Abstract
Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 μm below the pia. In addition, we found that RAMM could be used to simultaneously image activity from large (∼80) populations of apical dendrites and follow these dendrites down to their somata of origin.
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References
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Kerr, J.N. et al. Spatial organization of neuronal population responses in layer 2/3 of rat barrel cortex. J. Neurosci. 27, 13316–13328 (2007).
Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).
Sato, T.R., Gray, N.W., Mainen, Z.F. & Svoboda, K. The functional microarchitecture of the mouse barrel cortex. PLoS Biol. 5, e189 (2007).
Rothschild, G., Nelken, I. & Mizrahi, A. Functional organization and population dynamics in the mouse primary auditory cortex. Nat. Neurosci. 13, 353–360 (2010).
Dombeck, D.A., Graziano, M.S. & Tank, D.W. Functional clustering of neurons in motor cortex determined by cellular resolution imaging in awake behaving mice. J. Neurosci. 29, 13751–13760 (2009).
Kerr, J.N., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).
Hasan, M.T. et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, e163 (2004).
Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).
Wallace, D.J. et al. Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat. Methods 5, 797–804 (2008).
Lavis, L.D. & Raines, R.T. Bright ideas for chemical biology. ACS Chem. Biol. 3, 142–155 (2008).
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).
Greenberg, D.S., Houweling, A.R. & Kerr, J.N. Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat. Neurosci. 11, 749–751 (2008).
Helmchen, F., Svoboda, K., Denk, W. & Tank, D.W. In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nat. Neurosci. 2, 989–996 (1999).
Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399–405 (2010).
Mrsic-Flogel, T.D. et al. Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity. Neuron 54, 961–972 (2007).
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).
Nimmerjahn, A., Kirchhoff, F., Kerr, J.N. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).
Theer, P. & Denk, W. On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 3139–3149 (2006).
Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).
Kerr, J.N. & Denk, W. Imaging in vivo: watching the brain in action. Nat. Rev. Neurosci. 9, 195–205 (2008).
Theer, P. Combined Faculties for the Natural Sciences and for Mathematics, Vol. 100, Doctor of Natural Sciences. University of Heidelberg (2004).
Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J. & Charpak, S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J. Neurosci. Methods 111, 29–37 (2001).
Sawinski, J. et al. Visually evoked activity in cortical cells imaged in freely moving animals. Proc. Natl. Acad. Sci. USA 106, 19557–19562 (2009).
de Kock, C.P., Bruno, R.M., Spors, H. & Sakmann, B. Layer- and cell type–specific suprathreshold stimulus representation in rat primary somatosensory cortex. J. Physiol. (Lond.) 581, 139–154 (2007).
Meyer, H.S. et al. Number and laminar distribution of neurons in a thalamocortical projection column of rat vibrissal cortex. Cereb. Cortex 20, 2277–2286 (2010).
de la Rocha, J., Doiron, B., Shea-Brown, E., Josic, K. & Reyes, A. Correlation between neural spike trains increases with firing rate. Nature 448, 802–806 (2007).
Stuart, G.J. & Sakmann, B. Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367, 69–72 (1994).
Larkum, M.E., Nevian, T., Sandler, M., Polsky, A. & Schiller, J. Synaptic integration in tuft dendrites of layer 5 pyramidal neurons: a new unifying principle. Science 325, 756–760 (2009).
Polsky, A., Mel, B.W. & Schiller, J. Computational subunits in thin dendrites of pyramidal cells. Nat. Neurosci. 7, 621–627 (2004).
Stuart, G., Schiller, J. & Sakmann, B. Action potential initiation and propagation in rat neocortical pyramidal neurons. J. Physiol. (Lond.) 505, 617–632 (1997).
Margrie, T.W. et al. Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39, 911–918 (2003).
Tsai, P.S. et al. Spherical aberration correction in nonlinear microscopy and optical ablation using a transparent deformable membrane. Appl. Phys. Lett. 91, 191102 (2007).
Ji, N., Milkie, D.E. & Betzig, E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat. Methods 7, 141–147 (2010).
Rueckel, M., Mack-Bucher, J.A. & Denk, W. Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing. Proc. Natl. Acad. Sci. USA 103, 17137–17142 (2006).
Denk, W. Optical beam power controller using a tiltable birefringent plate. US patent 6,249,379 B1 (2001).
Shevtsova, Z., Malik, J.M., Michel, U., Bahr, M. & Kugler, S. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp. Physiol. 90, 53–59 (2005).
Zolotukhin, S. et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28, 158–167 (2002).
Acknowledgements
We would like to thank P. Theer for technical help with the RegA, J. Sawinski for help with detection system fabrication and D.S. Greenberg for dendrite morphology analysis. This work was supported by the Max Planck Society.
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W.M., D.J.W., W.D. and J.N.D.K. designed the research. W.M., D.J.W. and J.N.D.K. performed the research. D.J.W. and J.N.D.K. analyzed the data. U.C. performed the histological reconstructions. J.T.H., A.T.S. and L.L.L. produced the virus. W.M., D.J.W., J.N.D.K. and W.D. wrote the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–5 (PDF 3980 kb)
Supplementary Movie 1
Z-stack from pia to layer 5b (AVI 31015 kb)
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Mittmann, W., Wallace, D., Czubayko, U. et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat Neurosci 14, 1089–1093 (2011). https://doi.org/10.1038/nn.2879
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DOI: https://doi.org/10.1038/nn.2879
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