Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light

Journal name:
Nature Methods
Volume:
10,
Pages:
1013–1020
Year published:
DOI:
doi:10.1038/nmeth.2637
Received
Accepted
Published online

Abstract

Recent efforts in neuroscience research have been aimed at obtaining detailed anatomical neuronal wiring maps as well as information on how neurons in these networks engage in dynamic activities. Although the entire connectivity map of the nervous system of Caenorhabditis elegans has been known for more than 25 years, this knowledge has not been sufficient to predict all functional connections underlying behavior. To approach this goal, we developed a two-photon technique for brain-wide calcium imaging in C. elegans, using wide-field temporal focusing (WF-TeFo). Pivotal to our results was the use of a nuclear-localized, genetically encoded calcium indicator, NLS-GCaMP5K, that permits unambiguous discrimination of individual neurons within the densely packed head ganglia of C. elegans. We demonstrate near-simultaneous recording of activity of up to 70% of all head neurons. In combination with a lab-on-a-chip device for stimulus delivery, this method provides an enabling platform for establishing functional maps of neuronal networks.

At a glance

Figures

  1. Volumetric fluorescence imaging using wide-field two-photon light sculpting.
    Figure 1: Volumetric fluorescence imaging using wide-field two-photon light sculpting.

    (a) Schematic depicting of the light-sculpting microscope and microfluidic sample holder. The pulses at the bottom sketch the geometric dispersion in temporal focusing as a function of axial position. Top right, artistic rendering of a C. elegans head, indicating axially scanned light discs and the imaged region (red). URX and BAG neurons (pink) are also depicted. Scale bar, 15 μm. DC, dichroic mirror; OPA, optical parametric amplifier; sCMOS, scientific complementary metal-oxide semiconductor; NA, numerical aperture; Ti:Sa, titanium-sapphire oscillator. (b) Lateral characteristics of the excitation profile measured on a homogeneous fluorescent test sample showing Gaussian intensity pixel distribution. (c) Optical sectioning capability of the light disc generated by temporal focusing. Measured (solid dots) and fitted (line) intensity of an axially displaced, 100-nm thin fluorescent test sample. AU, arbitrary units. (d,e) Maximum-intensity projection of typical volumetric recordings in C. elegans with (d) and without (e) a high-gain image intensifier in front of the sCMOS camera. The red boxes indicate regions of interest used in determining the signal-to-noise ratio (SNR). A, anterior; P, posterior; D, dorsal; V, ventral. (f) SNR as a function of intensifier gain using a worm sample and exposure time identical to that in d,e. The same brightest neurons (n = 10) were used across recordings for the analysis. Error bars, s.e.m.

  2. In vivo characterization of NLS-GCaMP5K.
    Figure 2: In vivo characterization of NLS-GCaMP5K.

    (a) Schematic of NLS-GCaMP5K. M13, calmodulin-binding domain; cpEGFP, circular permuted enhanced GFP. (b,c) Maximum-intensity projections of fluorescence image stacks acquired with a spinning disk confocal microscope. Shown are head ganglia pan-neuronally expressing cytoplasmic GCaMP5K (b) versus nuclear GCaMP5K (c). Arrows indicate the locations of the nerve-ring neuropil. (dr) Ca2+ imaging of BAG and URX neurons by epifluorescence microscopy. (df) NLS-GCaMP5K fluorescence in stimulated chemosensory neurons. The color bar indicates scaled fluorescence intensities (IFL). (d) Baseline (21% O2) fluorescence levels in O2-sensing BAG and URX neurons. (e) O2 downshift–evoked response in BAG neurons. (f) O2 upshift–evoked response in URX neurons. (gr) Averaged Ca2+ transients in BAG and URX neurons and quantifications. Traces indicate mean fluorescence changes, ΔF/F0. Gray shading indicates s.e.m. White and blue backgrounds indicate periods at 21% O2 and 4% O2, respectively. The number of worms analyzed in each case is noted. (gi) O2 downshift–evoked Ca2+ transients in BAG neurons expressing cytoplasmic (g) or nuclear (h) GCaMP5K and quantification of mean peak responses (i). (jl) O2 upshift–evoked Ca2+ transients in URX neurons expressing cytoplasmic (j) or nuclear (k) GCaMP5K and quantification of mean peak responses (l). (mo) O2-regulated tonic Ca2+ signaling in BAG neurons as measured with cytoplasmic (m) or nuclear GCaMP5K (n) and quantification of mean response (o). (pr) O2-regulated tonic Ca2+ signaling in URX neurons as measured with cytoplasmic (p) or nuclear (q) GCaMP5K and quantification of mean response (r). Bar graphs show mean ΔF/F0 at time intervals indicated by red bars below the traces in the left and center panels. Asterisks indicate significance levels by t-test (*P = 0.0147; ***P = 0.0006; ns, not significant; P = 0.4017 (i); P = 0.8325 (l)). Scale bars, 10 μm.

  3. Brain-wide WF-TeFo Ca2+ imaging in C. elegans.
    Figure 3: Brain-wide WF-TeFo Ca2+ imaging in C. elegans.

    (a) Schematics of the microfluidic poly(dimethylsiloxane) (PDMS) device. Red and blue sketches indicate worm immobilization and the gas delivery channel, respectively. Right, cross-section (not to scale). Bottom, phase-contrast image of an immobilized worm inside the device; the head is at the bottom. The ventral side is shown by the vulva's location (blue arrow). (b) WF-TeFo imaging of the head region of a Punc-31double colonNLS-GCaMP worm. (b, i) Maximum-intensity projection of 14 z planes. Dashed lines outline the locations of the head ganglia in ii. Scale bar, 10 μm (i,iiiv). (b, ii) Schematic of the left anterior head ganglia, adapted from ref. 9 with permission from The Royal Society. Black lines outline neuronal nuclei. Gray lines outline the ganglia as indicated. Green indicates the pharynx. (b, iii) Single z plane (z = 2 μm). Dashed lines indicate yz and xz cross-sections shown in iv and v, respectively. Arrows in iiiv indicate example neurons each seen in two projections. A, anterior; P, posterior; D, dorsal; V, ventral; L, left; R, right. (c) Activity of 99 neurons from the worm in b, imaged volumetrically at 5 Hz for 200 s. Each row shows a time-series heat map of NLS-GCaMP5K fluorescence. Color indicates percent fluorescence changes (ΔF/F0); scaling is indicated by the color bar on the left. The x axis represents elapsed recording time. (d) Matrix showing correlation coefficient (R) calculated from all time series shown in c. Color indicates the degree of correlation; scaling is indicated by the color bar on the right. The data in c,d are grouped by agglomerative hierarchical clustering. Corresponding rows in c,d are aligned. Supplementary Figure 3 provides high-resolution images of panels c,d that indicate the neuron ID numbers consistent with Supplementary Data 1 and 5.

  4. Time-series correlations between neurons.
    Figure 4: Time-series correlations between neurons.

    (a) NLS-GCaMP5K fluorescence time series of active neurons from Figure 3c. Color indicates percent fluorescence changes (ΔF/F0); scaling is indicated by the color bar on the left. (b) Matrix of correlation coefficients (R). Color indicates the degree of correlation; scaling is indicated by the color bar on the right. Data in a,b are grouped by agglomerative hierarchical clustering. Corresponding rows in a,b are aligned. Supplementary Figure 6 provides high-resolution images of panels a,b. (c) Selected traces of neurons. Numbers (Neuron ID) in this and all subsequent panels identify neurons consistent with Supplementary Figures 3 and 6 and Supplementary Data 1 and 5. IDs are accompanied by C. elegans neuron names. Trace colors correspond to the clusters indicated by colored bars by the axes in a,b. Dashed lines indicate time points corresponding to images in d. (d) Examples for yz (iiii) and xz (iv) projections of some neurons shown in c. A, anterior; P, posterior; D, dorsal; V, ventral; L, left; R, right. (d, iiii) yz projections through pairs of correlated bilaterally symmetric neurons. The correlation coefficients are R5/87 = 0.986 (i), R12/96 = 0.893 (ii) and R92/43 = 0.8414 (iii). (d, iv) xz projection across the boundary between the lateral and ventral ganglia showing a group of six synchronously active neurons. Color bars show fluorescence intensities (IFL). (e) Schematic of the left head ganglia. Neuron colors match with the colors of traces in c and bars in a,b. Dashed lines outline the focal plane of each projection in d and are labeled by the corresponding panel number. Scale bars, 5 μm.

  5. WF-TeFo Ca2+ imaging in worms during chemosensory stimulation.
    Figure 5: WF-TeFo Ca2+ imaging in worms during chemosensory stimulation.

    (a) Activity of 69 neurons under consecutively changing O2 concentrations. Rows are heat plots of NLS-GCaMP5K fluorescence time series. Color indicates percent fluorescence changes (ΔF/F0); scaling is indicated by the color bar on the left. O2 concentrations were shifted as indicated by dashed lines. (b) Matrix showing correlation coefficients (R) calculated from data in a. Color indicates the degree of correlation; scaling is indicated by the color bar on the right. The data in a,b are grouped by agglomerative hierarchical clustering. Corresponding rows in a,b are aligned. Supplementary Figure 9 provides high resolution images of panels a,b that indicate the neuron ID numbers consistent with cf, Supplementary Figure 9 and Supplementary Data 2 and 5. (c) Fluorescence traces of oxygen sensory neurons. Colors correspond to the neurons labeled by colored bars by the axes in a,b. (d) Maximum-intensity xy projection of images at time point t = 45 s. Dashed lines indicate cut sections for the projections in e,f as indicated. A, anterior; P, posterior; D, dorsal; V, ventral; L, left; R, right. (e,f) Examples for xz (left) and yz (right) projections of BAG (e) and URX (f) neurons at indicated time points. Color bars show fluorescence intensities. Scale bars, 10 μm. Numbers in cf are Neuron IDs.

Videos

  1. Brain-wide Ca2imaging of basal activity in C. elegans.
    Video 1: Brain-wide Ca2+−imaging of basal activity in C. elegans.
    Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 s recording of basal activity at 21% O2. Frame rate of 70 frames per second equates to 5 volumes per second. See also Figures 3 and 4.
  2. Selected sections of brain-wide Ca2imaging of basal activity in C. elegans.
    Video 2: Selected sections of brain-wide Ca2+−imaging of basal activity in C. elegans.
    Selected transverse- (right) and coronal (bottom) sections plus maximum intensity projection through the left-right axis (center) of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. White and yellow lines indicate section planes and projection widths, respectively. Shown are 200 s recording of basal activity at constant 21% O2. Frame rate of 70 frames per second equates to 5 volumes per second. See also Fig. 3 and 4.
  3. Brain-wide Ca2imaging of neural activity upon repetitive O2 stimuli in C. elegans.
    Video 3: Brain-wide Ca2+−imaging of neural activity upon repetitive O2 stimuli in C. elegans.
    Maximum intensity projection of 16 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Recording time is 232 s. O2 concentrations consecutively shift between 21% and 4% as indicated in the movie. Frame rate of 70 frames per second equates to 4.362 volumes per second. See also Fig. 5.
  4. Selected sections of brain-wide Ca2imaging of neural activity upon repetitive O2 stimuli in C. elegans.
    Video 4: Selected sections of brain-wide Ca2+−imaging of neural activity upon repetitive O2 stimuli in C. elegans.
    Selected transverse- (right) and coronal (bottom) sections plus maximum intensity projection through the left-right axis (center) of 16 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. White and yellow lines indicate section planes and projection widths, respectively. Recording time is 232 s. O2 concentrations consecutively shift between 21% and 4% as indicated in the movie. The dynamic activity of both BAG and URX neurons (see Fig. 5E, ID 7, 42 and 38, 29) upon O2 shifts can be seen. Frame rate of 70 frames per second equates to 4.362 volumes per second. See also Fig. 5.

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Author information

  1. These authors contributed equally to this work.

    • Tina Schrödel &
    • Robert Prevedel

Affiliations

  1. Research Institute of Molecular Pathology, Vienna, Austria.

    • Tina Schrödel,
    • Robert Prevedel,
    • Karin Aumayr,
    • Manuel Zimmer &
    • Alipasha Vaziri
  2. Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

    • Robert Prevedel &
    • Alipasha Vaziri
  3. Research Platform Quantum Phenomena & Nanoscale Biological Systems (QuNaBioS), University of Vienna, Vienna, Austria.

    • Robert Prevedel &
    • Alipasha Vaziri
  4. Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria.

    • Karin Aumayr

Contributions

T.S. and R.P. designed and performed experiments and analyzed data; R.P. and A.V. designed and built the imaging system; T.S. and M.Z. designed and characterized NLS-GCaMP5K and designed and validated the microfluidic device; K.A. wrote analysis software and analyzed data. M.Z. and A.V. designed experiments and conceived of and led the project. T.S., R.P., M.Z. and A.V. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Supplementary information

Video

  1. Video 1: Brain-wide Ca2+−imaging of basal activity in C. elegans. (20.98 MB, Download)
    Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 s recording of basal activity at 21% O2. Frame rate of 70 frames per second equates to 5 volumes per second. See also Figures 3 and 4.
  2. Video 2: Selected sections of brain-wide Ca2+−imaging of basal activity in C. elegans. (2.54 MB, Download)
    Selected transverse- (right) and coronal (bottom) sections plus maximum intensity projection through the left-right axis (center) of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. White and yellow lines indicate section planes and projection widths, respectively. Shown are 200 s recording of basal activity at constant 21% O2. Frame rate of 70 frames per second equates to 5 volumes per second. See also Fig. 3 and 4.
  3. Video 3: Brain-wide Ca2+−imaging of neural activity upon repetitive O2 stimuli in C. elegans. (2.53 MB, Download)
    Maximum intensity projection of 16 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Recording time is 232 s. O2 concentrations consecutively shift between 21% and 4% as indicated in the movie. Frame rate of 70 frames per second equates to 4.362 volumes per second. See also Fig. 5.
  4. Video 4: Selected sections of brain-wide Ca2+−imaging of neural activity upon repetitive O2 stimuli in C. elegans. (2.57 MB, Download)
    Selected transverse- (right) and coronal (bottom) sections plus maximum intensity projection through the left-right axis (center) of 16 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. White and yellow lines indicate section planes and projection widths, respectively. Recording time is 232 s. O2 concentrations consecutively shift between 21% and 4% as indicated in the movie. The dynamic activity of both BAG and URX neurons (see Fig. 5E, ID 7, 42 and 38, 29) upon O2 shifts can be seen. Frame rate of 70 frames per second equates to 4.362 volumes per second. See also Fig. 5.

PDF files

  1. Supplementary Text and Figures (1,381 KB)

    Supplementary Figures 1–10 and Supplementary Table 1

Excel files

  1. Supplementary Data 1 (2,262 KB)

    The data sheets contain the neuron IDs, ΔF/F0 values, raw uncorrected fluorescence traces and elapsed time corresponding to Figures 3-4 and Supplementary Figure 3 (Rows correspond to neuron IDs. Columns correspond to time frames). The correction table assigns to each neuron ID (column A) a reference neuron (column B); see Online Methods.

  2. Supplementary Data 2 (1,609 KB)

    The data sheets contain the neuron IDs, ΔF/F0 values, raw uncorrected fluorescence traces and elapsed time corresponding to Figure 5 and Supplementary Figure 9. (Rows correspond to neuron IDs. Columns correspond to time frames). The correction table assigns to each neuron ID (column A) a reference neuron (column B); see Online Methods.

  3. Supplementary Data 3 (2,348 KB)

    The data sheets contain the neuron IDs, ΔF/F0 values, raw uncorrected fluorescence traces and elapsed time corresponding to Supplementary Figure 7 (Rows correspond to neuron IDs. Columns correspond to time frames). The correction table assigns to each neuron ID (column A) a reference neuron (column B); see Online Methods.

  4. Supplementary Data 4 (2,740 KB)

    The data sheets contain the neuron IDs, ΔF/F0 values, raw uncorrected fluorescence traces and elapsed time corresponding to Supplementary Figure 8 (Rows correspond to neuron IDs. Columns correspond to time frames). The correction table assigns to each neuron ID (column A) a reference neuron (column B); see Online Methods.

Zip files

  1. Supplementary Data 5 (6,021 KB)

    First frame positions of ROIs corresponding to all data sets. (1-4) Images are maximum intensity projections of all z-planes of one recording corresponding to the first acquired volume. Numbered regions indicate positions of all neuron IDs shown in Figures 3, 4, 5, Supplementary Figs 7 and 8. Note, that not all of the neurons are clearly visible at the shown first time point as their fluorescence intensity only increases later during the recording. In some cases regions were slightly moved in order to make all numbers readable. (1) ROIs of recording shown in Figures 3 and 4, Supplementary Figures 3 and 6 (2) ROIs of recording shown in Figure 5, Supplementary Figure 9 (3) ROIs of recording shown in Supplementary Figure 7. (4) ROIs of recording shown in Supplementary Figure 8.

Additional data