Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans

Abstract

The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output. We present a real-time, multimodal illumination technology that allows both tracking and recording the behavior of freely moving C. elegans while stimulating specific cells that express channelrhodopsin-2 or MAC. We used this system to optically manipulate nodes in the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals using optogenetics.

Figure 1

Illumination system for live animal tracking and optogenetic stimulation and quantification of behavior elicited by targeted illumination. (a) Optical configuration for using a projector for illumination. The normal epi-fluorescence optical train was replaced by a projector and a relay lens. Projector image planes are indicated, and a motorized X-Y translational stage is used to track animals. (b) Modification of the 3-color LCD projector to further narrow the spectrum is accomplished by the addition of filters into the individual RGB light paths. (c) Sequential frames from Supplementary Video 1 and 2 showing qualitative behavioral responses. Top, using the dorsal coiling effect to cause a worm to crawl in a triangle, and bottom, showing direct muscular control of a paralyzed worm. Images are falsely colored to show illumination pattern. (d) Illustration of the positions of the six sensory neurons, and a frame from Supplementary Video 3 showing the 20 μm bar of blue light, perpendicular to the animal’s longitudinal axis, which was scanned at a rate of 12.5% animal body length per second (~100 μm s⁻¹). (e) Two scanning schemes along the AP axis: head-to-tail and tail-to-head. (f) Histograms showing the distributions of positions along the AP axis at which point the blue light elicited a reversal response. Shown are the distribution of positions where accelerations elicited by the tail-to-head scan were observed (28 out of 52 animals showed an increase in speed two standard deviations greater than the average speed prior to illumination), and the distributions of the anatomical positions of the touch neurons in pmec-4::GFP animals. Scale bars are 100 μm.

Figure 2

Optical stimulation of anterior/posterior mechanosensory neurons or forward/backward command interneurons. (a) Illustration of the positions of neurons expressing ChR2 in pmec-4::ChR2 and pglr-1::ChR2 transgenic worms. (b) The touch circuit showing receptors, command neurons and the resulting behaviors. (c) Average velocity plots of pmec-4::ChR2 animals under illumination conditions (shown as a blue bar above). n = 13 (posterior illumination); n = 15 (anterior illumination). Error bars = s.e.m. (d) Average velocity plots of pglr-1::ChR2 animals under illumination conditions (shown as a blue bar above). n = 24 (posterior illumination); n = 12 (anterior illumination). Error bars = s.e.m.

Figure 3

Quantification of behavioral responses elicited by different anterior illumination intensities. (a) Patterns used for illumination location and their intensity. (b) Velocity plots from pooled data from animals receiving different illumination intensities (also see Supplementary Video 5). ‘NR’ = No Response; ‘Sl/P’ indicates a slowing or pausing of the animal with no negative velocity; ‘r’ is a small reversal; and ‘R’ is a large reversal. n = 40 for each of the three illumination levels. The number of animals showing NR, Sl/P, r, and R are 28, 14, 35, and 43 respectively. Error bars = s.e.m. (c) Distribution of the four responses observed at the three intensity levels.

Figure 4

Illumination patterns used to explore the integration of anterior/posterior signals and behavior generated from the stimulation (also see Supplementary Video 6). (a) Illumination locations and plot of the temporal variation of the intensity for the two patterns tested. Normalized intensity of 1 corresponds to blue light of intensity 1.17mW mm⁻². (b) Histogram distributions of intensity at which animals initiated a reversal under two illumination patterns: anterior alone, and anterior and posterior simultaneously. (n = 40 for each illumination scheme). (c) Distributions among the four response states for anterior illumination alone or simultaneous anterior/posterior illumination at the same intensity (1.17mW mm⁻²). (n = 40 for each).

Figure 5

Simultaneous two color illumination (also see Supplementary Video 8). (a) Illustrations of the two illumination schemes (b) Velocity plots of pmec-4::ChR2 and pglr-1::MAC::mCherry animals subjected to the illumination schemes in (a). Error bars = s.e.m; n = 19 for scheme 1; n = 12 for scheme 2. (c) The neural gentle touch circuit showing the neurons that are either stimulated or silenced and the resulting behaviors at different points in the two sets of experiments.