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Circadian remodeling of neuronal circuits involved in rhythmic behavior - PubMed

  • ️Tue Jan 01 2008

Circadian remodeling of neuronal circuits involved in rhythmic behavior

María Paz Fernández et al. PLoS Biol. 2008.

Abstract

Clock output pathways are central to convey timing information from the circadian clock to a diversity of physiological systems, ranging from cell-autonomous processes to behavior. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is unclear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the time of day information from core pacemaker cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Here we describe a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit, which display higher complexity during the day and significantly lower complexity at nighttime, both under daily cycles and constant conditions. In support to its circadian nature, cycling is lost in bona fide clockless mutants. We propose this clock-controlled structural plasticity as a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Daily Reorganization in the PDF Terminals at the Dorsal Protocerebrum

(A–F) pdf >mCD8-GFP wild-type brains were dissected at ZT2 and ZT14, that is 2 h after lights ON and light OFF, respectively. Brains were stained with anti-GFP (green) and anti-PDF (red) antibodies. sLNvs and lLNvs stand for small and large lateral neurons ventral. (A) Low magnification view comprising a brain hemisphere. The region subject to analysis is shown in the inset. (B) Representative confocal images taken during the early day and early night, underscoring the striking reorganization taking place within this area. (C) HRP staining on pdf >mCD8-GFP brains to qualitative confirm under DIC the anatomical properties of the daytime and nighttime conformation as seen by confocal microscopy. (D) Schematic diagram depicting how the quantitation of the complexity of the PDF axonal arbor on confocal images was carried out. (E) The total number of intersections between the concentric rings and the axonal projections was significantly different in wild-type brains dissected during the early day and early night (*** represent p < 0.0001, non parametric Mann-Whitney test). Immunohistochemistry was performed at least three times, each timepoint represents a minimum of 40 brains; quantitation was performed blind. (F) The complexity of the axonal arbors is consistently lower in the nighttime conformation. (G) Expression of the presynaptic marker synaptobrevin-GFP (pdf >syb-GFP) shows a differential arrangement of synaptic vesicles at these two timepoints. Flies were dissected at ZT2 and ZT14.

Figure 2
Figure 2. Changes in the Degree of Arborization of the PDF Circuit Are Preserved Under Constant Conditions

(A–C) pdf >mCD8-GFP wild-type brains were dissected at CT2 and CT14 during the second day in constant darkness (DD2). CT refers to the time that has passed from the last lights OFF/ON transition. CT2 and CT14 indicate subjective day and night, respectively. (A) Confocal images representative of the subjective day (upper) and subjective night (bottom panels) stained with anti-PDF (red) and anti-GFP (green) antibodies. (B) The number of intersections is consistently lower during subjective night, although the PDF circuit is not particularly shortened. (C) Total axonal crosses are significantly different during subjective day and night, and the difference persists in older flies. Immunohistochemistry was performed at least three times for each group. Each timepoint represents a minimum of 60 brains; quantitation was performed blind. *** represents p < 0.0001 and ** represents p < 0.0005, non parametric Mann-Whitney test.

Figure 3
Figure 3. Structural Plasticity Is Under the Control of Clock Genes

(A–C) The pdf >mCD8-GFP reporter was crossed into per01 (top panel) and tim01 (bottom panel) clockless mutants. Brains were dissected at ZT2 and ZT14 as explained in the legend to Figure 1. (A) Left: Low magnification view of a brain hemisphere in per01 or tim01 flies. Note the degree of defasciculation in tim01 axon tract towards the dorsal protocerebrum. Right: Representative confocal images taken during the early day and early night, highlighting the differential effect of these mutations on the structure of the circuit. Immunohistochemistry was performed twice, each timepoint represents 15–21 brains; quantitation was performed blind. (B) No significant differences between the daytime and nighttime configuration were observed (p > 0.5, non parametric Mann-Whitney test). (C) per01 flies display a less branched circuit of relatively normal length at all times; on the other hand, tim01 flies display an over branching phenotype, characteristically shorter than wild-type brains.

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