Optical interrogation of neural circuits in Caenorhabditis elegans - PubMed (original) (raw)

Optical interrogation of neural circuits in Caenorhabditis elegans

Zengcai V Guo et al. Nat Methods. 2009 Dec.

Abstract

The nematode Caenorhabditis elegans has a compact nervous system with only 302 neurons. Whereas most of the synaptic connections between these neurons have been identified by electron microscopy serial reconstructions, functional connections have been inferred between only a few neurons through combinations of electrophysiology, cell ablation, in vivo calcium imaging and genetic analysis. To map functional connections between neurons, we combined in vivo optical stimulation with simultaneous calcium imaging. We analyzed the connections from the ASH sensory neurons and RIM interneurons to the command interneurons AVA and AVD. Stimulation of ASH or RIM neurons using channelrhodopsin-2 (ChR2) resulted in activation of AVA neurons, evoking an avoidance behavior. Our results demonstrate that we can excite specific neurons expressing ChR2 while simultaneously monitoring G-CaMP fluorescence in several other neurons, making it possible to rapidly decipher functional connections in C. elegans neural circuits.

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Figures

Figure 1

Challenges facing an all-optical interrogation of neural circuits. (a) Excitation spectra of light-gated cation channels ChR2 and VChR1 (ref. 12) and of genetically encoded calcium sensor G-CaMP in the presence of 1 mM Ca2+ (ref. 7). (b) Schematic diagram showing the ASH neuron–mediated avoidance circuit: polymodal sensory neuron ASH detects repulsive stimuli; command interneurons AVA, AVD and AVE are critical for backward movement, AVB and PVC are critical for forward movement. RIM is an inter- or motorneuron with gap junctions to AVA and AVE. Direct chemical synapses and gap junctions based on electron microscopy reconstruction between these neurons are also shown.

Figure 2

Activation of ChR2 in ASH neurons by whole-body illumination. The fraction of worms of the indicated strains showing rapid reversal upon whole-body blue-light illumination is plotted. Thirty worms were tested for each strain, in the presence or absence of retinal. ASH::ChR2 represents sra-6p::ChR2. *P < 0.001.

Figure 3

Optical setup for ChR2 stimulation and simultaneous G-CaMP imaging. (a) The stimulation and imaging light paths are shown. In the stimulation light path, a high-power epifluorescence light source, used to excite ChR2, hits a digital light processing (DLP) mirror array before entering the objective. Each mirror in the DLP mirror array can be controlled independently. The number of mirrors turned on and their positions in the array determine the location and size of the spots on the sample. In the imaging light path, a low-power 488-nm laser coupled to a spinning disc and an electron-multiplying charge-coupled device camera was used to measure G-CaMP fluorescence. The light paths were independently controlled. (b) An image with nine 5 µm × 5 µm rectangular regions, each 5 µm apart from the other, illuminated using the DLP mirror array is shown (left). The intensity profile along the red line in the image (right, shaded regions indicate illuminated regions), showing sharp intensity differences between illuminated and dark regions, displaying the ability of the DLP mirrors to illuminate specific regions. Scale bar, 50 µm.

Figure 4

Validation of specific stimulation in vivo. (a) G-CaMP fluorescence traces (6 traces in 6 worms) upon low-power laser illumination in worms that express both G-CaMP and ChR2 in ASH neurons. Fold change is the relative fluorescence increase over the fluorescence intensity before stimulation. (b) The left ASH (ASHL) and ASI (ASIL) neurons from which traces were recorded are shown. Green (G-CaMP) and red (ChR2-mCherry) fluorescence images superposed on differential interference contrast (DIC) images show these neurons as well as the left AVA (AVAL) and AVD (AVDL) neurons. Raw and corrected fold changes in G-CaMP fluorescence traces of ASHL (middle) and ASIL (bottom) neurons are shown during ASHL neuron stimulation using the DLP mirror array. Blue light used for ChR2 stimulation also excited G-CaMP in ASHL to produce an intensity jump. The jump was removed to give a corrected trace. The gray region indicates the time period during which ASHL was illuminated with the stimulation light. Scale bar, 10 µm. (c) Corrected G-CaMP fluorescence traces of ASHL and ASIL in b, plotted together for comparison (ASHL stimulation; top). Fluorescence traces of ASHL and ASIL neurons during ASIL stimulation in the same worm as in b (bottom). (d) Compiled traces for either left or right ASH and ASI neurons during ASH stimulation in 7 worms. The traces from the worm shown in b are plotted in thick lines.

Figure 5

Stimulation of ChR2 in ASH neuron activates AVA and AVD neurons. (a) Representative G-CaMP traces in ASH, AVA and AVD neurons in lite-1(ce314) during either left or right ASH (ASHL or ASHR) neuron stimulation (bottom left). Stills from the same time-lapse experiment are shown to illustrate fluorescence intensity changes in neurons, AVA and AVD, from the same side of the body (top). The gray region indicates the time period during which ASH neuron was stimulated. Control traces were obtained with the stimulation light turned off. The green and red fluorescence images superposed on DIC images (right; as in Fig. 4) are shown for the worm from which the traces were recorded. (b–d) Similar recordings for wild-type (b), osm-3(p802) (c) and eat-4(ky5) (d) backgrounds. Scale bar, 10 µm.

Figure 6

Specific stimulation of ChR2 in RIM activates AVA and generates an avoidance response. (a) The fraction of worms that withdrew in response to blue light in the indicated strains. Thirty worms were tested in each genetic background in the presence or absence of retinal. RIM::ChR2 represents tdc-1p::ChR2. *P < 0.001. (b,c) Representative G-CaMP traces in RIM and AVA neurons from the same side of the body (left or right) in the wild-type (b) and lite-1(ce314) (c) background (left). The gray region indicates the time period during which either the left or right RIM (L or R) neuron was activated with the stimulation light. Control traces were obtained with the stimulation light turned off. The green and red fluorescence images superposed on DIC (right, as in Fig. 4) are shown for the worms from which the traces were recorded. Scale bar, 10 µm.

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Optical interrogation of neural circuits in Caenorhabditis elegans - PubMed (original) (raw)