Simultaneous optogenetic manipulation and calcium imaging in freely moving C-elegans

Understanding how an organism's nervous system transforms sensory input into behavioral outputs requires recording and manipulating its neural activity during unrestrained behavior. Here we present an instrument to simultaneously monitor and manipulate neural activity while observing behavior in a freely moving animal, the nematode Caenorhabditis elegans. Neural activity is recorded optically from cells expressing a calcium indicator, GCaMP3. Neural activity is manipulated optically by illuminating targeted neurons expressing the optogenetic protein Channelrhodopsin. Real-time computer vision software tracks the animal's behavior and identifies the location of targeted neurons in the nematode as it crawls. Patterned illumination from a DMD is used to selectively illuminate subsets of neurons for either calcium imaging or optogenetic stimulation. Real-time computer vision software constantly updates the illumination pattern in response to the worm's movement and thereby allows for independent optical recording or activation of different neurons in the worm as it moves freely. We use the instrument to directly observe the relationship between sensory neuron activation, interneuron dynamics and locomotion in the worm's mechanosensory circuit. We record and compare calcium transients in the backward locomotion command interneurons AVA, in response to optical activation of the anterior mechanosensory neurons ALM, AVM or both.