Calcium imaging of multiple neurons in freely behaving C. elegans

Abstract

Caenorhabditis elegans is a popular model organism to study how neural circuits and genes regulate behavior. To reliably correlate circuit function with behavior, it is important to record neuronal activity in freely behaving worms. As neural circuits are composed of multiple neurons that cooperate to process information, it is highly desirable to simultaneously record the activity of multiple neurons in the circuitry. However, such a system has not been available in C. elegans. Here, we report the CARIBN II (Calcium Ratiometric Imaging of Behaving Nematodes version II) system. This system provides smoother data collection and more importantly permits simultaneous imaging of calcium transients from multiple neurons in freely behaving worms. Using this system, we imaged the activity of AVA and RIM, two key neurons in the locomotion circuitry that regulate backward movement (reversal) in locomotion behavior. We found that AVA activity increases while RIM activity decreases during the same reversal events in spontaneous locomotion, consistent with the recent report that the AVA and RIM are involved in promoting the initiation of reversals. The CARIBN II system provides a valuable tool for dissecting the neural basis of behavior in C. elegans.

Figure 1. Hardware and software of CARIBN II

a, Schematic diagram of the hardware. b, Schematic diagram of the software.

Figure 2. Simultaneous imaging of AVA and RIM in freely-behaving nematodes expressing G-CaMP and DsRed

a, The autofocus function of CARIBN II automatically re-focus images. A day 1 transgenic worm expressing G-CaMP/DsRed2 under the nmr-1 promoter was tracked for AVA by CARIBN II. The focal plane was manually disturbed (left), which was automatically detected and corrected (right). b, CARIBN II transversely sections between the AVA (left panel) and RIM (right panel) focal planes. RIM was dim to be visible in the AVA focal plane. c-e, Calcium activity of AVA and RIM are associated with the same reversal events. c, AVA and RIM were imaged without enabling the z-sectioning function. The red bar denotes backward movement. The dotted lines represent the missing data due to bad quality of images. AVA activity increased while RIM activity decreased in the same reversal. d, AVA and RIM were imaged through z-sectioning. e, bar graph of peak/trough percentage of ratio change of G-CaMP/DsRed fluorescence . n≥7. Error bars: SEM.