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.
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Acknowledgements
We thank J. Akerboom and L. Looger (Howard Hughes Medical Institute, Janelia Farm Research Campus) for valuable information on characteristics of GCaMP variants and for constructs; M. Colombini for manufacturing of mechanical components; E. Sanchez, B. Bathellier, G. Haunert and D. Aschauer for helpful discussions and valuable input; M. Palfreyman, H. Kaplan, S. Kato and C. Bargmann for critically reading the manuscript; R. Latham, M. Sonntag, D. Grzadziela and S. Skora for technical support. R.P. acknowledges the VIPS Program of the Austrian Federal Ministry of Science and Research and the City of Vienna as well as the European Commission (Marie Curie, FP7-PEOPLE-2011-IIF). The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 281869 - elegans Neurocircuits, Vienna Science and Technology Fund (WWTF) project VRG10-11, Human Frontiers Science Program Project RGP0041/2012, Research Platform Quantum Phenomena and Nanoscale Biological Systems (QuNaBioS) and Research Institute of Molecular Pathology (IMP). The IMP is funded by Boehringer Ingelheim.
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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.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10 and Supplementary Table 1 (PDF 1349 kb)
Supplementary Data 1
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. (XLSX 2209 kb)
Supplementary Data 2
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. (XLSX 1572 kb)
Supplementary Data 3
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. (XLSX 2293 kb)
Supplementary Data 4
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. (XLSX 2676 kb)
Supplementary Data 5
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. (ZIP 5880 kb)
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. (MOV 21488 kb)
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. (MOV 2605 kb)
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. (MOV 2598 kb)
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. (MOV 2633 kb)
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Schrödel, T., Prevedel, R., Aumayr, K. et al. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat Methods 10, 1013–1020 (2013). https://doi.org/10.1038/nmeth.2637
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DOI: https://doi.org/10.1038/nmeth.2637
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