Suggested Title: Simultaneous optogenetic manipulation and calcium imaging in freely moving C. elegans

A fundamental goal of systems neuroscience is to probe the dynamics of neural activity that generate behavior. Here we present an instrument to simultaneously manipulate and monitor neural activity and behavior in the freely moving nematode Caenorhabditis elegans. We use the instrument to directly observe the relationship between sensory neuron activation, interneuron dynamics and locomotion in the mechanosensory circuit. Previously in this journal, we presented an optogenetic illumination system capable of real-time light delivery with high spatial resolution to stimulate or inhibit specified targets in freely moving C. elegans [1]. This " Colbert " system and others like it [2] have been instrumental in defining neural coding of several behaviors in C. elegans including chemotaxis [3], nociception [4] and the escape response [5]. Here we integrate the Colbert system with simultaneous monitoring of intracellular calcium activity. To our knowledge this is the first instrument of its kind. The integration of optogenetics, calcium imaging and behavioral analysis allows us to dissect neural circuit dynamics and correlate neural activity with behavior. In this system, custom computer vision software tracks animal behavior and identifies the location of targeted neurons in real-time. The software adjusts mirrors on a digital micromirror device (DMD) to project patterned illumination onto targeted neurons. We induce neural activity using Channelrhodopsin (ChR2) and monitor calcium dynamics by simultaneously measuring the fluorescence of an optical calcium indicator, GCaMP3, and a calcium-insensitive reference, mCherry. By patterning our illumination to independently target ChR2 and GCaMP3 expressing neurons, the system can continuously observe calcium dynamics from one neuron while transiently activating others (see Fig 1a and Supplementary methods). Unlike previous investigations in immobilized animals [6], here the manipulation and analysis of neural activity was directly correlated to behavioral output. We used the instrument to observe calcium transients in the backward locomotion command interneurons AVA, in response to activation of the anterior mechanosensory neurons ALM, AVM or both (Fig. 1b). Transgenic worms that co-expressed ChR2 in the mechanosensory neurons and a GCaMP3::SL2::mCherry operon in interneurons AVA were stimulated for 2.7s to induce a reversal (see representative trial, Fig 1c, Supplementary Fig 1 and Supplementary Movie 1). We found that AVA's calcium transients correlated with the worm's mean velocity (Pearson's correlation coefficient R=-0.83), irrespective of stimuli (Fig 1e). Averaging across trials, neither AVA's mean activity, the worm's mean reversal velocity nor the probability of a reversal varied significantly with stimulus (Fig 1d,e,g). Our system illuminates cell …