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Monitoring neural activity with bioluminescence during natural behavior

Nature Neuroscience volume 13, pages 513–520 (2010)Cite this article

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Abstract

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca2+-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ∼20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.

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Figure 1: Monitoring the neural activity of freely behaving zebrafish.
Figure 2: Neuroluminescence and behavior of Nβt–GFP-Aequorin zebrafish.
Figure 3: Targeted GFP-apoAequorin expression in Hypocretin neurons.
Figure 4: Activity in Hypocretin neurons during natural behavior.
Figure 5: Bioluminescent photons are generated by the GFP-Aequorin-targeted HCRT neurons.
Figure 6: Temporally gated detection for monitoring neuroluminescence during visual stimulation.

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References

  1. Kralik, J.D. et al. Techniques for long-term multisite neuronal ensemble recordings in behaving animals. Methods 25, 121–150 (2001).

    Article  CAS  Google Scholar 

  2. Miller, E.K. & Wilson, M.A. All my circuits: using multiple electrodes to understand functioning neural networks. Neuron 60, 483–488 (2008).

    Article  CAS  Google Scholar 

  3. Luo, L., Callaway, E.M. & Svoboda, K. Genetic dissection of neural circuits. Neuron 57, 634–660 (2008).

    Article  CAS  Google Scholar 

  4. Brustein, E., Marandi, N., Kovalchuk, Y., Drapeau, P. & Konnerth, A. In vivo monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging. Pflugers Arch. 446, 766–773 (2003).

    Article  CAS  Google Scholar 

  5. Douglass, A.D., Kraves, S., Deisseroth, K., Schier, A.F. & Engert, F. Escape behavior elicited by single, Channelrhodopsin-2–evoked spikes in zebrafish somatosensory neurons. Curr. Biol. 18, 1133–1137 (2008).

    Article  CAS  Google Scholar 

  6. Niell, C.M. & Smith, S.J. Functional imaging reveals rapid development of visual response properties in the zebrafish tectum. Neuron 45, 941–951 (2005).

    Article  CAS  Google Scholar 

  7. Ramdya, P. & Engert, F. Emergence of binocular functional properties in a monocular neural circuit. Nat. Neurosci. 11, 1083–1090 (2008).

    Article  CAS  Google Scholar 

  8. O'Malley, D.M., Kao, Y.H. & Fetcho, J.R. Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 17, 1145–1155 (1996).

    Article  CAS  Google Scholar 

  9. Higashijima, S., Masino, M.A., Mandel, G. & Fetcho, J.R. Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator. J. Neurophysiol. 90, 3986–3997 (2003).

    Article  Google Scholar 

  10. Orger, M.B., Kampff, A.R., Severi, K.E., Bollmann, J.H. & Engert, F. Control of visually guided behavior by distinct populations of spinal projection neurons. Nat. Neurosci. 11, 327–333 (2008).

    Article  CAS  Google Scholar 

  11. McLean, D.L., Fan, J., Higashijima, S., Hale, M.E. & Fetcho, J.R. A topographic map of recruitment in spinal cord. Nature 446, 71–75 (2007).

    Article  CAS  Google Scholar 

  12. Gahtan, E., Sankrithi, N., Campos, J.B. & O'Malley, D.M. Evidence for a widespread brain stem escape network in larval zebrafish. J. Neurophysiol. 87, 608–614 (2002).

    Article  Google Scholar 

  13. Dombeck, D.A., Khabbaz, A.N., Collman, F., Adelman, T.L. & Tank, D.W. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 43–57 (2007).

    Article  CAS  Google Scholar 

  14. Briggman, K.L., Abarbanel, H.D. & Kristan, W.B. Jr. Optical imaging of neuronal populations during decision-making. Science 307, 896–901 (2005).

    Article  CAS  Google Scholar 

  15. Clark, D.A., Gabel, C.V., Gabel, H. & Samuel, A.D. Temporal activity patterns in thermosensory neurons of freely moving Caenorhabditis elegans encode spatial thermal gradients. J. Neurosci. 27, 6083–6090 (2007).

    Article  CAS  Google Scholar 

  16. Baubet, V. et al. Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc. Natl. Acad. Sci. USA 97, 7260–7265 (2000).

    Article  CAS  Google Scholar 

  17. Daunert, S. & Deo, S.K. Photoproteins in Bioanalysis. (Wiley-VCH, Weinheim, Germany, 2006).

    Book  Google Scholar 

  18. Smith, S.J. & Zucker, R.S. Aequorin response facilitation and intracellular calcium accumulation in molluscan neurones. J. Physiol. (Lond.) 300, 167–196 (1980).

    Article  CAS  Google Scholar 

  19. Ashley, C.C. & Ridgway, E.B. Simultaneous recording of membrane potential, calcium transient and tension in single muscle fibers. Nature 219, 1168–1169 (1968).

    Article  CAS  Google Scholar 

  20. Hastings, J.W. & Johnson, C.H. Bioluminescence and chemiluminescence. Methods Enzymol. 360, 75–104 (2003).

    Article  CAS  Google Scholar 

  21. Shimomura, O., Musicki, B., Kishi, Y. & Inouye, S. Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium 14, 373–378 (1993).

    Article  CAS  Google Scholar 

  22. Curie, T., Rogers, K.L., Colasante, C. & Brulet, P. Red-shifted aequorin-based bioluminescent reporters for in vivo imaging of Ca2+ signaling. Mol. Imaging 6, 30–42 (2007).

    Article  CAS  Google Scholar 

  23. Martin, J.R., Rogers, K.L., Chagneau, C. & Brulet, P. In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila. PLoS One 2, e275 (2007).

    Article  Google Scholar 

  24. Rogers, K.L. et al. Non-invasive in vivo imaging of calcium signaling in mice. PLoS One 2, e974 (2007).

    Article  Google Scholar 

  25. Rogers, K.L. et al. Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters. Eur. J. Neurosci. 21, 597–610 (2005).

    Article  Google Scholar 

  26. Cheung, C.Y., Webb, S.E., Meng, A. & Miller, A.L. Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development. Int. J. Dev. Biol. 50, 561–569 (2006).

    Article  CAS  Google Scholar 

  27. Teranishi, K. & Shimomura, O. Solubilizing coelenterazine in water with hydroxypropyl-β-cyclodextrin. Biosci. Biotechnol. Biochem. 61, 1219–1220 (1997).

    Article  CAS  Google Scholar 

  28. Baraban, S.C. Emerging epilepsy models: insights from mice, flies, worms and fish. Curr. Opin. Neurol. 20, 164–168 (2007).

    Article  CAS  Google Scholar 

  29. Tallini, Y.N. et al. Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc. Natl. Acad. Sci. USA 103, 4753–4758 (2006).

    Article  CAS  Google Scholar 

  30. Park, H.C. et al. Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. Dev. Biol. 227, 279–293 (2000).

    Article  CAS  Google Scholar 

  31. Sakurai, T. The neural circuit of orexin (hypocretin): maintaining sleep and wakefulness. Nat. Rev. Neurosci. 8, 171–181 (2007).

    Article  CAS  Google Scholar 

  32. Prober, D.A., Rihel, J., Onah, A.A., Sung, R.J. & Schier, A.F. Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. J. Neurosci. 26, 13400–13410 (2006).

    Article  CAS  Google Scholar 

  33. Yokogawa, T. et al. Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants. PLoS Biol. 5, e277 (2007).

    Article  Google Scholar 

  34. Chemelli, R.M. et al. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98, 437–451 (1999).

    CAS  PubMed  Google Scholar 

  35. Lin, L. et al. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98, 365–376 (1999).

    Article  CAS  Google Scholar 

  36. Mileykovskiy, B.Y., Kiyashchenko, L.I. & Siegel, J.M. Behavioral correlates of activity in identified hypocretin/orexin neurons. Neuron 46, 787–798 (2005).

    Article  CAS  Google Scholar 

  37. Lee, M.G., Hassani, O.K. & Jones, B.E. Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle. J. Neurosci. 25, 6716–6720 (2005).

    Article  CAS  Google Scholar 

  38. Adamantidis, A.R., Zhang, F., Aravanis, A.M., Deisseroth, K. & de Lecea, L. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450, 420–424 (2007).

    Article  CAS  Google Scholar 

  39. Mank, M. & Griesbeck, O. Genetically encoded calcium indicators. Chem. Rev. 108, 1550–1564 (2008).

    Article  CAS  Google Scholar 

  40. Tricoire, L. et al. Calcium dependence of aequorin bioluminescence dissected by random mutagenesis. Proc. Natl. Acad. Sci. USA 103, 9500–9505 (2006).

    Article  CAS  Google Scholar 

  41. Roncali, E. et al. New device for real-time bioluminescence imaging in moving rodents. J. Biomed. Opt. 13, 054035 (2008).

    Article  Google Scholar 

  42. Drobac, E., Tricoire, L., Chaffotte, A.F., Guiot, E. & Lambolez, B. Calcium imaging in single neurons from brain slices using bioluminescent reporters. J. Neurosci. Res. 88, 695–711 (2009).

    Google Scholar 

  43. Pologruto, T.A., Yasuda, R. & Svoboda, K. Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J. Neurosci. 24, 9572–9579 (2004).

    Article  CAS  Google Scholar 

  44. Scott, E.K. et al. Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat. Methods 4, 323–326 (2007).

    Article  CAS  Google Scholar 

  45. Lillesaar, C., Tannhauser, B., Stigloher, C., Kremmer, E. & Bally-Cuif, L. The serotonergic phenotype is acquired by converging genetic mechanisms within the zebrafish central nervous system. Dev. Dyn. 236, 1072–1084 (2007).

    Article  CAS  Google Scholar 

  46. Wen, L. et al. Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev. Biol. 314, 84–92 (2008).

    Article  CAS  Google Scholar 

  47. Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

    Article  CAS  Google Scholar 

  48. Deisseroth, K. et al. Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26, 10380–10386 (2006).

    Article  CAS  Google Scholar 

  49. Plautz, J.D., Kaneko, M., Hall, J.C. & Kay, S.A. Independent photoreceptive circadian clocks throughout Drosophila. Science 278, 1632–1635 (1997).

    Article  CAS  Google Scholar 

  50. Flusberg, B.A. et al. High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat. Methods 5, 935–938 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank W. Hastings and T. Wilson for bountiful advice and discussion and generously providing an intensified CCD camera. We also thank L. Tricoire for the kind gift of the GFP-apoAequorin construct, M. Orger, A. Douglass, P. Ramdya, and members of the Engert and Schier laboratories for comments and advice, A. Douglass for Nβt-gal4 vectors, P. Ramdya for providing the nacre strains and B. Obama for his stimulation package. We thank S. Zimmerman, K. Hurley, and J. Miller for excellent zebrafish care. This work was funded by the McKnight Foundation (F.E.), the Harvard Mind, Brain and Behavior post-doctoral fellows program (A.R.K.), and the US National Institutes of Health (A.F.S. and D.A.P.).

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Author notes

  1. Eva A Naumann and Adam R Kampff: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA

    Eva A Naumann, Adam R Kampff, Alexander F Schier & Florian Engert

  2. Division of Biology, California Institute of Technology, Pasadena, California, USA

    David A Prober

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  1. Eva A Naumann
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Contributions

E.A.N. and A.R.K. designed the assay and performed the experiments. E.A.N., A.R.K. and F.E. analyzed the data. D.A.P. and A.F.S. generated the HCRT–GFP-apoAequorin transgenic line and assisted with behavioral analysis. E.A.N., A.R.K., D.A.P., A.F.S. and F.E. prepared the manuscript. E.A.N. suffered the most.

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Correspondence to Florian Engert.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 9118 kb)

Supplementary Video 1

Spontaneous and evoked neuroluminescence in freely swimming zebrafish. (WMV 1201 kb)

Supplementary Video 2

Neuroluminescence shortly after exposure to PTZ. (WMV 2487 kb)

Supplementary Video 3

Neuroluminescence after 20 min exposure to PTZ. (WMV 2974 kb)

Supplementary Video 4

Neuroluminescence after exposure to PTZ in paralysed zebrafish. (WMV 1708 kb)

Supplementary Video 5

Fluorescence changes in HuC:GCaMP2 zebrafish exposed to PTZ. (WMV 2451 kb)

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Naumann, E., Kampff, A., Prober, D. et al. Monitoring neural activity with bioluminescence during natural behavior. Nat Neurosci 13, 513–520 (2010). https://doi.org/10.1038/nn.2518

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