[
International Worm Meeting,
2019]
Identifying specific neurons within a neuronal circuit and linking their activity to a certain behavior remains one of the most challenging tasks in the field of neurobiology. Using an optimized system for fast automated x-,y-tracking of a fluorescent marker in freely moving C. elegans, we found that the GABAergic interneuron RIS, which is known to promote sleep during larval development, is reutilized in adulthood for orchestrating locomotion 'stop'. We could show that RIS exhibits compartmentalized calcium dynamics by performing axonal calcium imaging. By further expanding our setup using a Piezo scanner for simultaneous volumetric scanning we are able to analyze the neuronal network of RIS by recording calcium activity from two distinct neurons within the network at the same time. This is achieved by expressing the genetically encoded calcium indicators (GECIs) GCaMP6 and jRCaMP1b cell specifically in RIS and the neurons upstream (i.e. CEPs) and downstream (i.e. RIB & RIM) of RIS according to its connectome. The observed, possibly linked activities are verified by optogenetic stimulation or inhibition of RIS or the CEPs using microbial rhodopsins, and assessing the correlation of calcium activity in the downstream neuron. The scanner in our system requires less than 200 ms per volume, moving in a triangular scanning pattern, and enabling to account for fast calcium dynamics. The data analysis is based on open source and easy-to-use image analysis software. Image registration is done with ImageJ software using simple stack-slice manipulations and the Scale Invariant Feature Transform (SIFT). Subsequently all the data from the calcium and behavioral imaging is evaluated using customized MATLAB and Mathematica scripts. Measuring spontaneous or evoked calcium activity in one or two distinct neurons at high spatiotemporal resolution in soma, ventral nerve cord and nerve ring processes and extracting behavioral parameters like bending angles, body length and speed will enable deeper insights into the architecture and functional interplay of cells of the described neuronal network.