Abstract
Dendritic spines are small protrusions present postsynaptically at ∼90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (∼1 μm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.
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References
Gray, E.G. Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex. Nature 183, 1592–1593 (1959).
Matus, A., Ackermann, M., Pehling, G., Byers, H.R. & Fujiwara, K. High actin concentrations in brain dendritic spines and postsynaptic densities. Proc. Natl. Acad. Sci. USA 79, 7590–7594 (1982).
Fischer, M., Kaech, S., Knutti, D. & Matus, A. Rapid actin-based plasticity in dendritic spines. Neuron 20, 847–854 (1998).
Dunaevsky, A., Tashiro, A., Majewska, A., Mason, C. & Yuste, R. Developmental regulation of spine motility in the mammalian central nervous system. Proc. Natl. Acad. Sci. USA 96, 13438–13443 (1999).
Fischer, M., Kaech, S., Wagner, U., Brinkhaus, H. & Matus, A. Glutamate receptors regulate actin-based plasticity in dendritic spines. Nat. Neurosci. 3, 887–894 (2000).
Roelandse, M., Welman, A., Wagner, U., Hagmann, J. & Matus, A. Focal motility determines the geometry of dendritic spines. Neuroscience 121, 39–49 (2003).
Allison, D.W., Gelfand, V.I., Spector, I. & Craig, A.M. Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. J. Neurosci. 18, 2423–2436 (1998).
Matsuzaki, M., Honkura, N., Ellis-Davies, G.C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004).
Okamoto, K., Nagai, T., Miyawaki, A. & Hayashi, Y. Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat. Neurosci. 7, 1104–1112 (2004).
Heim, R., Cubitt, A.B. & Tsien, R.Y. Improved green fluorescence. Nature 373, 663–664 (1995).
Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).
Kaech, S., Ludin, B. & Matus, A. Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins. Neuron 17, 1189–1199 (1996).
Kaech, S., Parmar, H., Roelandse, M., Bornmann, C. & Matus, A. Cytoskeletal microdifferentiation: a mechanism for organizing morphological plasticity in dendrites. Proc. Natl. Acad. Sci. USA 98, 7086–7092 (2001).
Goldman, R. & Spector, D.L. Live Cell Imaging: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2005).
Lendvai, B., Stern, E.A., Chen, B. & Svoboda, K. Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature 404, 876–881 (2000).
Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).
Ehrengruber, M.U. et al. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc. Natl. Acad. Sci. USA 96, 7041–7046 (1999).
Griesbeck, O., Korte, M., Gravel, C., Bonhoeffer, T. & Thoenen, H. Rapid gene transfer into cultured hippocampal neurons and acute hippocampal slices using adenoviral vectors. Brain Res. Mol. Brain Res. 44, 171–177 (1997).
Moriyoshi, K., Richards, L.J., Akazawa, C., O'Leary, D.D. & Nakanishi, S. Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP. Neuron 16, 255–260 (1996).
Lo, D.C., McAllister, A.K. & Katz, L.C. Neuronal transfection in brain slices using particle-mediated gene transfer. Neuron 13, 1263–1268 (1994).
O'Brien, J.A. & Lummis, S.C.R. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat. Prot. 1, 977–981 (2006).
Wirth, M.J. & Wahle, P. Biolistic transfection of organotypic cultures of rat visual cortex using a handheld device. J. Neurosci. Methods 125, 45–54 (2003).
Fiala, J.C., Feinberg, M., Popov, V. & Harris, K.M. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1. J. Neurosci. 18, 8900–8911 (1998).
Kawamura, S-i., Negishi, H., Otsuki, S. & Tomosada, N. Confocal laser microscope scanner and CCD camera. Yokogawa Technical Report English Edition No. 33 〈http://www.yokogawa.com/rd/pdf/TR/rd-tr-r00033-005.pdf〉 (2002).
Gahwiler, B.H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342 (1981).
Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).
Gahwiler, B.H., Capogna, M., Debanne, D., McKinney, R.A. & Thompson, S.M. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471–477 (1997).
McAllister, A.K. Biolistic transfection of neurons. Sci. STKE 2000, PL1 (2000).
Kaech, S., Fischer, M., Doll, T. & Matus, A. Isoform specificity in the relationship of actin to dendritic spines. J. Neurosci. 17, 9565–9572 (1997).
De Paola, V., Arber, S. & Caroni, P. AMPA receptors regulate dynamic equilibrium of presynaptic terminals in mature hippocampal networks. Nat. Neurosci. 6, 491–500 (2003).
Roelandse, M. & Matus, A. Hypothermia-associated loss of dendritic spines. J. Neurosci. 24, 7843–7847 (2004).
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Dendritic spine mobility (MOV 98 kb)
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Verkuyl, J., Matus, A. Time-lapse imaging of dendritic spines in vitro. Nat Protoc 1, 2399–2405 (2006). https://doi.org/10.1038/nprot.2006.357
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DOI: https://doi.org/10.1038/nprot.2006.357
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