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. 2014 Mar 24;8:28.
doi: 10.3389/fncir.2014.00028. eCollection 2014.

Simultaneous Optogenetic Manipulation and Calcium Imaging in Freely Moving C. Elegans

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Free PMC article

Simultaneous Optogenetic Manipulation and Calcium Imaging in Freely Moving C. Elegans

Frederick B Shipley et al. Front Neural Circuits. .
Free PMC article

Abstract

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.

Keywords: behavior; calcium imaging; mechanosensation; optogenetics; sensorimotor transformation.

Figures

Figure 1
Figure 1
Schematic of the illumination and imaging systems. A worm moves freely on a motorized stage under infrared illumination (IR). A digital micromirror device (DMD) reflects blue and yellow laser light onto only targeted neurons. Three separate imaging paths simultaneously image the worm's behavior and record simultaneous red and green fluorescence images. Real-time computer vision software monitors the worm's posture and location and controls the DMD, lasers and stage. The DMD's illumination pattern is continuously updated to illuminate only targeted neurons.
Figure 2
Figure 2
(A) ChR2 is expressed in six soft-touch mechanosensory neurons, including ALM and AVM (dark blue). Calcium indicator GCaMP3 and fluorescent reference mCherry are expressed in command interneuron AVA (striped green and red). Light blue shaded regions indicate areas of illumination. “a” is anterior, “p” is posterior, “d” is dorsal, and “v” is ventral. (B) Intracellular calcium dynamics of AVA (green line) are measured before during and after optogenetic stimulation of the ALM touch neuron (2.7 s stimulation, blue shaded region). The velocity of the worms body bending waves is shown in gray.
Figure 3
Figure 3
(A) Mean of AVA activity and (B) worm velocity during a time window are shown (gray crosses) for trials when the worm reversed in response to optogenetic illumination of ALM (n = 8 trials), AVM (n = 6 trials) or both (n = 9). Mean across trials is shown (red squares). Error bars represent standard error of the mean. The dashed green line shows the mean plus standard deviation of the apparent calcium signal observed during reversals in worms expressing calcium-insensitive GFP instead of GCaMP3 (n = 5 trials).
Figure 4
Figure 4
(A) For each trial, the amplitude of AVA activity is plotted against the within-trial mean velocity during a trial-specific time window, (n = 39 trials). Both reversers (shaded points) and non-reversers (open points) are included. (B) The fraction, f, of worms that respond by reversing (reversers) are shown for each stimuli. Number, n, indicates total trials. Control worms grown without the ChR2 co-factor all-trans retinal (ATR (−)) are also shown. Error bars are standard error, σf=f(1f)/n, where f is the fraction of worms that reverse out of a total, n.

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