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

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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Figure 1: Volumetric fluorescence imaging using wide-field two-photon light sculpting.
Figure 2: In vivo characterization of NLS-GCaMP5K.
Figure 3: Brain-wide WF-TeFo Ca2+ imaging in C. elegans.
Figure 4: Time-series correlations between neurons.
Figure 5: WF-TeFo Ca2+ imaging in worms during chemosensory stimulation.

References

  1. Briggman, K.L. & Kristan, W.B. Multifunctional pattern-generating circuits. Annu. Rev. Neurosci. 31, 271–294 (2008).

    CAS  Article  Google Scholar 

  2. Bargmann, C.I. Beyond the connectome: how neuromodulators shape neural circuits. Bioessays 34, 458–465 (2012).

    CAS  Article  Google Scholar 

  3. Helmstaedter, M. et al. Connectomic reconstruction of the inner plexiform layer in the mouse retina. Nature 500, 168–174 (2013).

    CAS  Article  Google Scholar 

  4. Takemura, S.-y. et al. A visual motion detection circuit suggested by Drosophila connectomics. Nature 500, 175–181 (2013).

    CAS  Article  Google Scholar 

  5. Niessing, J. & Friedrich, R.W. Olfactory pattern classification by discrete neuronal network states. Nature 465, 47–52 (2010).

    CAS  Article  Google Scholar 

  6. Churchland, M.M. et al. Neural population dynamics during reaching. Nature 487, 51–56 (2012).

    CAS  Article  Google Scholar 

  7. Ahrens, M.B., Orger, M.B., Robson, D.N., Li, J.M. & Keller, P.J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).

    CAS  Article  Google Scholar 

  8. Panier, T. et al. Fast functional imaging of multiple brain regions in intact zebrafish larvae using Selective Plane Illumination Microscopy. Front. Neural Circuits 7, 65 (2013).

    Article  Google Scholar 

  9. White, J.G., Southgate, E., Thomson, J.N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).

    CAS  Article  Google Scholar 

  10. Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399–405 (2010).

    CAS  Article  Google Scholar 

  11. Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).

    CAS  Article  Google Scholar 

  12. Cheng, A., Gonçalves, J.T., Golshani, P., Arisaka, K. & Portera-Cailliau, C. Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat. Methods 8, 139–142 (2011).

    CAS  Article  Google Scholar 

  13. Turaga, D. & Holy, T.E. Organization of vomeronasal sensory coding revealed by fast volumetric calcium imaging. J. Neurosci. 32, 1612–1621 (2012).

    CAS  Article  Google Scholar 

  14. Keller, P.J., Schmidt, A.D., Wittbrodt, J. & Stelzer, E.H.K. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

    CAS  Article  Google Scholar 

  15. Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13, 1468–1476 (2005).

    Article  Google Scholar 

  16. Zhu, G., van Howe, J., Durst, M., Zipfel, W. & Xu, C. Simultaneous spatial and temporal focusing of femtosecond pulses. Opt. Express 13, 2153–2159 (2005).

    Article  Google Scholar 

  17. Dana, H. & Shoham, S. Numerical evaluation of temporal focusing characteristics in transparent and scattering media. Opt. Express 19, 4937–4948 (2011).

    Article  Google Scholar 

  18. Andrasfalvy, B.K., Zemelman, B.V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl. Acad. Sci. USA 107, 11981–11986 (2010).

    CAS  Article  Google Scholar 

  19. Vaziri, A., Tang, J., Shroff, H. & Shank, C. Multilayer three-dimensional super-resolution imaging of thick biological samples. Proc. Natl. Acad. Sci. USA 105, 20221–20226 (2008).

    CAS  Article  Google Scholar 

  20. Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).

    CAS  Article  Google Scholar 

  21. Vaziri, A. & Emiliani, V. Reshaping the optical dimension in optogenetics. Curr. Opin. Neurobiol. 22, 128–137 (2012).

    CAS  Article  Google Scholar 

  22. Katz, O., Small, E., Bromberg, Y. & Silberberg, Y. Focusing and compression of ultrashort pulses through scattering media. Nat. Photonics 5, 372–377 (2011).

    CAS  Article  Google Scholar 

  23. Papagiakoumou, E. et al. Functional patterned multiphoton excitation deep inside scattering tissue. Nat. Photonics 7, 274–278 (2013).

    CAS  Article  Google Scholar 

  24. Lyssenko, N.N., Hanna-Rose, W. & Schlegel, R.A. Cognate putative nuclear localization signal effects strong nuclear localization of a GFP reporter and facilitates gene expression studies in Caenorhabditis elegans. Biotechniques 43, 596–600 (2007).

    CAS  Article  Google Scholar 

  25. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    CAS  Article  Google Scholar 

  26. Bengtson, C.P., Freitag, H.E., Weislogel, J.-M. & Bading, H. Nuclear calcium sensors reveal that repetition of trains of synaptic stimuli boosts nuclear calcium signaling in CA1 pyramidal neurons. Biophys. J. 99, 4066–4077 (2010).

    CAS  Article  Google Scholar 

  27. Zimmer, M. et al. Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases. Neuron 61, 865–879 (2009).

    CAS  Article  Google Scholar 

  28. Busch, K.E. et al. Tonic signaling from O2 sensors sets neural circuit activity and behavioral state. Nat. Neurosci. 15, 581–591 (2012).

    CAS  Article  Google Scholar 

  29. Cáceres, I.C., Valmas, N., Hilliard, M.A. & Lu, H. Laterally orienting C. elegans using geometry at microscale for high-throughput visual screens in neurodegeneration and neuronal development studies. PLoS ONE 7, e35037 (2012).

    Article  Google Scholar 

  30. Chalasani, S.H. et al. Neuropeptide feedback modifies odor-evoked dynamics in Caenorhabditis elegans olfactory neurons. Nat. Neurosci. 13, 615–621 (2010).

    CAS  Article  Google Scholar 

  31. Gray, J.M., Hill, J.J. & Bargmann, C.I. A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 102, 3184–3191 (2005).

    CAS  Article  Google Scholar 

  32. Hendricks, M., Ha, H., Maffey, N. & Zhang, Y. Compartmentalized calcium dynamics in a C. elegans interneuron encode head movement. Nature 487, 99–103 (2012).

    CAS  Article  Google Scholar 

  33. Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 108, 17708–17713 (2011).

    CAS  Article  Google Scholar 

  34. Palero, J., Santos, S.I.C.O., Artigas, D. & Loza-Alvarez, P. A simple scanless two-photon fluorescence microscope using selective plane illumination. Opt. Express 18, 8491–8498 (2010).

    CAS  Article  Google Scholar 

  35. Denk, W. & Detwiler, P.B. Optical recording of light-evoked calcium signals in the functionally intact retina. Proc. Natl. Acad. Sci. USA 96, 7035–7040 (1999).

    CAS  Article  Google Scholar 

  36. Lockery, S.R. & Goodman, M.B. The quest for action potentials in C. elegans neurons hits a plateau. Nat. Neurosci. 12, 377–378 (2009).

    CAS  Article  Google Scholar 

  37. Liu, Q., Hollopeter, G. & Jorgensen, E.M. Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction. Proc. Natl. Acad. Sci. USA 106, 10823–10828 (2009).

    CAS  Article  Google Scholar 

  38. Chalasani, S.H. et al. Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63–70 (2007).

    CAS  Article  Google Scholar 

  39. Piggott, B.J., Liu, J., Feng, Z., Wescott, S.A. & Xu, X.Z.S. The neural circuits and synaptic mechanisms underlying motor initiation in C. elegans. Cell 147, 922–933 (2011).

    CAS  Article  Google Scholar 

  40. Chronis, N., Zimmer, M. & Bargmann, C.I. Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat. Methods 4, 727–731 (2007).

    CAS  Article  Google Scholar 

  41. Kawano, T. et al. An imbalancing act: gap junctions reduce the backward motor circuit activity to bias C. elegans for forward locomotion. Neuron 72, 572–586 (2011).

    CAS  Article  Google Scholar 

  42. Qi, Y.B. et al. Hyperactivation of B-type motor neurons results in aberrant synchrony of the Caenorhabditis elegans motor circuit. J. Neurosci. 33, 5319–5325 (2013).

    CAS  Article  Google Scholar 

  43. Ferkey, D.M. et al. C. elegans G protein regulator RGS-3 controls sensitivity to sensory stimuli. Neuron 53, 39–52 (2007).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Manuel Zimmer or Alipasha Vaziri.

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Competing interests

The authors declare no competing financial interests.

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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