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Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2

Abstract

A major long-term goal of systems neuroscience is to identify the different roles of neural subtypes in brain circuit function. The ability to causally manipulate selective cell types is critical to meeting this goal. This protocol describes techniques for optically stimulating specific populations of excitatory neurons and inhibitory interneurons in vivo in combination with electrophysiology. Cell type selectivity is obtained using Cre-dependent expression of the light-activated channel Channelrhodopsin-2. We also describe approaches for minimizing optical interference with simultaneous extracellular and intracellular recording. These optogenetic techniques provide a spatially and temporally precise means of studying neural activity in the intact brain and allow a detailed examination of the effect of evoked activity on the surrounding local neural network. Injection of viral vectors requires 30–45 min, and in vivo electrophysiology with optogenetic stimulation requires 1–4 h.

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Figure 1: AAV DIO ChR2-mCherry gives Cre-dependent and cell-type-specific expression of light-activated channels in vivo.
Figure 2: Neural activity evoked in vivo by activation of cell-type-specific expression of ChR2.
Figure 3: Elimination of light-induced artifacts in recordings of the local field potential.

References

  1. Ascoli, G.A. et al. Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nat. Rev. Neurosci. 9, 557–568 (2008).

    CAS  Article  PubMed  Google Scholar 

  2. Connors, B.W., Gutnick, M.J. & Prince, D.A. Electrophysiological properties of neocortical neurons in vitro . J. Neurophysiol. 48, 1302–1320 (1982).

    CAS  Article  PubMed  Google Scholar 

  3. Kawaguchi, Y. & Kubota, Y. GABAergic cell subtypes and their synaptic connections in rat frontal cortex. Cereb. Cortex 7, 476–486 (1997).

    CAS  Article  PubMed  Google Scholar 

  4. Markram, H. et al. Interneurons of the neocortical inhibitory system. Nat. Rev. Neurosci. 5, 793–807 (2004).

    CAS  Article  PubMed  Google Scholar 

  5. McCormick, D.A., Connors, B.W., Lighthall, J.W. & Prince, D.A. Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex. J. Neurophysiol. 54, 782–806 (1985).

    CAS  Article  PubMed  Google Scholar 

  6. Kuhlman, S.J. & Huang, Z.J. High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression. PLoS ONE 3, e2005 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

    CAS  Article  PubMed  Google Scholar 

  8. Sohal, V.S., Zhang, F., Yizhar, O. & Deisseroth, K. Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature 459, 698–702 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).

    CAS  Article  PubMed  Google Scholar 

  10. Petreanu, L., Mao, T., Sternson, S.M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Tsai, H.C. et al. Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324, 1080–1084 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Cardin, J.A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Han, X. et al. Millisecond-timescale optical control of neural dynamics in the nonhuman primate brain. Neuron 62, 191–198 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451, 61–64 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Gradinaru, V., Mogri, M., Thompson, K.R., Henderson, J.M. & Deisseroth, K. Optical deconstruction of parkinsonian neural circuitry. Science 324, 354–359 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Arenkiel, B.R. et al. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54, 205–218 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Marin, O. & Rubenstein, J.L. Cell migration in the forebrain. Annu. Rev. Neurosci. 26, 441–483 (2003).

    CAS  Article  PubMed  Google Scholar 

  18. Wonders, C. & Anderson, S.A. Cortical interneurons and their origins. Neuroscientist 11, 199–205 (2005).

    Article  PubMed  Google Scholar 

  19. Saito, T. & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237–246 (2001).

    CAS  Article  PubMed  Google Scholar 

  20. Tabata, H. & Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience 103, 865–872 (2001).

    CAS  Article  PubMed  Google Scholar 

  21. Tabata, H. & Nakajima, K. Neurons tend to stop migration and differentiate along the cortical internal plexiform zones in the Reelin signal-deficient mice. J. Neurosci. Res. 69, 723–730 (2002).

    CAS  Article  PubMed  Google Scholar 

  22. Tabata, H. & Nakajima, K. Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex. J. Neurosci. 23, 9996–10001 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Borrell, V., Yoshimura, Y. & Callaway, E.M. Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J. Neurosci. Methods 143, 151–158 (2005).

    CAS  Article  PubMed  Google Scholar 

  24. Taymans, J.M. et al. Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum. Gene Ther. 18, 195–206 (2007).

    CAS  Article  PubMed  Google Scholar 

  25. Nathanson, J.L., Yanagawa, Y., Obata, K. & Callaway, E.M. Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors. Neuroscience 161, 441–450 (2009).

    CAS  Article  PubMed  Google Scholar 

  26. Atasoy, D., Aponte, Y., Su, H.H. & Sternson, S.M. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 7025–7030 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    CAS  Article  PubMed  Google Scholar 

  28. Fu, H. et al. Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol. Ther. 8, 911–917 (2003).

    CAS  Article  PubMed  Google Scholar 

  29. Van Vliet, K.M., Blouin, V., Brument, N., Agbandje-McKenna, M. & Snyder, R.O. The role of the adeno-associated virus capsid in gene transfer. Methods Mol. Biol. 437, 51–91 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Jasnow, A.M., Rainnie, D.G., Maguschak, K.A., Chhatwal, J.P. & Ressler, K.J. Construction of cell-type specific promoter lentiviruses for optically guiding electrophysiological recordings and for targeted gene delivery. Methods Mol. Biol. 515, 199–213 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Tenenbaum, L. et al. Recombinant AAV-mediated gene delivery to the central nervous system. J. Gene Med. 6 (Suppl 1): S212–S222 (2004).

    CAS  Article  PubMed  Google Scholar 

  32. Aravanis, A.M. et al. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural. Eng. 4, S143–S156 (2007).

    Article  PubMed  Google Scholar 

  33. Ayling, O.G., Harrison, T.C., Boyd, J.D., Goroshkov, A. & Murphy, T.H. Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice. Nat. Methods 6, 219–224 (2009).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank members of the Tsai and Moore laboratories for discussions and comments on the paper and M.J. Higley for help with optics. This study was supported by grants to C.I.M. from Tom F. Petersen, the NIH and the NSF, and by the Simons Foundation Autism Research Initiative to L.-H.T. K.D. was supported by the NIH Pioneer Program. L.-H.T. is an Investigator of the Howard Hughes Medical Institute. J.A.C. was supported by a K99 from the NIH/NEI; M.C. and K.M. by postdoctoral fellowships from the Knut och Alice Wallenberg Foundation; M.C. by a NARSAD Young Investigator Award; and F.Z. by an NIH NRSA.

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Authors

Contributions

F.Z. and K.D. designed and cloned the AAV DIO ChR2-mCherry vector; M.C. and K.M. characterized the virus in vitro and in vivo and injected the animals; M.C. performed histological analyses; J.A.C. developed the experimental paradigm, and performed and analyzed the extracellular recordings; U.K. and J.A.C. performed the intracellular recordings; U.K. analyzed the intracellular data; and J.A.C., M.C., K.M., U.K., L.-H.T., and C.I.M. wrote the paper.

Corresponding authors

Correspondence to Karl Deisseroth, Li-Huei Tsai or Christopher I Moore.

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Cardin, J., Carlén, M., Meletis, K. et al. Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2. Nat Protoc 5, 247–254 (2010). https://doi.org/10.1038/nprot.2009.228

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