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In vivo single-cell excitability probing of neuronal ensembles in the intact and awake developing Xenopus brain

Nature Protocols volume 5, pages 841–848 (2010)Cite this article

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Abstract

Sensory experience can elicit long-lasting plasticity of both single neurons and ensemble neural circuit response properties during embryonic development. To investigate their relationship, one must image functional responses of large neuronal populations simultaneously with single-cell resolution. In this protocol, we describe a noninvasive approach to assay functional plasticity of individual neurons and neuronal populations in vivo using targeted infusion of calcium-sensitive dyes, two-photon microscopy and synchronized visual stimuli presentations. This technique allows visualization of ∼200 neurons while probing visual responses in the optic tectum of awake, immobilized Xenopus laevis tadpoles. The protocol includes visual training paradigms that elicit long-lasting potentiation or depression of functional responses, allowing investigations of population and single-neuron plasticity induced by natural sensory stimuli in the awake, intact, developing brain. Setup time for this protocol, including dye injection and chamber preparation, is ∼2 h. Excitability probing experiments can then be performed for at least 3 h.

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Figure 1: Single-cell excitability probing (SCEP) equipment setup.
Figure 2: Calcium-indicator loading of the tadpole optic tectum.
Figure 3: Scanning triggered visual stimuli.
Figure 4: In vivo single-cell excitability probing (SCEP) of wide-field visual stimuli in the awake, developing brain.

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References

  1. Sin, W.C., Haas, K., Ruthazer, E.S. & Cline, H.T. Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419, 475–480 (2002).

    Article  CAS  Google Scholar 

  2. Ruthazer, E.S., Akerman, C.J. & Cline, H.T. Control of axon branch dynamics by correlated activity in vivo. Science 301, 66–70 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Haas, K., Li, J.L. & Cline, H.T. AMPA receptors regulate experience-dependent dendritic arbor growth in vivo. Proc. Natl. Acad. Sci.USA 103, 12127–12131 (2006).

    Article  CAS  Google Scholar 

  5. Cline, H. & Haas, K. The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J. Physiol. (London) 586, 1509–1517 (2008).

    Article  CAS  Google Scholar 

  6. Aizenman, C.D., Akerman, C.J., Jensen, K.R. & Cline, H.T. Visually driven regulation of intrinsic neuronal excitability improves stimulus detection in vivo. Neuron 39, 831–842 (2003).

    Article  CAS  Google Scholar 

  7. Aizenman, C.D., Munoz-Elias, G. & Cline, H.T. Visually driven modulation of glutamatergic synaptic transmission is mediated by the regulation of intracellular polyamines. Neuron 34, 623–634 (2002).

    Article  CAS  Google Scholar 

  8. Zhang, L.I., Tao, H.W. & Poo, M. Visual input induces long-term potentiation of developing retinotectal synapses. Nat. Neurosci. 3, 708–715 (2000).

    Article  CAS  Google Scholar 

  9. Engert, F., Tao, H.W., Zhang, L.I. & Poo, M.M. Moving visual stimuli rapidly induce direction sensitivity of developing tectal neurons. Nature 419, 470–475 (2002).

    Article  CAS  Google Scholar 

  10. Espinosa, J.S., Wheeler, D.G., Tsien, R.W. & Luo, L. Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62, 205–17 (2009).

    Article  CAS  Google Scholar 

  11. Wiesel, T.N. & Hubel, D.H. Single cell responses in striate cortex of kittens deprived of vision in one eye. J. Neurophysiol. 26, 1003–1017 (1963).

    Article  CAS  Google Scholar 

  12. Gordon, J.A. & Stryker, M.P. Experience-dependent plasticity of binocular responses in the primary visual cortex of the mouse. J. Neurosci. 16, 3274–3286 (1996).

    Article  CAS  Google Scholar 

  13. Gaze, R.M., Keating, M.J. & Chung, S.H. The evolution of the retinotectal map during development in Xenopus. Proc. R. Soc. Lond., B Biol. Sci. 185, 301–330 (1974).

    Article  CAS  Google Scholar 

  14. Sretavan, D. & Shatz, C.J. Prenatal development of individual retinogeniculate axons during the period of segregation. Nature 308, 845–848 (1984).

    Article  CAS  Google Scholar 

  15. Sakaguchi, D.S. & Murphey, R.K. Map formation in the developing Xenopus retinotectal system: an examination of ganglion cell terminal arborizations. J. Neurosci. 5, 3228–3245 (1985).

    Article  CAS  Google Scholar 

  16. Vislay-Meltzer, R.L., Kampff, A.R. & Engert, F. Spatiotemporal specificity of neuronal activity directs the modification of receptive fields in the developing retinotectal system. Neuron 50, 101–114 (2006).

    Article  CAS  Google Scholar 

  17. Dunfield, D. & Haas, K. Metaplasticity governs natural experience-driven plasticity of nascent embryonic brain circuits. Neuron 64, 240–250 (2009).

    Article  CAS  Google Scholar 

  18. Zhou, Q., Tao, H.W. & Poo, M.M. Reversal and stabilization of synaptic modifications in a developing visual system. Science 300, 1953–1957 (2003).

    Article  CAS  Google Scholar 

  19. Wong, R.O. Calcium imaging and multielectrode recordings of global patterns of activity in the developing nervous system. Histochem. J. 30, 217–229 (1998).

    Article  CAS  Google Scholar 

  20. Takahashi, N., Sasaki, T., Usami, A., Matsuki, N. & Ikegaya, Y. Watching neuronal circuit dynamics through functional multineuron calcium imaging (fMCI). Neurosci. Res. 58, 219–225 (2007).

    Article  CAS  Google Scholar 

  21. Johenning, F.W. & Holthoff, K. Nuclear calcium signals during L-LTP induction do not predict the degree of synaptic potentiation. Cell Calcium 41, 271–283 (2007).

    Article  CAS  Google Scholar 

  22. Stocca, G., Schmidt-Hieber, C. & Bischofberger, J. Differential dendritic Ca2+ signalling in young and mature hippocampal granule cells. J. Physiol. 586, 3795–3811 (2008).

    Article  CAS  Google Scholar 

  23. 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 

  24. 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 

  25. Fetcho, J.R., Cox, K.J. & O'Malley, D.M. Monitoring activity in neuronal populations with single-cell resolution in a behaving vertebrate. Histochem. J. 30, 153–167 (1998).

    Article  CAS  Google Scholar 

  26. Ramdya, P., Reiter, B. & Engert, F. Reverse correlation of rapid calcium signals in the zebrafish optic tectum in vivo. J. Neurosci. Methods 157, 230–237 (2006).

    Article  CAS  Google Scholar 

  27. Yaksi, E. & Friedrich, R.W. Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging. Nat. Methods 3, 377–383 (2006).

    Article  CAS  Google Scholar 

  28. Sumbre, G., Muto, A., Baier, H. & Poo, M.-m. Entrained rhythmic activities of neuronal ensembles as perceptual memory of time interval. Nature 456, 102–106 (2008).

    Article  CAS  Google Scholar 

  29. Smetters, D., Majewska, A. & Yuste, R. Detecting action potentials in neuronal populations with calcium imaging. Methods 18, 215–221 (1999).

    Article  CAS  Google Scholar 

  30. Kerr, J.N.D., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).

    Article  CAS  Google Scholar 

  31. Greenberg, D.S., Houweling, A.R. & Kerr, J.N.D. Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat. Neurosci. 11, 749–751 (2008).

    Article  CAS  Google Scholar 

  32. Sasaki, T., Takahashi, N., Matsuki, N. & Ikegaya, Y. Fast and accurate detection of action potentials from somatic calcium fluctuations. J. Neurophysiol. 100, 1668–1676 (2008).

    Article  CAS  Google Scholar 

  33. Junek, S., Chen, T., Alevra, M. & Schild, D. Activity correlation imaging: visualizing function and structure of neuronal populations. Biophys. J. 96, 3801–3809 (2009).

    Article  CAS  Google Scholar 

  34. Zhang, L.I., Tao, H.W., Holt, C.E., Harris, W.A. & Poo, M. A critical window for cooperation and competition among developing retinotectal synapses. Nature 395, 37–44 (1998).

    Article  CAS  Google Scholar 

  35. Tao, H.W. & Poo, M.M. Activity-dependent matching of excitatory and inhibitory inputs during refinement of visual receptive fields. Neuron 45, 829–836 (2005).

    Article  CAS  Google Scholar 

  36. Mu, Y. & Poo, M.M. Spike timing-dependent LTP/LTD mediates visual experience-dependent plasticity in a developing retinotectal system. Neuron 50, 115–125 (2006).

    Article  CAS  Google Scholar 

  37. Nieuwkoop, P.D. & Faber, J. Normal Table of Xenopus Laevis (North Holland, Amsterdam, 1967).

  38. Chen, C. Heterosynaptic LTP in early development. Neuron 31, 510–512 (2001).

    Article  CAS  Google Scholar 

  39. Bhatt, D.H., Otto, S.J., Depoister, B. & Fetcho, J.R. Cyclic AMP-induced repair of zebrafish spinal circuits. Science 305, 254–258 (2004).

    Article  CAS  Google Scholar 

  40. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    Article  CAS  Google Scholar 

  41. Xu, C. Two-photon cross sections of indicators. in Imaging Neurons: A Laboratory Manual (ed. R. Yuste, F. Lanni & A. Konnerth) 191–199 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2000).

  42. Tao, H.W., Zhang, L.I., Engert, F. & Poo, M. Emergence of input specificity of ltp during development of retinotectal connections in vivo. Neuron 31, 569–580 (2001).

    Article  CAS  Google Scholar 

  43. Thévenaz, P., Ruttimann, U.E. & Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998).

    Article  Google Scholar 

  44. Sperry, R.W. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl. Acad. Sci. USA 50, 703–710 (1963).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Science and Engineering Council of Canada, the Canadian Institute of Health Research, the Michael Smith Foundation for Health Research, the Canadian Foundation for Innovation, The EJLB Foundation and the Human Early Learning Project.

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  1. Department of Cellular and Physiological Sciences and the Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada

    Derek Dunfield & Kurt Haas

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  1. Derek Dunfield
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Correspondence to Kurt Haas.

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Dunfield, D., Haas, K. In vivo single-cell excitability probing of neuronal ensembles in the intact and awake developing Xenopus brain. Nat Protoc 5, 841–848 (2010). https://doi.org/10.1038/nprot.2010.10

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