Figure 5. Restoring E cell PDFR rescues period, rhythmicity and firing-induced phase shifts.
(A) pdfr mutant flies show no morning peak and an advanced evening peak as expected (left panel). Rescue of PDFR expression with the Dv-E cell driver in this mutant background restores both morning and evening anticipation peaks, that is, these flies show an intact morning peak and more normal onset of evening peak activity (middle panel). Inhibition of rescue in CRY-positive E cells with Cry-GAL80 prevents the restoration of morning and evening activity peaks. n = 16–18 for each group. Genotypes are shown under each panel. The error bar indicates SEM. (B) CRY staining pattern in DvPdf-GAL4, Pdf-GAL80; UAS-mCD8::GFP brains. GFP (green) is expressed in 4 LNds (left and middle panel), and CRY signal (magenta) is visible in the large PDF cells (below and to the left of the arrow) as well as 3 LNds (left and right panel). The arrow shows the one CRY-positive DV-E cell. Magnified images of the upper dash line boxed area are shown in lower solid line box. (C) Adding a copy of Cry-GAL80 eliminates GFP staining of the CRY-positive Dv-E cells as well as their branches, which are adjacent to the PDF cell dorsal projections. Brains from DvPdf-GAL4/+; UAS-mCD8::GFP/+ (left panel) and DvPdf-GAL4/+; Cry-GAL80/UAS-mCD8::GFP (right panel) were stained with a GFP antibody (red). The upper arrow shows the typical projection from DvPdf -labeled CRY positive LNd neuron and the lower arrow points to likely branches from the 5th-sLNv. With Cry-GAL80, both of these projections are absent. Note that a copy of Cry-GAL80 can block GAL4 activity in CRY positive Dv-E cells but not in sLNv, the dorsal projections from sLNvs are still visible in DvPdf-GAL4/+; Cry-GAL80/UAS-mCD8::GFP brains (right panel). (D) Flies were exposed to a 30°C pulse for 2 hr at ZT15 (dark gray bars) or ZT21 (light gray bars). The expression pattern of UAS-dTrpA1 and UAS-PDFR is shown above the histogram. Genotypes shown below each histogram are (from left to right): DvPdf-GAL4/UAS-dTrpA1 (n = 32), pdfr−; DvPdf-GAL4/UAS-dTrpA1(n = 32), pdfr−; DvPdf-GAL4,UAS-dTrpA1/UAS-PDFR; Pdf-GAL80 (n = 24), pdfr−; DvPdf-GAL4, UAS-dTrpA1/UAS-PDFR; Cry-GAL80 (n = 24) and pdfr−; DvPdf-GAL4,UAS-dTrpA1/UAS-PDFR (n = 30). ‘***’ represents p<0.001 as determined by the student's t test and indicates a significant phase change. The error bars are SEM.