Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Significance Large-scale neural recordings in freely moving animals are important for understanding how patterns of activity across a population of neurons generates animal behavior. Previously, recordings have been limited to either small brain regions or to immobilized or anesthetized animals exhibiting limited behavior. This work records from neurons with cellular resolution throughout the entire brain of the nematode Caenorhabditis elegans during free locomotion. Neurons are found whose activity correlates with behaviors including forward and backward locomotion and turning. A growing body of evidence suggests that animal behavior is sometimes generated by the collective activity of many neurons. It is hoped that methods like this will provide quantitative datasets that yield insights into how brain-wide neural dynamics encode animal action and perception. The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.

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