Long-term imaging of circadian locomotor rhythms of a freely crawling C. elegans population

Abstract

Background Locomotor activity is used extensively as a behavioral output to study the underpinnings of circadian rhythms. Recent studies have required a populational approach for the study of circadian rhythmicity in Caenorhabditis elegans locomotion. New method We describe an imaging system for long-term automated recording and analysis of locomotion data of multiple free-crawling C. elegans animals on the surface of an agar plate. We devised image analysis tools for measuring specific features related to movement and shape to identify circadian patterns. Results We demonstrate the utility of our system by quantifying circadian locomotor rhythms in wild-type and mutant animals induced by temperature cycles. We show that 13 °C:18 °C (12:12 h) cycles are sufficient to entrain locomotor activity of wild-type animals, which persist but are rapidly damped during 13 °C free-running conditions. Animals with mutations in tax-2, a cyclic nucleotide-gated (CNG) ion channel, significantly reduce locomotor activity during entrainment and free-running. Comparison with existing method(s) Current methods for measuring circadian locomotor activity is generally restricted to recording individual swimming animals of C. elegans, which is a distinct form of locomotion from crawling behavior generally observed in the laboratory. Our system works well with up to 20 crawling adult animals, and allows for a detailed analysis of locomotor activity over long periods of time. Conclusions Our population-based approach provides a powerful tool for quantification of circadian rhythmicity of C. elegans locomotion, and could allow for a screening system of candidate circadian genes in this model organism.

A salt-induced kinase (SIK) is required for the metabolic regulation of sleep

“…The bacteria formed a circle of 0.6 cm diameter in the middle of the plate. Movies were taken on a custom-built imaging system and worm tracking software (Volumetry, version 8.a) [111] using a USB 2.0 monochrome machine vision cameras (Point Grey Research, CMLN-13SM-CS) equipped with a 12.5 mm focal length C-mount lenses (Fujinon, HF12.5HA-1B). All imaging was performed under dark field illumination using low angle red-light LED rings as a light source, such that worms appeared as white objects against a dark background.…”

Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter

“…Similar results regarding tax-2 involvement were described by van der Linden et al (19), who reported that a mutation in TAX-2 abolished the circadian expression of different transcripts entrained under either LD or WC conditions, indicating that TAX-2 is required for the transduction of both zeitgebers. In addition, tax-2 also was recently shown to be required for the transduction of thermal cues to the C. elegans circadian clock in locomotor activity rhythms (12). However, no evident role for lite-1 in light-entrained transcripts has been found previously (19), and to our knowledge there are no studies linking gur-3 to the input pathways of the clock.…”

“…Therefore our luciferase-based approach, which allows the recording of singlenematode molecular rhythms in real time and in intact animals, is a more sensitive and reliable method for revealing transcriptional rhythms. This fact is particularly relevant when working with C. elegans, in which circadian outputs do not appear to be particularly robust and there is a marked variability among nematodes (10)(11)(12)52).…”

“…However, the study of circadian rhythms in this model is far from complete, and the mechanism underlying the molecular basis of the central pacemaker remains elusive. Numerous circadian cycles have been described in the nematode at different levels, including behavior (locomotor activity) (10)(11)(12), physiology (defecation rate, pharyngeal pumping rate, and olfaction) (13,14), metabolism (abiotic and biotic stress tolerance, food and oxygen consumption) (15,16), protein activity and regulation (14,17,18), and gene expression (19). Recent exhaustive bioinformatics searches have identified proteins of relatively high homology to clock components from other species (20).…”

Increased food intake after starvation enhances sleep in Drosophila melanogaster

“…About 8 flies were individually placed in DAMS activity tubes, and their movement was recorded and video captured. Analysis of activity was performed using custom-written image analysis software within the Volumetry application environment as previously described (Winbush et al, 2015). Briefly, movies were background subtracted leaving only individual flies; an intensity threshold was then applied to adequately outline each fly.…”