Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans

Abstract

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 (∼50 frames per second) to provide high spatial resolution (∼30 μm). 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.

Figure 1

High resolution optogenetic control of freely moving C. elegans. (a) An individual worm swims or crawls on a motorized stage under red dark field illumination. A high-speed camera images the worm. Custom software instructs a DMD to reflect laser light onto targeted cells. (b) Images are acquired and processed at ~50 fps (scale bar is 100 µm). Each 1024×768 pixel image is thresholded and the worm boundary is found. Head and tail are located as maxima of boundary curvature (red arrows). Centerline is calculated from the midpoint of line segments connecting dorsal and ventral boundaries (blue bar), and is resampled to contain 100 equally spaced points. The worm is partitioned into segments by finding vectors (green arrows) from centerline to boundary, and selecting one that is most perpendicular to the centerline (orange arrow). Targets defined in worm coordinates are transformed into image coordinates and sent to the DMD for illumination (green bar). (c) Schematic shows body wall muscles. Anterior is to the left and dorsal is to the top. A swimming worm expressing Halo/NpHR in its body wall muscles was subjected to green light (10 mW mm⁻²) outside or inside the worm boundary (n = 5 worms, representative trace) and its bending wave speed is shown. (d) Schematic shows HSN. A swimming worm expressing ChR2 in HSN was subjected to blue light (5 mW mm⁻²). The histogram shows the position at which egg-laying occurred when a narrow stripe of light was slowly scanned along the worm’s centerline (n = 13 worms). Once an egg was laid, the worm was discarded.

Figure 2

Optogenetic inactivation of muscle cells. (a) A kymograph of time-varying body curvature along the centerline of a Pmyo3::Halo/NpHR::CFP transgenic worm. Between t = 0 s and t = 4 s, the worm is stimulated with green light (10 mW mm⁻²) in a region spanning the worm diameter and between 0.38 and 0.6 of the fractional distance along the centerline (n = 5 worms, representative trace). (b) For the kymograph shown in (a), time-varying curvature is shown at two points along the worm centerline, both anterior (upper panel) and posterior (lower panel) to the illuminated region.

Figure 3

Inhibition of motor neurons. (a) The schematic shows the positions of the cholinergic DB and VB motor neurons. Anterior is to the left and dorsal to the top. A kymograph of time-varying body curvature along the centerline of a Punc-17::Halo/NpHR::CFP transgenic worm illuminated by a stripe of green light (10 mW mm⁻²) along its ventral nerve cord between t = 0s and 1.6s. In the dorsal-ventral direction, the stripe width was equal to 50% of the worm diameter and centered on the ventral boundary. In the anterior-posterior direction, the stripe length was between 0.14 and 0.28 of the fractional distance along the body (n = 5 worms, representative trace). (b) For the kymograph shown in (a), time-varying curvature is shown at two points along the worm centerline, both anterior (upper panel) and posterior (lower panel) to the illuminated region. (c) Video sequence showing a worm illuminated by a long stripe of green light (10 mW mm⁻²) spanning the ventral nerve cord between t = 0 s and 1.8 s. Scale bar is ~100 micrometers (d) The bending wave speed of a swimming worm illuminated by a long stripe of green light (10 mW mm⁻²) lasting 1.8s and spanning the ventral nerve cord (upper panel) and dorsal nerve cord (lower panel) (n = 10 worms, representative trace).

Figure 4

Optogenetic analysis of mechanosensory neurons. The schematic shows the positions of anterior and posterior touch receptor cells. Anterior is to the left and dorsal to the top. Kymographs (left panels) show time-varying curvature of the centerline of worms expressing ChR2 in mechanosensory neurons (Pmec-4::ChR2::GFP) subjected to rectangles of blue light (5 mW mm⁻²) targeting different groups of touch receptor neurons. Plots of bending wave speed (right panels) indicate stimulus-evoked changes in direction or speed. (a) The AVM and ALM neurons are subjected to 1.5 s of stimulation (n = 5 worms, representative trace). Given a coordinate system where x specifies dorsal-ventral location (−1 is dorsal boundary, 0 is centerline and 1 is ventral boundary) and y defines fractional distance along the worm’s centerline (0 is head and 1 is tail), the rectangle of illumination has corners (x,y) = [(−1.1,0), (1.1,0.46)]. (b) The PVM and PLM neurons are subjected to 2.5 s of stimulation with a rectangular illumination (n = 5 worms, representative trace) with corners at (x,y) = [(−1.1,0.62), (1.1,0.99)]. (c) The ALM cell body is specifically stimulated by illuminating a small rectangle (n = 14 worms, representative trace) with corners at (x,y) = [(−0.3,0.38), (−0.9,0.46)]. (d) The AVM cell body is specifically stimulated by illuminating a small rectangle (n = 14 worms, representative trace) with corners at (x,y) = [(0.3,0.3), (0.9,0.38)].

Figure 5

Habituation of individual touch receptor neuronal types. (a,b) Schematic shows position of anterior and posterior touch receptor neurons. Anterior is to the left and dorsal to the top. A freely swimming animal expressing Kaede in touch receptor neurons was continuously tracked and illuminated with a small rectangle of 405 nm light ( 2 mW mm⁻² ) centered on either AVM or ALM as in (Fig. 4c,d) for 60 s total . Scale bars are 100 micrometers. (a) AVM was illuminated. Red and green fluorescence images show that photoconversion occurred in AVM but not ALM neurons. (b) ALM was illuminated. Photoconversion occurred in ALM neurons but not AVM. During tracking, a transient segmentation error owing to an omega turn by the animal caused the system to illuminate PLM and PVM for ~1 s, producing slight photoconversion in those neurons. (c–e) Individual ALM and AVM neurons were repeatedly stimulated with blue light (5 mW mm⁻²) for 1.5 s every 60 s for ~40 min, either (c,d) alone or (e) interleaved within each experiment (ALM, 30 s, AVM, 30 s, ALM, 30 s …). (c–e) Fractional response to stimulus of each neuronal type was fit to an exponential, a + b exp[−t /τ], using maximum likelihood estimator. Time constant for habituation, τ, was extracted from each fit. All error bars are s.e.m. (c) Fractional response of ALM when stimulated alone (n = 7 worms). (d) Fractional response of AVM when stimulated alone (n = 8 worms). (e) Fractional response of ALM (left panel) and AVM (right panel) during interleaved stimulation of both (n = 7 worms).