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Deep brain optical measurements of cell type–specific neural activity in behaving mice

Nature Protocols volume 9, pages 1213–1228 (2014)Cite this article

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Abstract

Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)–based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca2+ indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3–4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.

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Figure 1: General scheme for using TCSPC-based photometry to measure the fluorescence of genetically encoded biosensors in vivo.
Figure 2: In vivo measurement of GCaMP5G fluorescence using TCSPC-based photometry.
Figure 3: Potential applications of a TCSPC-based fiber optics system for ratiometric measurement in a dual-fluorophore system and lifetime measurement in FRET-based biosensors.
Figure 4: Surgical procedures for intra-striatal virus injection and optical fiber implantation.
Figure 5: Procedures to make a hybrid optical fiber probe.
Figure 6: A series of snapshots of time-resolved spectra acquired during the process of lowering the fiber probe into the striatum.
Figure 7: Examples of fluorescence changes in freely moving mice expressing GCaMPs.

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References

  1. Looger, L.L. & Griesbeck, O. Genetically encoded neural activity indicators. Curr. Opin. Neurobiol. 22, 18–23 (2012).

    Article  CAS  Google Scholar 

  2. Kerr, J.N. & Denk, W. Imaging in vivo: watching the brain in action. Nat. Rev. Neurosci. 9, 195–205 (2008).

    Article  CAS  Google Scholar 

  3. Ghosh, K.K. et al. Miniaturized integration of a fluorescence microscope. Nat. Methods 8, 871–878 (2011).

    Article  CAS  Google Scholar 

  4. Cui, G. et al. Concurrent activation of striatal direct and indirect pathways during action initiation. Nature 494, 238–242 (2013).

    Article  CAS  Google Scholar 

  5. Wang, K.H. et al. In vivo two-photon imaging reveals a role of arc in enhancing orientation specificity in visual cortex. Cell 126, 389–402 (2006).

    Article  CAS  Google Scholar 

  6. Lleres, D., Swift, S. & Lamond, A.I. Detecting protein-protein interactions in vivo with FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). Curr. Protoc. Cytometry 12, 12.10 (2007).

    Google Scholar 

  7. Kettling, U., Koltermann, A., Schwille, P. & Eigen, M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc. Natl. Acad. Sci. USA 95, 1416–1420 (1998).

    Article  CAS  Google Scholar 

  8. Gong, Y., Li, J.Z. & Schnitzer, M.J. Enhanced archaerhodopsin fluorescent protein voltage indicators. PLoS ONE 8, e66959 (2013).

    Article  CAS  Google Scholar 

  9. Grimley, J.S. et al. Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation. J. Neurosci. 33, 16297–16309 (2013).

    Article  CAS  Google Scholar 

  10. Vogel, S.S., Blank, P.S., Koushik, S.V. & Thaler, C. in Laboratory Techniques in Biochemistry and Molecular Biology Vol. 33 (ed. Gadella, T.W.J.) 351–394 (Elsevier, 2009).

    Google Scholar 

  11. Elangovan, M., Day, R.N. & Periasamy, A. Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J. Microsc. 205, 3–14 (2002).

    Article  CAS  Google Scholar 

  12. Klarenbeek, J.B., Goedhart, J., Hink, M.A., Gadella, T.W. & Jalink, K. A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range. PLoS ONE 6, e19170 (2011).

    Article  CAS  Google Scholar 

  13. Harvey, C.D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008).

    Article  CAS  Google Scholar 

  14. Hoffmann, C. et al. A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells. Nat. Methods 2, 171–176 (2005).

    Article  CAS  Google Scholar 

  15. Dombeck, D.A., Harvey, C.D., Tian, L., Looger, L.L. & Tank, D.W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat. Neurosci 13, 1433–1440 (2010).

    Article  CAS  Google Scholar 

  16. Yelin, D., Bouma, B.E., Yun, S.H. & Tearney, G.J. Double-clad fiber for endoscopy. Optics Lett. 29, 2408–2410 (2004).

    Article  CAS  Google Scholar 

  17. Dunn, R.C. Near-field scanning optical microscopy. Chem. Rev. 99, 2891–2928 (1999).

    Article  CAS  Google Scholar 

  18. Gong, S. et al. Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J. Neurosci. 27, 9817–9823 (2007).

    Article  CAS  Google Scholar 

  19. Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank V. Jayaraman, R.A. Kerr, D.S. Kim, L.L. Looger, and K. Svoboda from the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute for allowing us to use GCaMP6s vectors. We thank L.L. Looger, J. Akerboom and D.S. Kim from the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute for permission to use GCaMP5G vectors; L.L. Looger and Janelia Farm Research Campus of the Howard Hughes Medical Institute for permission to use GCaMP3 vectors; C.R. Gerfen for gifts of multiple bacterial artificial chromosome transgenic mouse lines; and K. Jalink for gifts of DNA constructs of EPAC (exchange proteins directly activated by cAMP) sensors. This work was supported by US government funding from the Division of Intramural Clinical and Biological Research of the National Institute on Alcohol Abuse and Alcoholism to D.M.L., S.S.V. and R.M.C., by the European Research Council (STG 243393), and by an International Early Career Scientist grant from the Howard Hughes Medical Institute to R.M.C.; by a National Research Foundation of Korea grant (2011-0029485, 2012-0004003) and a Smart IT Convergence System Research Center grant (SIRC-2011-0031866) from the Korean government (MEST) to S.B.J.; and by an Ellison Medical Foundation grant (AG-NS-0944-12) to X.J.

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Authors and Affiliations

  1. Section on In Vivo Neural Function, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, USA

    Guohong Cui, Xin Jin, Guoxiang Luo, Michael D Pham, David M Lovinger & Rui M Costa

  2. Department of Electronics Engineering, Ewha Womans University, Seoul, Korea

    Sang Beom Jun

  3. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA

    Xin Jin

  4. Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, USA

    David M Lovinger

  5. Section on Cellular Biophotonics, Laboratory for Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, USA

    Steven S Vogel

  6. Champalimaud Neuroscience Programme at Instituto Gulbenkian de Ciência, Oeiras, Portugal

    Rui M Costa

  7. Champalimaud Centre for the Unknown, Lisbon, Portugal

    Rui M Costa

Authors
  1. Guohong Cui
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  7. Steven S Vogel
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Contributions

R.M.C. and S.S.V. conceived the original idea of using the TCSPC technique for optical equipment, and optimized procedures for in vivo optical recording. G.C. and S.B.J. carried out the in vivo experiments and analyzed data. X.J. helped with programming and data analysis. G.L. performed the DNA transfection in HEK cells and helped in maintaining mouse lines. M.D.P. performed initial in vitro experiments by using the TCSPC system and analyzed data. G.C., D.M.L., S.S.V. and R.M.C. wrote the paper.

Corresponding authors

Correspondence to Steven S Vogel or Rui M Costa.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 An alternative design of the optical pathway using a single fiber probe to convey excitation laser light and collect emitted photons.

1. AR-coated multimode fibre cable with 2 SMA connectors (e.g. Thorlabs cat. no. M200L02S-A). 2. Fibre coupler for SMA connectors (e.g.Thorlabs cat.no. PAF-SMA-11-A). 3. Fibre coupler adapter with threaded central bore for C mount (e.g. Thorlabs cat. no. HCL). 4. Adapter with external C mount and SM1 threads (e.g. Thorlabs cat. no. SM1A39). 5. Dichroic cube (e.g. Thorlabs cat. no. CM1-DCH). 6. Dichroic mirror for GFP (e.g. Thorlabs cat. no. MD498). 7. Fibre coupler for FC/PC connectors (e.g. Thorlabs cat. no. PAFA-X-4-A). 8. Optical fibre patchcord with a FC/PC connector at proximal end and a 1.25 (or 2.5) mm diameter ceramic ferule at distal end (e.g. Thorlabs cat. no. M83L01). 9. Ceramic mating sleeve for 1.25 (or 2.5) mm ferules (e.g. Thorlabs cat. no. ADAL1). 10. Implantable fibre optic cannula with 1.25 (or 2.5) mm ceramic ferrule (e.g. Thorlabs cat. no. CFMLC12U-20). 11. Stacked lens tube couplers (e.g. Thorlabs cat. no. SM1T2 and SM1T1). 12. End cap (e.g. Thorlabs cat.no. SM1CP2M). Inset a: A picture of assembled single-fibre system (corresponding to the parts in the blue rectangle) using the listed parts. Inset b: A time-resolved spectrum of GCaMP3 measured by the single-fibre system from the striatum of an A2A-Cre mouse. Note: In addition to commercially available parts for 8 and 10 listed here, users can also custom make them using optical fibres with smaller diameter such as the 105 μm core/ 125 μm cladding multimode fibres (e.g. Thorlabs cat. no. AFS105/125Y). To further reduce the excitation and collection volume, parts 8 and 10 can also be fabricated using single mode fibres (e.g. Thorlabs cat. no. 460HP), which provide additional advantages over multimode fibres such as less bending-induced light loss, smaller temporal and spectral dispersion of the laser pulse, and more even beam profile at the end. When ferrule-ferrule connection is included in the optical pathway, we find it essential to apply index matching material (e.g. Zeiss Immersol 518F) between the ferrules (part 8 and part 10 in the drawing). All animal protocols used in this study were approved by the US National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.

Supplementary Figure 2 Connections between equipment to synchronize optical recordings with behavior events.

Supplementary Figure 3 An example method to test the accuracy of timing in a 60-s-long optical file.

Top: Timestamps of commands programmed in MeD PC IV. Bottom: Illustration of expected change in recorded light intensity. In this experiment, the optical probe is placed in the operant chamber to measure the ambient light in the chamber. The 'acquisition start' signal will trigger the optical recording, which run on its own clock. The houselight on and off changes in the recorded optical file should match the timing of those commands programmed in MED PC IV.

Supplementary Figure 4 Example settings in SPCM for optical data acquisition.

Supplementary information

Supplementary Figure 1

An alternative design of the optical pathway using a single fiber probe to convey excitation laser light and collect emitted photons. (PDF 457 kb)

Supplementary Figure 2

Connections between equipment to synchronize optical recordings with behavior events. (PDF 671 kb)

Supplementary Figure 3

An example method to test the accuracy of timing in a 60-s-long optical file. (PDF 154 kb)

Supplementary Figure 4

Example settings in SPCM for optical data acquisition. (PDF 177 kb)

Supplementary Methods (PDF 113 kb)

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Cui, G., Jun, S., Jin, X. et al. Deep brain optical measurements of cell type–specific neural activity in behaving mice. Nat Protoc 9, 1213–1228 (2014). https://doi.org/10.1038/nprot.2014.080

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