Functional analysis of circadian pacemaker neurons in Drosophila melanogaster

Abstract

The molecular mechanisms of circadian rhythms are well known, but how multiple clocks within one organism generate a structured rhythmic output remains a mystery. Many animals show bimodal activity rhythms with morning (M) and evening (E) activity bouts. One long-standing model assumes that two mutually coupled oscillators underlie these bouts and show different sensitivities to light. Three groups of lateral neurons (LN) and three groups of dorsal neurons govern behavioral rhythmicity of Drosophila. Recent data suggest that two groups of the LN (the ventral subset of the small LN cells and the dorsal subset of LN cells) are plausible candidates for the M and E oscillator, respectively. We provide evidence that these neuronal groups respond differently to light and can be completely desynchronized from one another by constant light, leading to two activity components that free-run with different periods. As expected, a long-period component started from the E activity bout. However, a short-period component originated not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as "main" oscillator controlling M and E activity bouts in Drosophila.

Figure 1.

Circadian pacemaker neurons in the brain of the fruit fly. The three clusters of dorsal neurons (DN₁, DN₂, and DN₃ cells) are shown in blue colors, the three clusters of lateral neurons are shown in red (PDF-positive s-LN_v and l-LN_v cells) or orange (LN_d cells), and the PDF-negative 5th s-LN_v cell is depicted in green. The square in the right brain hemisphere indicates the area of the sections shown in Figures 4A–D, 6, and 7.

Figure 2.

Constant dim light induces arrhythmicity and/or desynchronization in wild-type flies. Actograms of four wild-type flies (A–D) and cli^eya;cry^b mutants (E) are shown that were recorded under an LD cycle and subsequently under LL conditions. The light program is indicated as white and black bars on top. The actograms are shown as double plot to better see the activity pattern under LL. During LD, light intensity was 500 μW/cm² for all flies; during LL, light intensity was kept at 500 μW/cm² for the cli^eya;cry^b mutant (E) and the wild-type fly (A); for the other wild-type flies, light intensity was reduced to 0.25 μW/cm². Under 500 μW/cm² light, the wild-type fly became arrhythmic immediately after transfer into LL (A), whereas the cli^eya;cry^b mutant remained rhythmic (E). At 0.25 μW/cm², the wild-type flies showed internal desynchronization into two free-running components, one with a longer period and the other with a shorter period (see Table 1). Long and short components stayed together after the first cross-over in the fly shown in B, whereas they showed full cross-over for two times in the fly shown in C. In B and D, the short-period component appears to originate from the morning peak and the long-period component from the evening peak.

Figure 3.

Internal desynchronization in cry^b mutants. Actogram of a typical cry^b fly recorded for 7 d under an LD and for an additional 20 d under LL. The light program is indicated as white and black bars on top. Light intensity was 500 μW/cm². The actogram is shown as double plot and reveals two free-running rhythm components, one with a short period (τ of ∼22 h) and the other with a long period (τ of ∼25 h). On day 1 in LL (d1), both rhythms were in-phase and the average ± SE activity profile calculated from all 40 flies showed one single activity bout (top right). On day 5 in LL (d5), the long and short components were 12 h out-of-phase, with two activity bouts visible in the average activity profile (middle right). On day 11, the two components crossed and therefore appear as a single peak in the average activity profile (bottom right). The flies were fixed on day 1 and day 5 at the indicated times (time points a and b, gray arrows on day 1; time points c and d, open arrows on day 5).

Figure 4.

PER, TIM, and PDF expression in the lateral neurons of wild-type flies. For clarity, single labeling is shown in gray, and double labeling is shown in color. A–C, E–G, H–K, and L–O show the same optical sections, respectively. Brains were stained 1 h before lights on in an LD cycle. At this time point, PER and TIM were predominantly nuclear in all lateral neurons: the LN_d (E–G), the l-LN_v (H–K), the 5th s-LN_v (arrowhead in A–D, H–K), and the s-LN_v (L–O). The strongest PER and TIM labeling was found close to the nuclear membrane, whereby TIM was always located slightly lateral of PER (see arrows in G and O). PDF was exclusively found in the cytoplasm of the l-LN_v (J) and s-LN_v (N), as well as in their arborizations (e.g., the network on the surface of the medulla in D). Note that the nuclei of all groups of lateral neurons were more or less of similar size, but the cytoplasmic area was larger in the l-LN_v. The 5th PDF-negative s-LN_v (arrowhead) could be distinguished from the other cells because it lacked PDF. It was located among the l-LN_v (A–D, H–K). The LN_d were usually grouped around the anterior optic tract (ant opt tr; E–G) at the place in which it enters the central brain. One cell of the LN_d was usually more prominently stained than the others and appeared slightly larger (double arrowhead, A–C, E–G). In the brain shown in D, the LN_d were displaced dorsally toward the DN₃ (not in the focal plane). Furthermore, two l-LN_v and three s-LN_v cells (2 of the latter are below the displaced l-LN_v and are hard to see) were displaced toward the original location of the LN_d. In A–C, one l-LN_v was displaced toward the LN_d. Note that the extra LN_d (double arrowhead), the 5th s-LN_v (arrowhead), and the PDF-positive s-LN_v are the cells that are most prominently stained by anti-PER in A. In A–D, 10 confocal sections of 5 μm were combined; E–O consist of five confocal sections of 1 μm. A–D and E–O are of the same magnification, respectively. Scale bars, 20 μm.

Figure 5.

The dorsal neurons of cry^b flies stained with anti-PER (green), anti-TIM (red), and anti-PDF (blue) 1 h before lights on in an LD cycle (A–C) and on the fifth day of LL at time point c (D, E). One hour before lights on (A–C), PER and TIM were nuclear in most dorsal neurons (the DN₁, DN₂, and DN₃ cells) except for a few DN₃ cells (arrow in A). The DN₁ consisted of up to 17 cells that showed a different intensity of PER and TIM labeling; half of the cells were strongly labeled, and the other half were only weakly labeled (B, C). The two DN₂ cells were always close to the terminals of the s-LN_v (blue), and they showed rather weak TIM labeling. On the fifth day of LL, TIM labeling was very weak in all dorsal neurons and was restricted to the cytoplasm (D, E). PER labeling was nuclear in some DN₁ cells and cytoplasmic in others. Note that nuclear PER labeling was found in two DN₁ cells that showed no TIM labeling at all (arrowheads in D). A consists of 12 confocal sections (5 μm). B and C are composed of five confocal sections (2 μm) and are of the same magnification. Scale bars, 20 μm.

Figure 6.

Lateral neurons of internally synchronized cry^b flies stained on day 1 of LL with anti-PER, anti-TIM, and anti-PDF at the time points of low and high locomotor activity. Single labeling is shown in gray, and double labeling is shown in color. Top, PDF (blue) is used as marker for the four s-LN_v and all l-LN_v cells plus their neurites. At time point a (activity trough, see Fig. 3), strong PER and TIM staining was found in the nuclei of all neurons, except the l-LN_v cells, in which TIM is predominantly cytoplasmic. At time point b (activity peak, see Fig. 3), PER and TIM staining was weaker in most cells, and both proteins were in the cytoplasm. In the l-LN_v cells, PER remained nuclear and TIM was found in the cytoplasm, as was the case in time point a. Double arrowheads point to the extra LN_d that showed the highest staining index among the LN_d at time point a and appears to have a larger cytoplasmic area than the other LN_d cells, as visible at time point b. Scale bar, 20 μm. Bottom, Quantification of the staining intensity for PER and TIM in all neurons at the time points a (gray columns) and b (black columns). Localization of proteins: n, nuclear; c, cytoplasmic; n + c, nuclear in some cells and cytoplasmic in other cells. Error bars represent SEs, and asterisks indicate significant differences in staining index between the two time points (p < 0.001).

Figure 7.

Lateral neurons of internally desynchronized cry^b flies stained on day 5 of LL with anti-PER, anti-TIM, and anti-PDF at the activity trough points of the long-period (time point c) and the short-period (time point d) components (labeling as in Fig. 6). Top, At time point c, strong PER and TIM staining was found in the nuclei of the LN_d cells (especially the extra LN_d cell; double arrowhead) and the PDF-negative 5th s-LN_v cell. Note that the LN_d cells are differently stained for PER and TIM (details in Results). At time point d, strong PER and TIM staining was found in the nuclei of the PDF-positive s-LN_v cells. The l-LN_v cells showed nuclear PER and cytoplasmic TIM at both time points (compare with Fig. 6). Bottom, Quantification of the staining intensity for PER and TIM at the time points c and d. Note that staining indices for TIM are rather low, and the protein is mainly cytoplasmic in all three DN groups. Error bars indicate SE.

Figure 8.

Quantification of the staining intensity for PER and TIM in eyeless cli^eya;cry^b flies on the fifth day in LL at the times of high and low activity (labeling as in Fig. 6). Note that all neurons that show significant differences (asterisks) in staining intensity appear to cycle in-phase. Error bars indicate SE.

Figure 9.

Actograms of cry^b flies showing a short-period component originating from the M activity bout. The fly shown in A was kept in the usual LD cycle for the first 7 d and then transferred to LL; the other three flies (B–D) received dim light during the night and thus had a light/moonlight cycle (LM) (night, gray bars). A, A second short-period rhythm appears on day 8 in LL that could be extrapolated back to the morning activity bout (arrow). From day 10 onward in LL, the fly free-ran with the short period. B–D, The morning activity bout started to free-run with a short period already during the LM cycle (filled arrowhead). At the same time, a short-period component also detached from the E activity bout (open arrowhead). In LL, the short-period components originating from the M and E activity bouts continued to free-run, indicating that the neurons free-running with short period under LL control M and E activity bouts simultaneously.