In Vivo Optical Recording of Action Potentials In C. elegans Muscles using Arclight, a Genetically Expressed Voltage Sensitive Fluorescent Protein

Abstract

The locomotory system of the roundworm C. elegans is an attractive model for elucidating the genetic, cellular, and neural circuit bases of behavior. How the motor neurons and body wall muscles (BWMs) coordinate the worm's locomotion remain poorly understood. Electrophysiological studies have found that BWMs fire action potentials, both spontaneously and in response to nerve stimulation (Liu et al., 2011, Gao and Zhen, 2011). However, these recordings require dissection of the animal and can only be obtained from one or at most two cells simultaneously. To measure electrical activity throughout the intact animal, we have introduced the voltage sensitive fluorescent protein, Arclight-A242 (Jin et al., 2012) into body wall muscle cells under the myo-3 promoter. Arclight is trafficked efficiently to the muscle cell membrane and does not cause any obvious behavioral defect. We record activity of muscles in immobilized worms at 100 frames per second throughout the worm by using a 5.5 megapixel sCMOS camera and LED illumination (475 nm), and find oscillations in fluorescence intensity similar to bursts of action potentials reported previously (Liu et al., 2010) with average frequency of approximately 3 Hz. These oscillations are particularly prominent in the head (anterior BWM). We observe that activity in dorsal and ventral muscle cells are anticorrelated, consistent with the muscles functioning in opposition to each other. These methods will allow us to investigate the mechanisms of nervous system coordination of BWM activity. Supported by U24 NS057631 (JRAW and BMS), R01 NS040966 (BMS), the University of Pennsylvania Comprehensive Neuroscience Center (LH), and the Alfred P. Sloan Research Foundation (CFY). We are grateful to Stanford Photonics, Inc. for the loan of the camera and Piper software. JRAW and LH contributed equally.