Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health inC. elegans

Abstract

Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematode C. elegans. In this study, we report a new assay format for engaging C. elegans in burrowing that enables rapid assessment of nematode neuromuscular health. In contrast to agar environments that pose specific drawbacks for characterization of C. elegans burrowing ability, here we use the optically transparent and biocompatible Pluronic F-127 gel that transitions from liquid to gel at room temperature, enabling convenient and safe handling of animals. The burrowing assay methodology involves loading animals at the bottom of well plates, casting a liquid-phase of Pluronic on top that solidifies via a modest temperature upshift, enticing animals to reach the surface via chemotaxis to food, and quantifying the relative success animals have in reaching the chemoattractant. We study the influence of Pluronic concentration, gel height and chemoattractant choice to optimize assay performance. To demonstrate the simplicity of the assay workflow and versatility, we show its novel application in multiple areas including (i) evaluating muscle mutants with defects in dense bodies and/or M-lines (pfn-3, atn-1, uig-1, dyc-1, zyx-1, unc-95 and tln-1), (ii) tuning assay conditions to reveal changes in the mutant gei-8, (iii) sorting of fast burrowers in a genetically-uniform wild-type population for later quantitation of their distinct muscle gene expression, and (iv) testing proteotoxic animal models of Huntington and Parkinson's disease. Results from our studies show that stimulating animals to navigate in a dense environment that offers mechanical resistance to threedimensional locomotion challenges the neuromuscular system in a manner distinct from standard crawling and thrashing assays. Our simple and high throughput burrowing assay can provide insight into molecular mechanisms for maintenance of neuromuscular health and facilitate screening for therapeutic targets.

Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration

“…The C. elegans body wall muscle is the tissue most directly involved in long-term exercise training and thus we sought to characterize additional aspects of body wall muscle performance and health consequent to exercise. We recently developed a burrowing assay, in which nematodes are stimulated to move through a Pluronic F-127 gel (a hydrogel that transitions from liquid to gel at safe temperatures for C. elegans survival) toward a chemoattractant (26). We loaded liquid-phase Pluronic F-127 on top of C. elegans in 12-well plates, and allowed the Pluronic F-127 to quickly gel at room temperature, trapping animals under the gel.…”

“…2 A ). In this 3D gel environment, animals with a better neuromuscular performance reach the surface faster and in higher numbers (26). After a 3+3+2+2 training regimen, Ad5 exercised nematodes reached the gel surface at a significantly higher proportion during the 3-h burrowing assay window as compared to their nonexercised control counterparts (Fig.…”

Alcohol induces mitochondrial fragmentation and stress responses to maintain normal muscle function in Caenorhabditis elegans

“…Burrowing assay. The burrowing assay was performed as described by Lesanpezeshki et al with some modifications (14). Briefly, we placed fifteen age-matched (24 h post L4) naïve or ethanol-exposed animals at the bottom center of a 12 well plate.…”

“…To better simulate the natural locomotion, we performed a modified burrow assay (14), in which naïve control and ethanol-exposed animals are allowed to migrate across a solidified Pluronic F-127 gel layer from the bottom of a 12 well plate to an olfactory attractant on the surface. This assay has been shown to effectively detect even a slight deficit in neuromuscular function (14). We found that compared to naïve animals ethanol-exposed animals had difficulties in reaching the surface (Fig.…”

Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles

“…For all samples, we performed a modified gel burrowing assay (Figure 4a) by transferring the nematodes and casting them in Pluronic F127 in a well plate. 33 The surface was coated with E. coli OP-50 lysate as a food source and to attract the nematodes, and the number of C. elegans that penetrated to the surface was counted. Nematode mobility was quantified based on their total track coverage using edge detection (Figures 4b−d, S9, and S10).…”