Abstract
In vivo 2-photon imaging of neurons that have been bulk-loaded with fluorescent calcium indicator dyes is permitting many fundamental principles of neural circuit organization and development to be uncovered. In this article, we describe the materials and procedures that we have used in our investigations of ferrets, tree shrews, and mice. Special attention is given to the design and construction of custom stereotaxic devices and the prevention of stray light from entering the 2-photon microscope during vision experiments.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76
Göppert-Mayer M (1931) Über Elementarakte mit zwei Quantensprüngen. Ann Phys 401(3):273–294. doi:10.1002/andp.19314010303
Denk W, Svoboda K (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18(3):351–357
Piston DW (1999) Imaging living cells and tissues by two-photon excitation microscopy. Trends Cell Biol 9(2):66–69
Takahashi A, Camacho P, Lechleiter JD, Herman B (1999) Measurement of intracellular calcium. Physiol Rev 79(4):1089–1125
Euler T, Detwiler PB, Denk W (2002) Directionally selective calcium signals in dendrites of starburst amacrine cells. Nature 418(6900):845–852, 10.1038/nature00931
Svoboda K, Denk W, Kleinfeld D, Tank DW (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385(6612):161–165. doi:10.1038/385161a0
Yuste R, Denk W (1995) Dendritic spines as basic functional units of neuronal integration. Nature 375(6533):682–684. doi:10.1038/375682a0
Yuste R, Tank DW (1996) Dendritic integration in mammalian neurons, a century after Cajal. Neuron 16:701–716
Grienberger C, Konnerth A (2012) Imaging calcium in neurons. Neuron 73(5):862–885. doi:10.1016/j.neuron.2012.02.011
Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100(12):7319–7324
Tsien RY (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290(5806):527–528
Probes M (2010) Acetoxymethyl (AM) and acetate esters. http://tools.invitrogen.com/content/sfs/manuals/g002.pdf
Kara P, Boyd JD (2009) A micro-architecture for binocular disparity and ocular dominance in visual cortex. Nature 458(7238):627–631. doi:10.1038/nature07721
Ohki K, Chung S, Ch'ng YH, Kara P, Reid RC (2005) Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433(7026):597–603
Ohki K, Chung S, Kara P, Hubener M, Bonhoeffer T, Reid RC (2006) Highly ordered arrangement of single neurons in orientation pinwheels. Nature 442(7105):925–928
Huber D, Gutnisky DA, Peron S, O'Connor DH, Wiegert JS, Tian L, Oertner TG, Looger LL, Svoboda K (2012) Multiple dynamic representations in the motor cortex during sensorimotor learning. Nature 484(7395):473–478. doi:10.1038/nature11039
Li Y, Vanhooser SD, Mazurek M, White LE, Fitzpatrick D (2008) Experience with moving visual stimuli drives the early development of cortical direction selectivity. Nature 456(7224):952–956, doi:nature07417 [pii]10.1038/nature07417
Van Hooser SD, Li Y, Christensson M, Smith GB, White LE, Fitzpatrick D (2012) Initial neighborhood biases and the quality of motion stimulation jointly influence the rapid emergence of direction preference in visual cortex. J Neurosci 32(21):7258–7266. doi:10.1523/JNEUROSCI.0230-12.2012
Kerlin AM, Andermann ML, Berezovskii VK, Reid RC (2010) Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex. Neuron 67(5):858–871. doi:10.1016/j.neuron.2010.08.002
Kuhlman SJ, Tring E, Trachtenberg JT (2011) Fast-spiking interneurons have an initial orientation bias that is lost with vision. Nat Neurosci 14(9):1121–1123. doi:10.1038/nn.2890
Runyan CA, Schummers J, Van Wart A, Kuhlman SJ, Wilson NR, Huang ZJ, Sur M (2010) Response features of parvalbumin-expressing interneurons suggest precise roles for subtypes of inhibition in visual cortex. Neuron 67(5):847–857. doi:10.1016/j.neuron.2010.08.006
Sohya K, Kameyama K, Yanagawa Y, Obata K, Tsumoto T (2007) GABAergic neurons are less selective to stimulus orientation than excitatory neurons in layer II/III of visual cortex, as revealed by in vivo functional Ca2+ imaging in transgenic mice. J Neurosci 27:2145–2149
Li Y, Lu H, Cheng PL, Ge S, Xu H, Shi SH, Dan Y (2012) Clonally related visual cortical neurons show similar stimulus feature selectivity. Nature 486(7401):118–121. doi:10.1038/nature11110
Jarosiewicz B, Schummers J, Malik WQ, Brown EN, Sur M (2012) Functional biases in visual cortex neurons with identified projections to higher cortical targets. Curr Biol 22(4):269–277. doi:10.1016/j.cub.2012.01.011
Ko H, Hofer SB, Pichler B, Buchanan KA, Sjostrom PJ, Mrsic-Flogel TD (2011) Functional specificity of local synaptic connections in neocortical networks. Nature 473(7345):87–91. doi:10.1038/nature09880
Bock DD, Lee WC, Kerlin AM, Andermann ML, Hood G, Wetzel AW, Yurgenson S, Soucy ER, Kim HS, Reid RC (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471(7337):177–182. doi:10.1038/nature09802
Briggman KL, Helmstaedter M, Denk W (2011) Wiring specificity in the direction-selectivity circuit of the retina. Nature 471(7337):183–188. doi:10.1038/nature09818
Chen X, Leischner U, Varga Z, Jia H, Deca D, Rochefort NL, Konnerth A (2012) LOTOS-based two-photon calcium imaging of dendritic spines in vivo. Nat Protoc 7(10):1818–1829. doi:10.1038/nprot.2012.106
Grienberger C, Adelsberger H, Stroh A, Milos RI, Garaschuk O, Schierloh A, Nelken I, Konnerth A (2012) Sound-evoked network calcium transients in mouse auditory cortex in vivo. J Physiol 590(Pt 4):899–918. doi:10.1113/jphysiol.2011.222513
Jia H, Rochefort NL, Chen X, Konnerth A (2010) Dendritic organization of sensory input to cortical neurons in vivo. Nature 464(7293):1307–1312, doi:nature08947 [pii]10.1038/nature08947
Jia H, Rochefort NL, Chen X, Konnerth A (2011) In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons. Nat Protoc 6(1):28–35. doi:10.1038/nprot.2010.169
Garaschuk O, Milos RI, Grienberger C, Marandi N, Adelsberger H, Konnerth A (2006) Optical monitoring of brain function in vivo: from neurons to networks. Pflugers Arch 453(3):385–396. doi:10.1007/s00424-006-0150-x
Garaschuk O, Milos RI, Konnerth A (2006) Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1(1):380–386, doi:nprot.2006.58 [pii]10.1038/nprot.2006.58
Golshani P, Portera-Cailliau C (2008) In vivo 2-photon calcium imaging in layer 2/3 of mice. J Vis Exp 13. doi:10.3791/681
Helmchen F, Waters J (2002) Ca2+ imaging in the mammalian brain in vivo. Eur J Pharmacol 447(2–3):119–129
Kleinfeld D, Denk W (2000) Two-photon imaging of neocortical microcirculation. In: Yuste R, Konnerth A, Lanni F (eds) Imaging neurons: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY
Mostany R, Portera-Cailliau C (2008) A method for 2-photon imaging of blood flow in the neocortex through a cranial window. J Vis Exp 12. doi:10.3791/678
Ohki K, Reid RC (2011) In Vivo Two-Photon Calcium Imaging in the Visual System. In: Helmchen F, Konnerth A (eds) Imaging in neuroscience: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 511–528
Rochefort NL, Grienberger C, Konnerth A (2011) In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading of Fluorescent Indicators. In: Helmchen F, Konnerth A (eds) Imaging in neuroscience: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 491–500
Shih AY, Mateo C, Drew PJ, Tsai PS, Kleinfeld D (2012) A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J Vis Exp 61. doi:10.3791/3742
Svoboda K, Tank DW, Stepnoski RA, Denk W (2000) Two-photon Imaging of Neuronal Function in the Neocortex In Vivo. In: Yuste R, Konnerth A, Lanni F (eds) Imaging neurons: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY
Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. doi:10.1038/nmeth818
Helmchen F, Denk W (2002) New developments in multiphoton microscopy. Curr Opin Neurobiol 12(5):593–601
Svoboda K, Yasuda R (2006) Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50(6):823–839
Margrie TW, Brecht M, Sakmann B (2002) In vivo, low-resistance, whole cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Arch 444:491–498
Judkewitz B, Rizzi M, Kitamura K, Hausser M (2009) Targeted single-cell electroporation of mammalian neurons in vivo. Nat Protoc 4(6):862–869. doi:10.1038/nprot.2009.56
Nimmerjahn A (2011) Two-Photon Imaging of Neuronal Structural Plasticity in Mice during and after Ischemia. In: Helmchen F, Konnerth A (eds) Imaging in neuroscience: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 961–980
Nauhaus I, Nielsen KJ, Callaway EM (2012) Nonlinearity of two-photon Ca2+ imaging yields distorted measurements of tuning for V1 neuronal populations. J Neurophysiol 107(3):923–936. doi:10.1152/jn.00725.2011
Schummers J, Yu H, Sur M (2008) Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex. Science 320(5883):1638–1643
Nauhaus I, Nielsen KJ, Disney AA, Callaway EM (2012) Orthogonal micro-organization of orientation and spatial frequency in primate primary visual cortex. Nat Neurosci 15(12):1683–1690. doi:10.1038/nn.3255
Probes M (2013) Fluorescence SpectraViewer. http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html
Xu C (2000) Two-photon Cross Sections of Indicators. In: Yuste R, Konnerth A, Lanni F (eds) Imaging neurons: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY
Johnson EN, Van Hooser SD, Fitzpatrick D (2010) The representation of S-cone signals in primary visual cortex. J Neurosci 30(31):10337–10350, doi:30/31/10337 [pii]10.1523/JNEUROSCI.1428-10.2010
Histed MH, Bonin V, Reid RC (2009) Direct activation of sparse, distributed populations of cortical neurons by electrical microstimulation. Neuron 63(4):508–522, doi:S0896-6273(09)00545-5 [pii]10.1016/j.neuron.2009.07.016
Kerr JN, Greenberg D, Helmchen F (2005) Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci U S A 102(39):14063–14068
Rochefort NL, Narushima M, Grienberger C, Marandi N, Hill DN, Konnerth A (2011) Development of direction selectivity in mouse cortical neurons. Neuron 71(3):425–432. doi:10.1016/j.neuron.2011.06.013
Sato TR, Gray NW, Mainen ZF, Svoboda K (2007) The functional microarchitecture of the mouse barrel cortex. PLoS Biol 5(7):e189. doi:10.1371/journal.pbio.0050189
Acknowledgments
We thank Prakash Kara, Tom Mrsic-Flogel, Aaron Kerlin, and Clay Reid for their valuable advice as we were learning to perform 2-photon imaging. We also thank David Fitzpatrick and Leonard E. White for support and mentoring. We thank Frank Mello, machinist at Brandeis University, Don Pearce of the Medical Instrument Shop at Duke University Medical Center, and Janet Patterson of the Physics Machine Shop at Duke University for their creative input and expertise in building the stereotaxic devices we describe here. This work was supported by the National Institutes of Health, National Science Foundation, and the John Merck Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Van Hooser, S.D. et al. (2015). Practical Methods for In Vivo Cortical Physiology with 2-Photon Microscopy and Bulk Loading of Fluorescent Calcium Indicator Dyes. In: Arenkiel, B. (eds) Neural Tracing Methods. Neuromethods, vol 92. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1963-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1963-5_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1962-8
Online ISBN: 978-1-4939-1963-5
eBook Packages: Springer Protocols