R1 Neural Circuitry on Ellipsoid Body Promotes Sleep in Drosophila Melanogaster

Abstract

This research identified sleep promoting R1 neurons on Drosophila ellipsoid body, but did not locate PPM3 neurons as upstream signal input in this sleep promoting R1 neural circuit. Two GAL4 labeled R1 neuron lines, VT058968-GAL4 and R28E01-GAL4, were selected and identified as sleep promoting ellipsoid body neurons, by utilizing Drosophila heat activated transient receptor ion channel, dTRPA1, to activate ellipsoid body R1 neurons (Brody 2015). The research also facilitates the tetanus toxin light chain (TNT) to inhibit the R1 neural activities constantly, and look at locomotor assays on flies (Umezaki et al. 2011). Then we considered dopaminergic PPM3 neurons as the possible candidate for upstream neural input in R1 sleep promoting circuit. PPM3 neurons are dopaminergic neurons and they project to dorsal fan shape body and R2 neurons on ellipsoid body to mediate sleep promotion. The anatomical connectivity between PPM3 neurons and R1 neurons were confirmed by RFP and GFP confocal imaging, and R1 neurons were identified as dopaminergic, as they were activated by dopamine bath in GCamp6f and EPAC assays. Behavioral assays were performed on two GAL4 labeled PPM3 neurons, VT040016-GAL4 and VT024624-GAL4, which were activated by dTRPA1 at sensitive temperature and inhibited by constant expression of TNT. The locomotor assays indicated activated PPM3 neurons participated in sleep promotion during evening and early night time. However, TNT inhibition on PPM3 did not induce sleep reduction or any other sleep phenotype in transgenetic flies. As PPM3 also innervates other sleep promoting circuit, the partial inhibition of PPM3 neurons still have intact sleep promotion circuit in brains. Therefore, PPM3 neurons are still considered as upstream neurons that mediates R1 sleep promoting circuit on ellipsoid body.This research identified sleep promoting R1 neurons on Drosophila ellipsoid body, but did not locate PPM3 neurons as upstream signal input in this sleep promoting R1 neural circuit. Two GAL4 labeled R1 neuron lines, VT058968-GAL4 and R28E01-GAL4, were selected and identified as sleep promoting ellipsoid body neurons, by utilizing Drosophila heat activated transient receptor ion channel, dTRPA1, to activate ellipsoid body R1 neurons (Brody 2015). The research also facilitates the tetanus toxin light chain (TNT) to inhibit the R1 neural activities constantly, and look at locomotor assays on flies (Umezaki et al. 2011). Then we considered dopaminergic PPM3 neurons as the possible candidate for upstream neural input in R1 sleep promoting circuit. PPM3 neurons are dopaminergic neurons and they project to dorsal fan shape body and R2 neurons on ellipsoid body to mediate sleep promotion. The anatomical connectivity between PPM3 neurons and R1 neurons were confirmed by RFP and GFP confocal imaging, and R1 neurons were identified as dopaminergic, as they were activated by dopamine bath in GCamp6f and EPAC assays. Behavioral assays were performed on two GAL4 labeled PPM3 neurons, VT040016-GAL4 and VT024624-GAL4, which were activated by dTRPA1 at sensitive temperature and inhibited by constant expression of TNT. The locomotor assays indicated activated PPM3 neurons participated in sleep promotion during evening and early night time. However, TNT inhibition on PPM3 did not induce sleep reduction or any other sleep phenotype in transgenetic flies. As PPM3 also innervates other sleep promoting circuit, the partial inhibition of PPM3 neurons still have intact sleep promotion circuit in brains. Therefore, PPM3 neurons are still considered as upstream neurons that mediates R1 sleep promoting circuit on ellipsoid body.