and influence various life phenomena such as behavior pattern, cellular metabolism, and physiological process. So most animal have abilities to find appropriate temperature for surviving and maintaining body temperature, and to avoid harsh temperature such as freezing and burning. However, the molecular mechanism mediating temperature preference has been pooly decribed. Among techniques, calcium imaging has become a standard technique in diverse areas of biology in general, and neuroscience in particular, reflecting the many cellular roles and actions of this ion as a second messenger and as a carrier of charge. Calcium can stimulate biological cascades and changes in gene transcription, mediate synaptic potentials, and trigger vesicle release. Various genetically encoded fluorescent sensors can monitor change in intracellular calcium concentration. circularly permuted GFP variants are singly sensitive to physiologically calcium ions. We use a circularly permuted green fluorescent protein(cpEYEP). These sensors show very low fluorescence in the unbound state, but increase emission intensity considerably after binding calcium. By using the Gal4-UAS system in Drosophila, we can make one fly strain, carrying the inversepericam DNA under the control of an upstream activator sequences(UAS). When another fly strain expressing Gal4 in a cell type-specific manner is crossed with UAS strain, the progeny will express the inversepericam, GFP signals in the specific region reflecting the expression pattern of the Gal4 line. By using the device for assaying preference for temperature, the fly(MB247 Gal4/UAS-TNT-E) showed abnormal temperature preference. That result revealed Mushroom bodies plays important roles on the temperature sensing. So we designed the $Ca^{2+}$ imaging on mushroom bodies during temperature fluctuation. We compared the result in Mushroom bodies with other parts in larvas. Mushroom bodies and specific...