Figure 3. Synthetic PDF application suppresses and/or delays Ca²⁺ activity in vivo.

(A) Left, schematic illustrating yoked pairs of WT and pdfr mutant flies for pharmacological tests. Right, representative images of LNd and s-LNv pacemakers in such Drosophila pairs responding differentially to 10⁻⁰⁵M synthetic PDF. Axis above denotes the circadian time of recordings and the CT7 time point of synthetic PDF application.

(B) Averaged Ca²⁺ transients of M pacemakers (s-LNv, orange) and E pacemakers (LNd, blue) responding to (top) PDF or (bottom) vehicle from (left) WT or (right) pdfr mutant flies. Gray traces represent individual cells in trials (PDF: n = 5 pairs; vehicle: n= 4 pairs).

(C–D) Ca²⁺ signal changes by (C) PDF treatment or (D) vehicle treatment measured at the point that WT pacemakers displayed a maximal reduction in response to PDF (*p<0.01: single-sample t-test). The average increase in Ca²⁺ signals in WT LNd with vehicle treatment represents their normal daily Ca²⁺ peak that occurs during these recording periods (cf. Figure 1C).

(E) Ca²⁺ activity responses in pdf⁰¹ mutants to application of vehicle saline at the time indicated by vertical arrows under DD (n = 6 flies).

(F) Ca²⁺ activity responses in pdf⁰¹ mutants to single synthetic PDF application under DD (n = 5 flies). PDF was present in the initial saline bath as indicated by the vertical orange arrow at a peak concentration of 10⁻⁵ M. This initial time point represents the peak time of Ca²⁺ in s-LNv (CT0 – orange arrow). PDF was slowly washed out by perfusion (gray curve below the orange arrow denotes PDF concentration).

(G) Ca²⁺ activity responses in pdf⁰¹ mutants to single synthetic PDF application at CT5 under DD (n = 6 flies).

(H) Ca²⁺ activity responses to two serial applications of synthetic PDF in pdf⁰¹ mutants under DD (n = 6 flies). The first dose was at CT0 (as in panel F), and the second at CT5 (~ at the l-LNv Ca²⁺ peak time) both denoted by vertical orange arrows; constant perfusion followed each application.

(I) Quantification of Ca²⁺ phase shifts produced by synthetic PDF; the mean phase of each group in pdf⁰¹ mutants under DD is set to zero (*p<0.05: Watson-Williams test).