Dopamine Can Regulate Period of the Drosophila Circadian Clock. Karol Cichewicz¹, Emma Garren¹, Magali Iché-Torres², Serge Birman², Jay Hirsh¹. 1) Biology, University of Virginia, Charlottesville, VA; 2) CNRS, ESPCI, Paris.

   Dopamine (DA) is important for many physiological functions including locomotor activity, learning and memory and light sensitivity. We have developed a new genetic background lacking DA in the CNS, which allows us to study complex DA modulation by rescuing its expression in specific neurons. Here we show that spatial imbalance of DA expression results in altered period of the circadian clock. Drosophila tyrosine hydroxylase (DTH, ple), encoding the rate limiting enzyme in DA biosynthesis, expresses in the CNS and hypoderm, with tissue-specific alternative splice forms. A recent study from our laboratories (Riemensperger et al, 2010) shows that a modified DTHg FS+/- gene that selectively expresses in the hypoderm rescues the lethality of a DTH null mutation (ple²), generating healthy flies with normal lifespan. These flies were constructed with the GAL4:UAS binary expression system, such that further genetic manipulations are difficult. Using the general approach from the previous study, we generated a genetic background lacking DA in the CNS without using binary expression tools. The DTH FS+/- mutations were recombineered into a genomic BAC, which was integrated site-specifically into a 3^rd chromosome, and then recombined onto a ple² mutant background. BAC plasmids containing the wild type 20kb DTH gene or the DTH FS+/- rescued ple² lethality, showing that all DTH cis-regulatory elements are contained within this segment. This DTH FS+/- ple² genetic background allows the study of DA signaling by restoring its expression in a spatial and temporal manner using Gal4 and LexA drivers. Restoration of DTH expression in the DA neurons labeled by TH-Gal4 produced a 27h period of locomotor activity in constant darkness, whereas total loss of DA, or expression driven by both TH-Gal4 and DDC-Gal4 produced a normal 24h rhythm. These results indicate an important circadian role for DA in the non-overlapping TH-Gal4 and DDC-Gal4 neurons. Since TH-GAL4 prominently lacks expression in PAM DA neurons, these are candidates for this role. We are studying the mechanism by which this occurs.