Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Resource
  • Published:

Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice

Abstract

To facilitate a functional analysis of neuronal connectivity in a mammalian nervous system that is tightly packed with billions of cells, we developed a new technique that uses inducible genetic manipulations in fluorescently labeled single neurons in mice. Our technique, single-neuron labeling with inducible Cre-mediated knockout (SLICK), is achieved by coexpressing a drug-inducible form of Cre recombinase and a fluorescent protein in a small subsets of neurons, thus combining the powerful Cre recombinase system for conditional genetic manipulation with fluorescent labeling of single neurons for imaging. Here, we demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we applied SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals and demonstrate a Cre-loxP compatible system for dissecting gene functions in single identifiable neurons.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Strategy used for coexpression of YFP and Cre in SLICK transgenic mice.
Figure 2: YFP labeling of distinct neuronal populations in SLICK transgenic mice.
Figure 3: Genetic manipulation in SLICK transgenic mice.
Figure 4: Inhibition of neurotransmission in subsets of motor neurons through Chat knockout in SLICK-A mice.

Similar content being viewed by others

References

  1. International Mouse Knockout Consortium. Collins, F.S., Rossant, J. & Wurst, W. A mouse for all reasons. Cell 128, 9–13 (2007).

    Article  CAS  Google Scholar 

  2. Brecht, M. et al. Novel approaches to monitor and manipulate single neurons in vivo. J. Neurosci. 24, 9223–9227 (2004).

    Article  CAS  Google Scholar 

  3. Callaway, E.M. A molecular and genetic arsenal for systems neuroscience. Trends Neurosci. 28, 196–201 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Marek, K.W. & Davis, G.W. Controlling the active properties of excitable cells. Curr. Opin. Neurobiol. 13, 607–611 (2003).

    Article  CAS  Google Scholar 

  6. Miesenbock, G. Genetic methods for illuminating the function of neural circuits. Curr. Opin. Neurobiol. 14, 395–402 (2004).

    Article  CAS  Google Scholar 

  7. Miyawaki, A. Fluorescence imaging of physiological activity in complex systems using GFP-based probes. Curr. Opin. Neurobiol. 13, 591–596 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Young, P. & Feng, G. Labeling neurons in vivo for morphological and functional studies. Curr. Opin. Neurobiol. 14, 642–646 (2004).

    Article  CAS  Google Scholar 

  10. Zong, H., Espinosa, J.S., Su, H.H., Muzumdar, M.D. & Luo, L. Mosaic analysis with double markers in mice. Cell 121, 479–492 (2005).

    Article  CAS  Google Scholar 

  11. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

  12. Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    Article  CAS  Google Scholar 

  13. Kasthuri, N. & Lichtman, J.W. Structural dynamics of synapses in living animals. Curr. Opin. Neurobiol. 14, 105–111 (2004).

    Article  CAS  Google Scholar 

  14. Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).

    Article  CAS  Google Scholar 

  15. Mizrahi, A. & Katz, L.C. Dendritic stability in the adult olfactory bulb. Nat. Neurosci. 6, 1201–1207 (2003).

    Article  CAS  Google Scholar 

  16. Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752–757 (1997).

    Article  CAS  Google Scholar 

  17. Branda, C.S. & Dymecki, S.M. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004).

    Article  CAS  Google Scholar 

  18. Metzger, D. & Chambon, P. Site- and time-specific gene targeting in the mouse. Methods 24, 71–80 (2001).

    Article  CAS  Google Scholar 

  19. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).

    Article  CAS  Google Scholar 

  20. Burrone, J. & Murthy, V.N. Synaptic gain control and homeostasis. Curr. Opin. Neurobiol. 13, 560–567 (2003).

    Article  CAS  Google Scholar 

  21. Hua, J.Y. & Smith, S.J. Neural activity and the dynamics of central nervous system development. Nat. Neurosci. 7, 327–332 (2004).

    Article  CAS  Google Scholar 

  22. Misgeld, T. et al. Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron 36, 635–648 (2002).

    Article  CAS  Google Scholar 

  23. Brandon, E.P. et al. Aberrant patterning of neuromuscular synapses in choline acetyltransferase–deficient mice. J. Neurosci. 23, 539–549 (2003).

    Article  CAS  Google Scholar 

  24. Chen, C. & Tonegawa, S. Molecular genetic analysis of synaptic plasticity, activity-dependent neural development, learning, and memory in the mammalian brain. Annu. Rev. Neurosci. 20, 157–184 (1997).

    Article  CAS  Google Scholar 

  25. Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999).

    Article  CAS  Google Scholar 

  26. Wang, X. et al. Prolongation of evoked and spontaneous synaptic currents at the neuromuscular junction after activity blockade is caused by the upregulation of fetal acetylcholine receptors. J. Neurosci. 26, 8983–8987 (2006).

    Article  CAS  Google Scholar 

  27. Wang, X. et al. Activity-dependent presynaptic regulation of quantal size at the mammalian neuromuscular junction in vivo. J. Neurosci. 25, 343–351 (2005).

    Article  CAS  Google Scholar 

  28. Son, Y.J. & Thompson, W.J. Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells. Neuron 14, 133–141 (1995).

    Article  CAS  Google Scholar 

  29. Johns, D.C., Marx, R., Mains, R.E., O'Rourke, B. & Marban, E. Inducible genetic suppression of neuronal excitability. J. Neurosci. 19, 1691–1697 (1999).

    Article  CAS  Google Scholar 

  30. Sweeney, S.T., Broadie, K., Keane, J., Niemann, H. & O'Kane, C.J. Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341–351 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148 (2007).

    Article  CAS  Google Scholar 

  33. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  34. Lewandoski, M. Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743–755 (2001).

    Article  CAS  Google Scholar 

  35. Sauer, B. Inducible gene targeting in mice using the Cre/lox system. Methods 14, 381–392 (1998).

    Article  CAS  Google Scholar 

  36. Caroni, P. Overexpression of growth-associated proteins in the neurons of adult transgenic mice. J. Neurosci. Methods 71, 3–9 (1997).

    Article  CAS  Google Scholar 

  37. Hogan, B., Constantini, F. & Lacey, E. Production of transgenic mice. in Manipulating the Mouse Embryo: A Laboratory Manual (eds Hogan, B., Constantini, F. & Lacey, E.) 217–252 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1994).

    Google Scholar 

Download references

Acknowledgements

We thank J. Sanes for providing the Chat conditional-knockout mice and the antibody to β-galactosidase. We thank A. Mizrahi for performing two-photon imaging of SLICK-3 mice and R. Kotloski for advice regarding tamoxifen administration. We also thank the Duke Neurotransgenic Core Facility for generating the SLICK mice. We thank J. McNamara, M.D. Ehlers and M. McCaffrey for access to confocal microscopes. We are grateful to B. Arenkiel, M.D. Ehlers, A. West, K. Dean and members of the Feng laboratory for critically reading and discussing the manuscript. This work was supported by the Ruth K. Broad Biomedical Research Foundation, the Science Foundation Ireland and the US National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

P.Y. and G.F. conceived the SLICK method, designed the experiments and wrote the manuscript. P.Y. and J.G. generated the SLICK transgenic mice. P.Y., L.Q., S.Z. and D.W. characterized the SLICK mice. P.Y. and S.Z. carried out the quantification and data analysis.

Corresponding authors

Correspondence to Paul Young or Guoping Feng.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Methods and Discussion (PDF 6860 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Young, P., Qiu, L., Wang, D. et al. Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice. Nat Neurosci 11, 721–728 (2008). https://doi.org/10.1038/nn.2118

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2118

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing