We present an imaging system for panneuronal recording in crawling C. elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ~100 neurons that co-express cytoplasmic calcium indicator GCaMP6 and nuclear localized RFP at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal’s posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of worms subjected to thermosensory inputs. We uncover a strikingly sparse representation of thermosensory perception involving two neurons (AFDL and AFDR), and a broadly distributed representation of the motor state across the nervous system. Our imaging setup and analysis pipeline, panneuronal imaging in roaming animals, or PANERA, provides a new platform to establish functional maps of sensory to motor transformation in behaving C. elegans and Drosophila larva.
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