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. 2010 Apr 13;20(7):591-9.
doi: 10.1016/j.cub.2010.02.056. Epub 2010 Apr 1.

DN1(p) circadian neurons coordinate acute light and PDF inputs to produce robust daily behavior in Drosophila

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DN1(p) circadian neurons coordinate acute light and PDF inputs to produce robust daily behavior in Drosophila

Luoying Zhang et al. Curr Biol. .

Abstract

Background: Daily behaviors in animals are determined by the interplay between internal timing signals from circadian clocks and environmental stimuli such as light. How these signals are integrated to produce timely and adaptive behavior is unclear. The fruit fly Drosophila exhibits clock-driven activity increases that anticipate dawn and dusk and free-running rhythms under constant conditions. Flies also respond to the onset of light and dark with acute increases in activity.

Results: Mutants of a novel ion channel, narrow abdomen (na), lack a robust increase in activity in response to light and show reduced anticipatory behavior and free-running rhythms, providing a genetic link between photic responses and circadian clock function. We used tissue-specific rescue of na to demonstrate a role for approximately 16-20 circadian pacemaker neurons, a subset of the posterior dorsal neurons 1 (DN1(p)s), in mediating the acute response to the onset of light as well as morning anticipatory behavior. Circadian pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) are especially important for morning anticipation and free-running rhythms and send projections to the DN1(p)s. We also demonstrate that DN1(p)Pdfr expression is sufficient to rescue, at least partially, Pdfr morning anticipation defects as well as defects in free-running rhythms, including those in DN1 molecular clocks. Additionally, these DN1 clocks in wild-type flies are more strongly reset to timing changes in PDF clocks than other pacemaker neurons, suggesting that they are direct targets.

Conclusions: Taking these results together, we demonstrate that the DN1(p)s lie at the nexus of PDF and photic signaling to produce appropriate daily behavior.

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Figures

Figure 1
Figure 1. Expression Pattern of two DN1p Specific Clk-GAL4 Lines
(A and B) Maximum projections of confocal sections taken in representative adult Clk4.5F-GAL4/UAS-nGFP (A, green) and Clk4.1M-GAL4/UAS-nGFP (B, green) brains labeled with PER antibody (red). (C and D) Maximum projections of confocal sections taken in representative adult UAS-mGFP/+;Clk4.5F-GAL4/+ (C) and UAS-mGFP/+;Clk4.1M-GAL4/+ (D) brains labeled with GFP (green) and PER antibody (red). The DN1s are indicated by lines.
Figure 2
Figure 2. Rescue of na Expression in a Subset of DN1 Cells Restores Morning Anticipation, Lights-On Arousal Response, and DD1 Evening Anticipation
Normalized activity plots for adult male populations averaged across either (A-D) 4 days of LD or (E-H) the first day of DD. For (A-D), light and dark phases are indicated by horizontal white and black bars, respectively. For (E-H), subjective light and dark phases are indicated by horizontal gray and black bars, respectively. White arrows indicate LD evening behavior, gray arrows indicate DD1 evening behavior, and black arrows indicate morning behavior. Genotypes shown are (A,E) nahar;;UAS-na/Clk4.1M-GAL4, (B,F) nahar;;UAS-na/+, (C,G) UAS-na/+, and (D,H) Clk4.1M-GAL4/+. n=49-76. Error bars indicate standard error of mean (SEM). See also figure S2 for additional data.
Figure 3
Figure 3. PDFR Expression in the DN1ps is Sufficient for Robust Morning Anticipatory Behavior
(A-D) Normalized activity plots for adult male populations averaged over four days of 12 hour light: 12 hour dark entrainment. The light phase is indicated by white bars, while the dark phase is indicated by black bars. (E-H) Normalized activity plots of adult male populations over the last 6 hours of LD (ZT18-CT0) followed by the first 18 hours of DD (CT0-18). Subjective light phase (CT0-12) is indicated by dark gray bars while subjective dark phase is indicated by black bars. (A and E) UAS-Pdfr/+; (B and F) Pdfrhan5304;;UAS-Pdfr/+; (C and G) Pdfrhan5304;;Clk4.5F-GAL4/UAS-Pdfr; (D and H) Pdfrhan5304;;Clk4.1M-GAL4/UAS-Pdfr. Error bars represent standard error of the mean (n=25-53). White arrows indicate evening behavior and black arrows indicate morning behavior. See also figure S2 and table S1 for additional data.
Figure 4
Figure 4. PDFR Expression in the DN1ps Alters the Molecular Oscillation in the DN1s of Pdfr Mutants
(A) Maximum projections of confocal sections taken in representative adult Pdfrhan5304;;UAS-Pdfr/+ and Pdfrhan5304;;Clk4.1M-GAL4/UAS-Pdfr brains labeled with PER antibody. Sections contain the DN1s at CT1, 7, 12, and 18. (B) Maximum projections of confocal sections taken in representative adult Pdfrhan5304;;UAS-Pdfr/+ and Pdfrhan5304;;Clk4.1M-GAL4/UAS-Pdfr brains labeled with with PER and PDF antibodies. Sections contain the LNs at CT1, 7, 12, and 18. The PDF(+) s-LNvs and LNds are in boxes, while the 5th s-LNv is indicated by line. (C) Plots of average normalized pixel intensity versus circadian time for each pacemaker cell group. See experimental procedures for details of quantification method. Error bars represent standard error of mean. The results are a combination of two independent experiments: s-LNv, n=32-57; LNd, n=56-91; 5th s-LNv, n=8-17; DN1, n=143-226. Asterisks mark significant differences between genotypes (Student's t-test, *p<0.05, **p<0.01, ***p<0.001).
Figure 5
Figure 5. Manipulating the Clock in the PDF(+) s-LNvs Alters the Clock in the DN1s but Not the Other E-cell Subsets
(A and B) Maximum projections of confocal sections taken in representative adult UAS-CK2βRNAi/+ and Pdf-GAL4,UAS-CK2βRNAi/+ brains labeled with with PER and PDF antibodies. Sections contain the LNs at CT1, 7, 14, and 20. The PDF(+) s-LNvs and LNds are in boxes, while the 5th s-LNv is indicated by line. (B) Maximum projections of confocal sections taken in representative adult UAS-CK2βRNAi/+ and Pdf-GAL4,UAS-CK2βRNAi/+ brains labeled with PER antibody. Sections contain the DN1s at CT1, 7, 14, and 20. (C) Plots of average normalized pixel intensity versus circadian time for each pacemaker cell group. See experimental procedures for details of quantification method. Error bars represent standard error of mean. The results are a combination of three independent experiments: s-LNv, n=32-76; LNd, n=54-94; 5th s-LNv, n=10-18; DN1, n=104-189. Asterisks mark significant differences between genotypes (Student's t-test, *p<0.05, **p<0.01, ***p<0.001). See also figures S4 and S5 and table S2 for additional data.

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