In vivo application of upconverting force sensors to elucidate neuromuscular pump action in C. elegans

DIPC Seminars

PhD. Student Alice Lay, Stanford University, Department of materials science and engineering
Donostia International Physics Center
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In vivo application of upconverting force sensors to elucidate neuromuscular pump action in C. elegans The feeding behavior of _C. elegans_ is a strong indicator of health; changes indicate environmental toxins, scarce or abundant food resources, aging, and neurodegenerative disease. In particular, the pharyngeal pump action is a rhythmic contraction and relaxation of muscles that allows the worm _ _ to pull in and concentrate bacteria, crush and chew them in the grinder, and then pass them through the intestinal tract [1]. Because the pump action is regulated by motor neurons (MC, M3, M4) [2-5], it serves as a model system for neuromuscular pumps like the heart. Here, we investigate the magnitude of forces exerted by muscles in the pharynx, combining extracellular electrophysiological recordings, or electrophargyngograms (EPGs), with optical force measurements from upconverting nanoparticles (UCNPs). Sub-25 nm Mn2+-doped NaYF4:Er,Yb UCNPs provide a photostable and consistent color response to stress [6]. The nano- to micro-Newton sensitivity of these nanoparticles relies on the energetic coupling between the crystal field sensitive _d-_ metal and upconverting lanthanides, which under stress, yields a positive or negative change in the red to green Er3+ emission ratio for cubic- and hexagonal-phase NaYF4, respectively. Further, we investigate new geometries (e.g. core-shell) for more efficient and force-sensitive nanoparticles. We demonstrate the first _in vivo _capabilities of these nanosensors to image and quantify forces exerted along the pharynx. First, we incubate the worms with water-soluble UCNPs (5 mg/mL) overnight for feeding, which yields no significant chronic cytotoxicity effects on their fertility. Then, we load the worms in a microfluidic device and collect _ _ upconversion spectra at key anatomical features. Based on ratiometric differences in emission peaks, we find that forces exerted in the grinder (~10 uN) are nearly an order of magnitude higher than those exerted at the pharyngeal-intestinal valve (~1 uN). Furthermore, we compare these optical force measurements to muscle contraction and relaxation events, characterized by voltage spikes in the EPGs. We determine pump action parameters (e.g., duration, frequency, amplitude) and muscular forces in wild-type and neurotransmitter-treated (5 mM serotonin) worms. From these _ _ results, we work towards mapping neuromuscular pump dynamics and providing the first in-vivo determination of the forces required for healthy function in _C. elegans_. [1] Fang-Yen, C., L. Avery, and S. Aravinthan. _PNAS_ (2009) [2] Raizen, D.M. and L. Avery. _Neuron_ (1994) [3] Niacaris, T. and L. Avery. _Journal of Experimental Biology_ (2003) [4] Trojanowski, N.F., D.M. Raizen, and C. Fang-Yen. _Scientific reports_ (2016) [5] Lee, K.S. et al. _Nature Communications _(2017) [6] Lay, A. et al. _Nano Letters _(2017)