VOLTAGE-SENSITIVE DYE IMAGING FOR LARGE-SCALE FUNCTIONAL MAPPING IN THE <EM>LYMNAEA</EM> CNS

The pond snail, Lymnaea stagnalis, is a well-established model for detailed investigation of neural mechanisms underlying the central generation of rhythmic behaviours such as feeding, locomotion, and respiration. It lends itself to circuit interrogation thanks to its large and uniquely identifiable neurons, which enable the use of targeted intracellular recordings to monitor neural activity and resolve connectivity. However, the number of electrodes that can be positioned in target cells ultimately limits the characterization of circuit operation at scale across the Lymnaea brain. Here we address this issue by establishing a voltage-sensitive dye recording approach that captures neuronal activity in hundreds to thousands of neurons in parallel. Using a commercial photo-induced electron transfer (PeT)-based dye, FluoVolt™, coupled with time-lapse imaging (100-200 Hz) using a sCMOS camera, we demonstrate the faithful tracking of intracellular voltage changes – including subthreshold deflections – to gain a large-scale neural activity readout that is directly registered to anatomical organization. Moreover, this method can be readily integrated with targeted intracellular electrophysiology and stimulation approaches permitting detailed characterization and causal manipulation in parallel. We demonstrate the application of this method to profile behavioural circuit priorities across the brain and to identify a novel output circuit underlying rhythmic locomotion. Overall, this approach substantially expands the experimental toolkit available for systems-level interrogation of neural circuits in the Lymnaea brain.