Impaired clock output by altered connectivity in the circadian network

Abstract

Substantial progress has been made in elucidating the molecular processes that impart a temporal control to physiology and behavior in most eukaryotes. In Drosophila, dorsal and ventral neuronal networks act in concert to convey rhythmicity. Recently, the hierarchical organization among the different circadian clusters has been addressed, but how molecular oscillations translate into rhythmic behavior remains unclear. The small ventral lateral neurons can synchronize certain dorsal oscillators likely through the release of pigment dispersing factor (PDF), a neuropeptide central to the control of rhythmic rest-activity cycles. In the present study, we have taken advantage of flies exhibiting a distinctive arrhythmic phenotype due to mutation of the potassium channel slowpoke (slo) to examine the relevance of specific neuronal populations involved in the circadian control of behavior. We show that altered neuronal function associated with the null mutation specifically impaired PDF accumulation in the dorsal protocerebrum and, in turn, desynchronized molecular oscillations in the dorsal clusters. However, molecular oscillations in the small ventral lateral neurons are properly running in the null mutant, indicating that slo is acting downstream of these core pacemaker cells, most likely in the output pathway. Surprisingly, disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit. Our observations demonstrate that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity.

Fig. 1.

Neuronal rescue of SLO function restores rhythmic rest-activity cycles. Newly eclosed males were synchronized and then released in DD (black arrow). Experiments were repeated three to nine times. (A) Representative double-plotted actograms from CS, slo⁴, B52H;slo⁴, and M131;slo⁴. Open boxes, day; black boxes, night; gray boxes, subjective day. (B) Circadian phenotypes in various slo rescues. R, rhythmic; WR, weakly rhythmic; AR, arrhythmic. Period values were determined only on rhythmic individuals. The mean power Fast Fourier Transform (FFT) (a quantification of the strength of the rhythm) for those flies is shown. Statistical analysis included one-way ANOVA with the Bonferroni correction. slo⁴, ash2¹⁸/slo⁴, and M131;slo⁴ are significantly different from CS (P < 0.001).

Fig. 2.

SLO expression in the context of circadian structures. (A–F) tim-Gal4/UAS-CD8GFP (timGFP, A–C) and pdf-Gal4/UAS-CD8GFP (pdfGFP, D–F) brains were stained with anti GFP (green) and pSLO (red). Images with no label are the result of an overlay. Images shown in A, D, G, L, and M are projections, whereas the remaining ones are single optical scans. Arrows indicate colocalization. (A) SLO expression in a brain hemisphere displaying GFP+ tim neurons. (B) Inset from a dorsal region indicated in A, displaying axons from DNs juxtaposed to SLO+ neurites. (C) Inset from the same region of a different brain displaying three consecutive scans to illustrate how GFP+ axons reach a region where a cluster of SLO+ somas is located. (D) Extensive ramification of PDF and SLO+ neurites in the optic lobe. (E) Inset from D showing colocalization between both signals. (F) A higher magnification of a dorsalmost segment of the small LNvs projections from another brain showing SLO+ somas in the region where these PDF+ projections reach their targets. (G–K) Wild-type brains stained for PDFR (green) and SLO (red), revealing a widely distributed punctate signal. Symbols depict the anatomical regions from which the single focal planes were taken. Each image corresponds to a different brain. (H and I) Magnified views of a region similar to that indicated in G, revealing colocalization of SLO and PDFR in some of the cell bodies in which the two are expressed. (J and K) Images display punctate-like signal along the midline and the main commissure, respectively. (L and M) The specificity of the pSLO antibody was assessed comparing the signal in the optic lobe (L) and the dorsal area (M) between CS and slo⁴. (N–P) PDFR signal in slo⁴ brains in the regions shown in H–J, respectively.

Fig. 3.

Molecular oscillations in pacemaker neurons are not affected in slo⁴. Newly eclosed CS and slo⁴ adult flies were synchronized, and samples were taken in DD3. Whole-mount brain immunohistochemistry was performed to follow TIM (red) or PER (green) and proPDF (white) accumulation at CT5, CT11, CT17, and CT23. A minimum of 12 brains per genotype were analyzed at each time point. (A) Representative confocal stacks focused on the small LNvs in wild-type flies (Left) and slo⁴ (Right). Time courses were analyzed blind and repeated three to five times with identical results. (B) Ratio between nuclear and cytoplasmic PER/TIM signal. Error bars represent the SE of the mean. No statistical differences were found between both genotypes (P > 0.05). See Materials and Methods for details on quantitation methods and statistics.

Fig. 4.

Dorsal clusters are out of phase in the absence of synchronizing signals. (A) (Right) Whole-mount brain immunocytochemistry on the DN1 region of CS and slo⁴ flies taken in DD3 at CT5, CT11, CT17, and CT23 stained for TIM (red), PER (green), and ELAV (data not shown). Results were consistent in the brains (n = 7–21) analyzed at each time point in 2–4 experiments. (Left) Representative images of low magnification at CT11 and CT23. (B) Quantitation of TIM distribution in DN1s. Black bars, nuclear (N); white bars, cytoplasmic (C); gray bars, nonlocalized (N + C) TIM signal. χ² analysis of the frequency distribution retrieved a significantly different distribution throughout the time course between CS and slo⁴ brains (P < 0.0001). Between 4–12 DN1 cells could be counted at each time point. Lack of TIM signal in CS flies at CT5 reflects that most brains showed no TIM, and those that did were dim enough to forfeit any attempt of localization. (C) Example of how localization was assessed: C, absence of colocalization with ELAV (white arrows); N, absolute colocalization of both signals (black arrows); N + C (or nonlocalized), cells where the signal could not be attributed to any single compartment (gray arrows).

Fig. 5.

Mutation in slo affects normal axonal arborization of pacemaker cells. (A) (Left) PDF secretion or accumulation is altered in slo⁴. Whole-mount brain immunohistochemistry of CS and slo⁴ flies stained for PDF. Brains <1 day old were fixed at the times indicated in the figure (CT samples were taken on DD2). Experiments were repeated at least three times, and a minimum of 50 brains were analyzed per time point. (Right) Magnified views of the PDF+ projections in the dorsal region. Box shows the area quantified in B. PI, pars intercerebralis; POT, posterior optic tract. (B) Quantitation of the average intensity of the LNvs dorsal projections. Error bars represent the SE of the mean. No circadian differences in PDF intensity in slo⁴ were found (P > 0.05). See Materials and Methods for details on quantitation methods and statistics. (C) (Upper) Evaluation of the structure of the PDF projections in pdfGFP and pdfGFP;slo⁴ flies stained for PDF (red) and GFP (green). (Lower) Higher magnification view of the dorsalmost segment.