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Timing of Locomotor Activity Circadian Rhythms in Caenorhabditis elegans

  • Sergio H. Simonetta,

    Affiliation Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes and National Research Council (CONICET), Buenos Aires, Argentina

  • María Laura Migliori,

    Affiliation Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes and National Research Council (CONICET), Buenos Aires, Argentina

  • Andrés Romanowski,

    Affiliation Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes and National Research Council (CONICET), Buenos Aires, Argentina

  • Diego A. Golombek

    dgolombek@unq.edu.ar

    Affiliation Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes and National Research Council (CONICET), Buenos Aires, Argentina

Timing of Locomotor Activity Circadian Rhythms in Caenorhabditis elegans

  • Sergio H. Simonetta, 
  • María Laura Migliori, 
  • Andrés Romanowski, 
  • Diego A. Golombek
PLOS
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Abstract

Circadian rhythms are driven by endogenous biological clocks and are synchronized to environmental cues. The chronobiological study of Caenorhabditis elegans, an extensively used animal model for developmental and genetic research, might provide fundamental information about the basis of circadian rhythmicity in eukaryotes, due to its ease of use and manipulations, as well as availability of genetic data and mutant strains. The aim of this study is to fully characterize the circadian rhythm of locomotor activity in C. elegans, as well as a means for genetic screening in this nematode and the identification of circadian mutants. We have developed an infrared method to measure locomotor activity in C. elegans and found that, under constant conditions, although inter-individual variability is present, circadian periodicity shows a population distribution of periods centered at 23.9±0.4 h and is temperature-compensated. Locomotor activity is entrainable by light-dark cycles and by low-amplitude temperature cycles, peaking around the night-day transition and day, respectively. In addition, lin-42(mg152) or lin-42(n1089) mutants (bearing a mutation in the lin-42 gene, homolog to the per gene) exhibit a significantly longer circadian period of 25.2±0.4 h or 25.6±0.5 h, respectively. Our results represent a complete description of the locomotor activity rhythm in C. elegans, with a methodology that allowed us to uncover three of the key features of circadian systems: entrainment, free-running and temperature compensation. In addition, abnormal circadian periods in clock mutants suggest a common molecular machinery responsible for circadian rhythmicity. Our analysis of circadian rhythmicity in C. elegans opens the possibility for further screening for circadian mutations in this species.

Introduction

Circadian rhythms of gene expression and behavior are ubiquitous in nature. They are generated by endogenous clocks that are entrained by environmental cues, such as light or temperature, and have been described in several animal models. Indeed, the use of the adequate model systems accelerates research and might facilitate the search for the genetic basis of behavior and disease. The molecular central pacemaker consists of a series of feedback loops that regulate the expression of specific clock genes (such as period or timeless), as well as post-translational events that finely tune the dynamics of the cycle. Diverse chronobiological models have contributed to the understanding of the properties of the circadian system and the molecular machinery of the biological clock, emphasizing common mechanisms in organisms as diverse as fungi, flies and mammals [1], [2].

In this sense, the chronobiological study of Caenorhabditis elegans, an extensively used animal model for developmental and genetic research, might provide fundamental information about the basis of circadian rhythmicity in eukaryotes, due to its ease of use and manipulations, as well as availability of genetic data and mutant strains. Interestingly, not much is known about circadian behaviors or putative clock genes in this nematode. In particular, rhythms in swimming behavior [3] and response to osmotic stress [4], [5] have been reported in L1 larvae and adults, as well as preliminary results in locomotor activity of adults [6], while several putative homologs to clock genes that have been characterized in other systems have also been proposed [7], [8]. However, several of these genes have been described as being involved in other regulatory mechanisms, such as developmental processes, including lin-42 (a period homolog) that is expressed during molting in several cells [9], or a timeless homolog which appears to be involved in chromosomal cohesion [10], [11]. The relationship of these genes to circadian rhythmicity is currently unknown.

On the other hand, ultradian rhythms (i.e., with a period shorter than 20 h, including cycles in the second-to-hours range) have been extensively described in C. elegans, specifically for defecation cycles [12][14]. Some of the genes which act in temperature compensation of this ultradian behavior might be responsible for the modulation of circadian behavior [15].

The finding of a robust circadian behavior in C. elegans, which could be recorded automatically [6], [16], would contribute a powerful tool for the study of the biological clock in this model, as well as a screening procedure for the elucidation of its molecular basis. The aim of this study is to fully characterize the circadian rhythm of locomotor activity in adult C. elegans, including free-running and entrained conditions, as well as a means for genetic screening in this nematode.

Results

As we have previously shown, locomotor activity of individual nematodes can be recorded with an infrared microbeam system [6]. In order to increase the number of nematodes to be recorded at the same time, we have modified the system adding a microcontroller to track simultaneously 48 nematodes per plate (figure 1a). This modification plus the optimization of culture conditions allowed us to track locomotor activity for more than 20 days (figure 1b). As a control of the system a slow-moving strain (clk-1 (qm30) mutant) was recorded, as well as the lethal effects of azide addition (average activity on day 3 of adult stage: N2 (wild type)  = 267 bins/day (SEM = 27, n = 42) ; clk-1 (qm30)  = 133 bins/day (SEM = 10, n = 47); N2+azide  = 0 bins/day (SEM = 0, n = 46)) . Our system is then capable of recording locomotor activity corresponding to the probability of the worm interrupting the light microbeam, which correlates with the activity pattern of the animal.

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Figure 1. Nematode tracking system.

A) Schematic diagram of the tracking system. From bottom to top: 1. IR emitter; 2. Microhole filter; 3. well plate where nematodes are held with 20 µl CeMM + FuDR 40 µM + AB 1x, 20 µl silicon oil (optional to avoid medium evaporation), tape; 4. Phototransistor. The light intensity output is converted to digital format and acquired by a microcontroller system. B) Whole-life recording of locomotor activity in wild-type (N2) and mutant clk-1(qm30) (slow moving strain) nematodes. The graph shows average activity of 48 nematodes. FuDR prevented the birth of larvae throughout the experiment (see inset picture).

https://doi.org/10.1371/journal.pone.0007571.g001

Locomotor rhythms are entrained by light

When entrained to light-dark cycles, the nematodes showed an activity pattern of 24 hours. However, we observed individual nematodes entrained at different phases with respect to the zeitgeber (external time cue); to deal with this problem we conducted population studies described with average plots and phase and frequency histograms.

Nematodes were synchronized to white light (400 lux) with a normal distribution of acrophases (peak time), exhibiting higher activity levels around the dark-to-light transition and a minimum at dusk; moreover, nematodes reentrained to the LD cycle after a 6-h shift in the photoperiod (figure 2a). Average activity patterns did not show a clear anticipation to the light or dark phase of the cycle, while overt locomotor behavior appeared to be dampened during the light phase (figure 2b). Since nematodes have been reported to be responsive to light in the 520 to 600 nm bandwidth (green/yellow light) [17], as well as to blue/ultraviolet wavelengths [18], [19], and with the intention to control any possible artifact due to changes in the incubator temperature, we also studied the effect of red light on circadian activity. While circadian rhythms were synchronized by white light – dark cycles (400:0 lux), red light - dark cycles were unable to synchronize activity (Figure S1), suggesting this is a specific effect of white light and is not due to artifactual effects of the stimulus, such as changes in the temperature of the cultures.

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Figure 2. Light synchronization.

A) Phase histogram of nematodes entrained to a light-dark cycle (12 h∶12 h 400 lux, 18°C), before and after a 6 h shift of the photoperiod. The middle panel shows the circular statistical analysis (Rayleigh test) for both situations (LD1: lights on at 0900 h; lights off at 2100 h; p<0.05, Rayleigh’s test, n = 80; LD2: lights on at 1500 h, lights off at 0300 h, p<0.05, Rayleigh’s test, n = 81) (the dotted circle inside the plot represents the significance threshold (p = 0.05), to compare with the resultant vector amplitude). The actogram on the right corresponds to average activity of the population. Gray shaded areas represent dark phases of the photoperiod. B) Average daily activity plot corresponding to 3 days of data from 20 nematodes synchronized to a 12 h∶12 h light-dark cycle. The analysis of 3 days of average activity indicates a significant difference between diurnal and nocturnal values (Maximum value  = 1.35±0.10 (10 h); Minimum value  = 0.68±0.09 (20 h); ANOVA test: p<0.0001). c) Comparison of locomotor activity acrophases between LD and the initial days under constant dark (DD) conditions, each triangle corresponds to the mean acrophase (cosinor analysis) of individual nematodes, calculated with the last 3 days of the pre-entrained condition (X axis) vs. the mean acrophase of the 3 first days of the constant dark condition (Y axis). Inset: Rayleigh test showing the phase angle of the population in entrainment (dark circles; r = 0.3 phi = 1.9 (7.3 h) n = 24) and the beginning of the constant condition post entrainment (white circles) (Rayleigh test: r = 0.3 phi = 2.0 (7.6 h) n = 24).

https://doi.org/10.1371/journal.pone.0007571.g002

When nematodes were released into continuous darkness after 5 days of preentrainment (LD white light 400:0lux), their initial phase in DD could be predicted by the previous phase under entrainment conditions (Figure 2c), suggesting the light cycle was indeed synchronizing the circadian rhythm of locomotion. The correlation between the phase of locomotor activity under LD and DD conditions indicates entrainment; if light were simply masking behavior, a random phase would be expected when animals were placed under constant darkness.

Locomotor activity rhythms are entrained by temperature cycles

We then considered the role of temperature in synchronizing C. elegans circadian rhythms. When nematodes were entrained to cycles of 17°C∶16°C (T∶t 12∶12 h) under constant dark conditions, we observed a normal distribution of acrophases peaking in the beginning / middle of the day, which was reentrained after a 6 h shift in the Tt cycle (considering “day” as the phase with higher temperature; figure 3a). Locomotor activity appeared to be increased during the high temperature phase (figure 3b). Moreover, under a larger temperature variation (20°C∶16°C T∶t 12∶12 h) the amplitude of rhythms w