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. 2013 Mar 1;36(3):385-95.
doi: 10.5665/sleep.2456.

The microarchitecture of C. elegans behavior during lethargus: homeostatic bout dynamics, a typical body posture, and regulation by a central neuron

Shachar Iwanir  1 , Nora Tramm, Stanislav Nagy, Charles Wright, Daniel Ish, David Biron
Affiliations

The microarchitecture of C. elegans behavior during lethargus: homeostatic bout dynamics, a typical body posture, and regulation by a central neuron

Shachar Iwanir et al. Sleep. .

Abstract

Study objectives: The nematode C. elegans develops through four larval stages before it reaches adulthood. At the transition between stages and before it sheds its cuticle, it exhibits a sleep-like behavior during a stage termed lethargus. The objectives of this study were to characterize in detail behavioral patterns and physiological activity of a command interneuron during lethargus.

Measurements and results: We found that lethargus behavior was composed of bouts of quiescence and motion. The duration of individual bouts ranged from 2 to 100 seconds, and their dynamics exhibited local homeostasis: the duration of bouts of quiescence positively correlated with the duration of bouts of motion that immediately preceded them in a cAMP-dependent manner. In addition, we identified a characteristic body posture during lethargus: the average curvature along the body of L4 lethargus larvae was lower than that of L4 larvae prior to lethargus, and the positions of body bends were distributed non-uniformly along the bodies of quiescent animals. Finally, we found that the AVA interneurons, a pair of backward command neurons, mediated locomotion patterns during L4 lethargus in similar fashion to their function in L4 larvae prior to lethargus. Interestingly, in both developmental stages backward locomotion was initiated and terminated asymmetrically with respect to AVA intraneuronal calcium concentration.

Conclusions: The complex behavioral patterns during lethargus can be dissected to quantifiable elements, which exhibit rich temporal dynamics and are actively regulated by the nervous system. Our findings support the identification of lethargus as a sleep-like state.

Citation: Iwanir S; Tramm N; Nagy S; Wright C; Ish D; Biron D. The microarchitecture of C. elegans behavior during lethargus: homeostatic bout dynamics, a typical body posture, and regulation by a central neuron. SLEEP 2013;36(3):385-395.

Keywords: Behavior; C. elegans; homeostasis; lethargus.

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Figures

Figure 1
Figure 1
(A) The mean fraction of quiescence (red) and overall motion (blue) calculated by subtracting consecutive frames (Δt = 500ms) and smoothing using a 10-min sliding window. N = 17 animals. Error lines indicate ± SEM. (B) A visualization of the level of motion of a single animal in an “artificial dirt” chamber, quantified as the ratio between the area where pixel values changed during an interval of 2 sec and the area of the body of the animal. Top: a late L4int larva (about 30 min prior to L4leth). Bottom: the same animal during early L4leth. During L4leth the duration of quiescence bouts (QBs, red valleys) and the motion bouts (MBs, blue peaks) ranges between 1-100 sec. (C) Typical postures associated with the L4int stage (top) and with a QB during the L4leth stage. (D-E) The microarchitecture of quiescence and motion of a single hermaphrodite during L4leth. Black curve: the fraction of quiescence (black) smoothed using a 10-min sliding window. Vertical lines: the durations of individual QBs (D, red) or MB (E, blue) positioned at their respective time of occurrence.
Figure 2
Figure 2
(A) The mean durations of QBs exhibited by hermaphrodite wild-type animals during 10 equally spaced intervals that spanned the L4leth stage. The mean value of bout durations was calculated for each animal separately, and the single animal means were then averaged. Dashed line: the mean durations of intervals 2-10 were fitted to a sigmoidal (hyperbolic tangent) curve with an exponentially decaying tail (see methods). The resulting decay constant was τ = 0.19 ± 0.08, corresponding to 33.8 ± 13.9 minutes. Inset: the sigmoidal fit was compared to a linear fit and was found to better explain the data (P < 2×10-4). N = 51 animals. Error bars indicate ± SEM. The number of QBs that were averaged in each bar was 533, 845, 972, 1,042, 1,214, 1,392, 1,525, 1,399, 1,023, and 721, respectively. (B) The average duration of MBs (calculated similarly to the QB means) during L4leth. The mean duration of MBs exceeded 100 sec during the final 10 percentile of L4leth. N = 51 animals (the number of bouts that were averaged is shown within each bar). Error bars indicate ± SEM. The number of MBs that were averaged in each bar was 516, 830, 971, 1,049, 1,200, 1,413, 1,526, 1,403, 1,129, and 699, respectively. (C) The coefficients of variance (standard deviation divided by the mean) corresponding to the QBs depicted in (A). N = 51 animals. Error bars indicate ± SEM.
Figure 3
Figure 3
(A) In order to graphically visualize the positive correlation between durations of MB/QB pairs, MBs were grouped according to their duration (with a resolution of 1 sec). For every group of MBs with a given duration, the mean duration of their respective subsequent QBs was plotted. A linear regression between MB/QB pairs during the 10-50th percentile of L4leth (slope of 0.56 ± 0.05, P < 10-6) was overlaid on the plot as a guide to the eye: the thick dashed line represents the linear regression line and the thin dashed lines represent the 95% confidence interval. The slope of the linear fit was found to be significantly different from zero within the 95% confidence interval. N = 3,953 bouts from 51 animals. (B) Same as (A) for consecutive MBs that were separated by a single QB during the 10-50th percentiles of L4leth. Regression performed on individual pairs of duration yielded a slope of -0.09 ± 0.04, P < 10-6 and R = -0.09. N = 3,902 bouts from 51 animals.
Figure 4
Figure 4
(A) The mean fraction of quiescence and overall motion of acy-1(ce2) mutants and wild-type controls, calculated as in Figure 1A. Nwild-type = 35, Nacy-1(ce2) = 30. Error lines indicate ± SEM. (B-C) A graphical representation of the correlations between MB/QB pairs of acy-1(ce2) mutants and a control dataset of wild-type animals. The correlations were calculated as in Figure 3A, i.e., for the 10-50th percentiles of L4leth. Statistically significant correlations were detected with the wild-type animals (R = 0.32, P < 0.001, Nwild-type = 4,072 bouts from 37 animals), but the R-value of the acy-1(ce2) mutants was not significantly different from zero (Nacy-1(ce2) = 2,738 bouts from 32 animals).
Figure 5
Figure 5
(A) The mean total bend, summed over all sharp body bends of hermaphrodites. All pairs of categories were compared: the categories “L4int” and “dwelling during L4int” were indistinguishable. All comparisons denoted by dashed lines and ** were distinct (P < 0.01). N = 42. Error bars indicate ± SEM. (B-C) The positions of large bends along the body of L4int larvae (B) and during QBs of L4leth larvae (C). Quiescent L4leth larvae exhibited large bends preferentially in the anterior half of the body. The two distributions of positions of body bends were found to be statistically distinct (P < 10-6). N = 42 animals. (D) The total bend, summed over all sharp body-bends of hermaphrodites, and then averaged using a continuous 10-min sliding window. L4leth starts at t = 0 and ends approximately at t = 180 min. N = 42 animals. Error bars indicate ± SEM.
Figure 6
Figure 6
(A) A typical trace of AVA::GCaMP3 fluorescence before (t < 0, white background) and during (t > 0, gray background) L4leth. Physiological activity in AVA was correlated with backward locomotion in awake and lethargic animals. Scale bar represents a 50% deviation from the average baseline fluorescence. The colors superposed on the calcium traces denote the locomotion behavior, which was independently manually scored (see methods). (B) An example of AVA::GCaMP3 fluorescence during a backward-rich epoch during L4int. Backward locomotion (blue) correlated with secondary peaks in the calcium transients. (C) The percentage of time spent in forward locomotion (green), backward locomotion (blue), dwelling (black) and quiescence (red) during late L4int and early L4leth. The L4leth stage (gray shading) starts at time t = 0. The fraction of time spent in forward locomotion was 3-fold higher than the fraction of time spent in backward locomotion during late L4int but not during early L4leth. Comparisons denoted by ** indicate significant differences (P < 0.01). N = 6 animals. Error bars indicate ± SEM.
Figure 7
Figure 7
(A) A 10-min interval of AVA::GCaMP3 fluorescence. Blue segments indicated backward locomotion (see methods). (B) The linear combination of fluorescence level and fluorescence slope that was found to be the optimal predictor of backward locomotion. Intervals of backward locomotion are generally centered on peaks of the predictor, but not the original trace. (C-D) Transition-triggered averages of AVA::GCaMP3 fluorescence at the initiation and the termination of backward locomotion of an L4int larvae. Animals were moving backward in the gray areas, i.e., t > 0 for (C) and t < 0 for (D). When the traces were aligned at the initiation of backward locomotion, they typically exhibited a rise from baseline levels at the time of the initiation of the motion. In contrast, when the traces were aligned at the termination of backward locomotion, they typically exhibited a decrease in fluorescence from a higher-than-baseline level. (E-F) the same as (C-D) for L4leth larvae. N = 7 animals, 40-90 min of imaging per animal, 40-60 backward locomotion intervals per animal were scored. Shaded error bars indicate ± SEM.
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Comment in

  • Do C. elegans sleep? A closer look.
    Singh K, Huang H, Hart AC. Singh K, et al. Sleep. 2013 Mar 1;36(3):307-8. doi: 10.5665/sleep.2436. Sleep. 2013. PMID: 23450901 Free PMC article. No abstract available.

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