A fundamental goal of systems neuroscience is to probe the dynamics of neural activity that drive behavior. Here we present an instrument to simultaneously manipulate neural activity via Channelrhodopsin, monitor neural response via GCaMP3, and observes behavior in freely moving C. elegans. We use the instrument to directly observe the relation between sensory stimuli, interneuron activity and locomotion in the mechanosensory circuit. Now published as: Front Neural Circuits 8:28, doi:10.3389/fncir.2014.00028

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.

Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a Galphao pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed ( approximately 50 frames per second) to provide high spatial resolution ( approximately 30 mum). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.