Studying circadian rhythms in Drosophila melanogaster

Abstract

Circadian rhythms have a profound influence on most bodily functions: from metabolism to complex behaviors. They ensure that all these biological processes are optimized with the time-of-day. They are generated by endogenous molecular oscillators that have a period that closely, but not exactly, matches day length. These molecular clocks are synchronized by environmental cycles such as light intensity and temperature. Drosophila melanogaster has been a model organism of choice to understand genetically, molecularly and at the level of neural circuits how circadian rhythms are generated, how they are synchronized by environmental cues, and how they drive behavioral cycles such as locomotor rhythms. This review will cover a wide range of techniques that have been instrumental to our understanding of Drosophila circadian rhythms, and that are essential for current and future research.

Keywords: Behavior; Circadian rhythms; Clock neurons; Drosophila.

Figure 1. The transcriptional feedback loop of the Drosophila circadian clock

CLK/CYC drive expression of their own repressors PER and TIM. PER/TIM go through various modifications during the day, until they are eventually turned over to release CLK/CYC from repression, starting the next cycle.

Figure 2. Locomotor activity monitoring

(A) Fly locomotion is detected when a fly breaks an infra-red beam crossing the small glass tube in which it is housed (B) Double-plotted actogram showing the activity of flies entrained to a 12/12-hr LD cycle and then released in constant darkness for period determination. Each day is plotted twice, first on the right and duplicated on the left half of the next line, except for the first day. Note the progressive drift of circadian behavior in constant conditions in per mutant flies, corresponding to long and short periods (C) Eduction plot of fly activity after entrainment to an LD cycle. The Morning (M) and Evening (E) anticipatory behavior driven by the circadian clock are shown with arrows.

Figure 3. Analyzing phase-shifts

(A) Flies are entrained to an LD cycle and pulsed with a brief light pulse during the early or late night (white arrow). The phase in pulsed flies is compared to non-pulsed controls (Δϕ) (B) A Phase-response curve (PRC) can be generated by plotting phase delays (−) and phase advances (+) as a function of time of light pulse (or any other stimuli).

Figure 4. Luciferase recording

Individual flies are housed in wells of a multiplate containing food with luciferin. The light generated by the luciferase activity is recorded over several days with a photomultiplier-based device. The data is plotted to show the phase, amplitude and period length of the rhythm.

Figure 5. Targeting circadian neurons

(A) The circadian neurons of Drosophila and their best-known functions. Projection from the s-LNvs are shown in green. (B) Important driver and repressor transgenes and their expression patterns in circadian neurons. References are indicated in brackets.