Controlling interneuron activity in Caenorhabditis elegans to evoke chemotactic behaviour

Abstract

Animals locate and track chemoattractive gradients in the environment to find food. With its small nervous system, Caenorhabditis elegans is a good model system in which to understand how the dynamics of neural activity control this search behaviour. Extensive work on the nematode has identified the neurons that are necessary for the different locomotory behaviours underlying chemotaxis through the use of laser ablation, activity recording in immobilized animals and the study of mutants. However, we do not know the neural activity patterns in C. elegans that are sufficient to control its complex chemotactic behaviour. To understand how the activity in its interneurons coordinate different motor programs to lead the animal to food, here we used optogenetics and new optical tools to manipulate neural activity directly in freely moving animals to evoke chemotactic behaviour. By deducing the classes of activity patterns triggered during chemotaxis and exciting individual neurons with these patterns, we identified interneurons that control the essential locomotory programs for this behaviour. Notably, we discovered that controlling the dynamics of activity in just one interneuron pair (AIY) was sufficient to force the animal to locate, turn towards and track virtual light gradients. Two distinct activity patterns triggered in AIY as the animal moved through the gradient controlled reversals and gradual turns to drive chemotactic behaviour. Because AIY neurons are post-synaptic to most chemosensory and thermosensory neurons, it is probable that these activity patterns in AIY have an important role in controlling and coordinating different taxis behaviours of the animal.

Figure 1. Asymmetric component of the odor signal controls gradual turning

a, Trajectory of a nematode’s nose tip overlaid on modeled exponential profile (decay length 1 cm) of the gradient of chemoattractants (pseudocolor) from the bacterial lawn. Plus signs: positions of animal at indicated times. a.u.: arbitrary units. b, Illustration of an animal crawling perpendicular to (above) and along (below) the odor gradient. h: head-bending angle; D: dorsal (h > 0); V: ventral (h < 0). c, Odor signal at nose tip, I(t), vs. head-bending angle, h(t), over the last (black) and sequentially prior (grey) head swings for the nose-tip trajectory in (a). d, Plot of the mean (black line) and s.d. (colored bar) of the asymmetric (A(t)) and symmetric (S(t)) components of I(t) in (c). |A|: magnitude of A(t). e, Dorsal asymmetric odor stimulation (see supplementary method). f–g, Sample trajectories of center of mass of the animals upon (f) dorsal and ventral asymmetric, (g) symmetric odor stimulation. Grey bar: mean turning angle; D: dorsal; V: ventral; F: front; B: back; angles (0, 90, −90, 180) define the turning angles with respect to initial orientation of animal. Mean (grey bar), error bar: s.d. over n=10; **, p<0.05, two-sample t-test.

Figure 2. Asymmetric and symmetric excitation of AIY control gradual turning and reversal frequency

a, Setup for closed-loop single neuron stimulation (see supplementary method). b, Sample trajectories of center of mass of animals upon dorsal or ventral asymmetric excitation of AIY (n=10, pttx-3::ChR2). c, Fluorescence image overlaid on the bright field image of a nematode co-expressing ChR2 and mKO in AIY, AIZ and RME (ser-2prom2::mKO and ser-2prom2::ChR2). d, Asymmetric dorsal stimulation of the animal in (c). Upon dorsal head-bending (top, left), ChR2 and mKO were excited in AIY using setup in (a) (top, right, higher fluorescence in AIY) but not upon ventral head-bending (bottom, right, decreased fluorescence in AIY). Left: 1× dark-field image of animal; right: 15× fluorescent images of neurons in that animal. e, Sample trajectories of nose tip of animals upon dorsal (left) or ventral (right) asymmetric excitation of AIY (n=10, ser-2prom2::ChR2). f, Turning angle of animals upon asymmetric stimulation of AIY::ChR2 (n=10), AIY::Arch (n=10) and AIB::Arch (n=7). Dorsal: asymmetric dorsal stimulation. No light: unstimulated. Ventral: asymmetric ventral stimulation. No ATR: Control without all-trans retinal (ATR). g, Reversal frequencies upon symmetric stimulation of AIY::ChR2 (n=10), AIY::Arch (n=19), AIB::ChR2 (n=14), and AIB::Arch (n=11) (f–g mean: white and blue bars, error bars: 1 s.d over n animals, white bars: no ATR; **, p<0.05, two-sample t-test).

Figure 3. Asymmetric AIY excitation modulates head-bending angle to cause turning

a, Representative head-bending angle (horizontal lines: means of maximum and minimum h during each full head swing) of an AIY::ChR2 animal forced to first turn ventrally and then dorsally (positive: dorsal, negative: ventral). b, Histogram of head-bending angle bias (max(h)+min(h)) during dorsal and ventral turn caused by asymmetric AIY excitation (error bar, s.d. over n=5). c–d, Sample tracks of animals upon dorsal (left) or ventral (right) asymmetric excitation (c) of AIZ::ChR2 (ser-2prom2::ChR2), (d) of SMB::ChR2 (podr-2(18)::ChR2) animals. e, Turning angle upon asymmetric stimulation with odor (n=10) and optically of AWC^ON::Arch (n=10), AWC^ON::ChR2 (n=9), AIY::ChR2 (n=10), AIZ::ChR2 (n=5), SMB::ChR2 (n=5), and RME::ChR2 (n=5). Dorsal: asymmetric dorsal stimulation; no light: unstimulated; ventral: asymmetric ventral stimulation (error bar: s.d. over n, **, p<0.05, two-sample t-test). f, The monadic (red) and polyadic (orange) synapses between AWC^ON, AIY, AIB, AIZ, SMB and RME (thickness: proportional to synapse number). Triangle: sensory neuron; hexagon: interneuron; circle: motor neuron.

Figure 4. Controlling AIY activity is sufficient to evoke chemotactic behavior

a, Virtual light gradient algorithm (see supplementary method). At each time t, AIY::ChR2 animals (pttx-3::ChR2) are stimulated with 480 nm blue light with an intensity (I(n_x,n_y)) of the virtual gradient at the nose-tip position (n_x,n_y). b, Trajectories of AIY::ChR2 animals moving in a virtual light gradient (as in (a)) with a gradient direction at 45 degrees (black tick: mean direction of trajectory, grey bar: s.d., n=10). c, Top: A sample trajectory of an animal in (b). Bottom: Snapshots of the animal making a gradual turn to reorient itself to the gradient direction (pseudocolor: same as (a)). d, Magnitude of A_I (black), and S_I (blue, right axis), over a head swing, as a function of numbers of head swings during a gradual turn (n=5, from trajectories in (b), error bar: 1 s.d.). e, Model for chemotaxis in the virtual light gradient. f, Trajectories of center of mass of animals when the gradient direction was suddenly rotated (at times when the animal reached the red dots) by 180, 120 or 90 degrees.