Discriminating between sleep and exercise-induced fatigue using computer vision and behavioral genetics

Abstract

Following prolonged swimming, Caenorhabditis elegans cycle between active swimming bouts and inactive quiescent bouts. Swimming is exercise for C. elegans and here we suggest that inactive bouts are a recovery state akin to fatigue. It is known that cGMP-dependent kinase (PKG) activity plays a conserved role in sleep, rest, and arousal. Using C. elegans EGL-4 PKG, we first validate a novel learning-based computer vision approach to automatically analyze C. elegans locomotory behavior and an edge detection program that is able to distinguish between activity and inactivity during swimming for long periods of time. We find that C. elegans EGL-4 PKG function impacts timing of exercise-induced quiescent (EIQ) bout onset, fractional quiescence, bout number, and bout duration, suggesting that previously described pathways are engaged during EIQ bouts. However, EIQ bouts are likely not sleep as animals are feeding during the majority of EIQ bouts. We find that genetic perturbation of neurons required for other C. elegans sleep states also does not alter EIQ dynamics. Additionally, we find that EIQ onset is sensitive to age and DAF-16 FOXO function. In summary, we have validated behavioral analysis software that enables a quantitative and detailed assessment of swimming behavior, including EIQ. We found novel EIQ defects in aged animals and animals with mutations in a gene involved in stress tolerance. We anticipate that further use of this software will facilitate the analysis of genes and pathways critical for fatigue and other C. elegans behaviors.

Keywords: C. elegans; Fatigue; computer vision; locomotion; quiescence; swimming.

Figure 1:

Visual representations of poseEIQ (A) and edgeEIQ (B). (A) poseEIQ uses a shape-consistent flexible mixture of parts model to track C. elegans locomotion. Image adapted from Guo et al. (2018). (B) edgeEIQ compares edge overlap between consecutive frames to determine activity level.

Figure 2:

Locomotion analysis of egl-4 mutant animals during prolonged swimming. Evaluation of parameters (A) Wave Initiation Rate (B) Activity Index (C) Curling (D) Stretch (E) Attenuation (F) Brush Stroke (G) Body Wave Number (H) Asymmetry; each indicative of different aspects of locomotion over two windows of 30 minutes each. For a detailed explanation of these parameters, please refer to Restif et al. (2014). The ‘early’ time point is at the very beginning of the 6-hour long behavioral assay while the ‘late’ time point is approximately at the 4 hour mark (228.25 ± 4.28 minutes). The choice of these time points was motivated by the ethograms of Figure 3A. Behavioral parameters for 12 animals from each of the three genotypes: wild type (WT, black), egl-4(n477lf) (egl-4(lf), red), and egl-4(ad450gf) (egl-4(gf), blue) are shown here. The within-group mean temporal course of each parameter is shown as insets in the respective panel, with the early time point on the top left and the late time point on the top right. The average parameter value (over the 30 minute window) for each individual animal is shown in the respective panel; corresponding early/late points are connected by dashed/straight lines. Activity index and brush stroke were normalized to body size (calculated in pixels). Statistical analysis available in Table 1.

Figure 3:

Analysis of exercise-induced quiescence in egl-4 mutant animals. 24 animals per genotype. (A) Left panel: Ethograms generated using an unsupervised Hidden Markov Model for wild type, egl-4(n477lf), and egl-4(ad450gf) animals. Each row represents the latent states of a single animal over the course of the 6 hour experiment. Right panel: Ethograms constructed from manually thresholded binary activity data. Filled (or empty) regions can be interpreted as an ‘active’ (or ‘inactive’) state, respectively. (B) On average, the egl-4(n477lf) animals showed decreased fractional quiescence (fraction of each hour spent in quiescent bouts) compared to wild type animals during hours 4 through 6. egl-4(ad450gf) animals showed increased average fractional quiescence at all time points compared to wild type animals. (C) egl-4(n477lf) animals showed decreased average number of bouts (per hour) in hours 3 through 6, compared to wild type animals. egl-4(ad450gf) animals showed increased average number of bouts for hours 1 and 2, and decreased average number of bouts in hours 4 through 6, compared to wild type. (D) Average duration of quiescent bouts did not differ between wild type and the egl-4(n477lf) animals. egl-4(ad450gf) animals showed increased average bout duration during hours 3, 4, and 6. Animals from three independent biological replicates (3 different days). 2-way ANOVA and Dunnett’s multiple comparisons test. (E) egl-4(ad450gf) animals initiated quiescent bouts earlier than wild type animals, while egl-4(n477lf) animals were not different. Kruskal-Wallis test and Dunn’s multiple comparisons test. Error bars indicate ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 4:

Exercise-induced quiescent bouts are not sleep. (A) Behavior of wild type animals during quiescent bouts was classified as ‘pumping’ (exhibited pharyngeal pumping throughout a quiescent bout), ‘mixed’ (began a quiescent bout without pumping, and resumed pumping midway through the bout), and ‘not pumping’ (no pharyngeal pumping). Left panel: The majority of quiescent bouts were classified as pumping, regardless of when they occurred. Right panel: When classified based on bout duration, most bouts lasting 3 minutes or less were classified as not pumping, while bouts longer than 4 minutes were usually classified as mixed bouts. 199 total quiescent bouts classified drawn from 5 animals. Loss of function aptf-1(gk794lf) and aptf-1(tm3287lf) animals did not differ from wild type in fractional quiescence (A), bout number (B), or bout duration (C), with the exception of fractional quiescence (B) and bout duration (B) of aptf-1(tm3287lf) animals at hour 5. 2-way ANOVA and Dunnett’s multiple comparisons test. (E) Average time to first bout was slightly sooner in aptf-1(tm3287lf), but not aptf-1(gk794lf), animals versus wild type. Kruskal-Wallis test and Dunn’s multiple comparisons test. n = 24 per genotype. Loss of function ceh-17(np1lf) showed no difference in fractional quiescence (F), bout number (G), and bout duration (H), with the exception of increased fractional quiescence at hour 5 (F). 2-way ANOVA and Dunnett’s multiple comparisons test. (I) No difference in time to first bout was observed between ceh-17(np1lf) animals and wild type. Kruskal-Wallis test and Dunn’s multiple comparisons test. n = 24 per genotype. Error bars indicate ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 5:

Analysis of exercise-induced quiescence in daf-16 mutant animals. (A) Loss of function daf-16(mgDf50lf) animals showed increased average fractional quiescence, compared to wild type at hours 3, 4, and 6. This difference was not repeated in daf-16(mu86lf) animals. (B) daf-16(mu86lf) animals showed increased average number of bouts per hour, compared to wild type in hour 4, and daf-16(mgDf50lf) animals showed an increase during hours 3 and 4. (C) daf-16(mgDf50lf) animals showed increased average bout duration, compared to wild type during hours 3 and 4, while daf-16(mu86lf) animals showed no difference. 2-way ANOVA and Dunnett’s multiple comparisons test. (D) The daf-16(mgDf50lf) and daf-16(mu86lf) animals both showed decreased average time to first bout, compared to wild type. Kruskal-Wallis test and Dunn’s multiple comparisons test. n = 32 per genotype. Error bars indicate ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 6:

Analysis of exercise-induced quiescence in aged animals. Wild type animals were aged 1, 2, 3, 4, 7, and 10 days into adulthood. (A) Day 3 adult animals showed increased average fractional quiescence, compared to day 1 adult animals at hour 1. Compared to day 1 adult animals, day 7 adult animals showed increased average fractional quiescence at all timepoints, while day 10 animals showed increased average fractional quiescence at hours 2 through 5. (B) An increase in average number of bouts per hour, compared to day 1 adult animals, was observed at day 7 adult animals during hours 2, 3, and 6. Day 10 adult animals also showed increased average number of bouts (per hour) during hours 1, 2, 3, 5, and 6, compared to day 1 adult animals. (C) No difference in average bout duration was found amongst the different age groups, with the exception of increased average bout duration in day 3 adult animals at hour 1 and at day 7 adult animals at hours 1 and 3. 2-way ANOVA and Dunnett’s multiple comparisons test. (D) Day 3, 4, 7, and 10 adult animals all showed decreased average time to first bout, compared to day 1 adult animals. Kruskal-Wallis test and Dunn’s multiple comparisons test. n = 36 per group. Error bars indicate ± S.E.M. * P < 0.05; ** P < 0.01; *** P < 0.001.