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Experimental & Translational Stroke Medicine
Published in: Experimental & Translational Stroke Medicine 1/2013

Open Access 01-12-2013 | Review

An experimental protocol for in vivo imaging of neuronal structural plasticity with 2-photon microscopy in mice

Authors: Christian Stetter, Markus Hirschberg, Bernhard Nieswandt, Ralf-Ingo Ernestus, Manfred Heckmann, Anna-Leena Sirén

Published in: Experimental & Translational Stroke Medicine | Issue 1/2013

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Abstract

Introduction

Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex.

Methods

Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45–60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100–200 μm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis.

Results

Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging.

Conclusions

Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury.
Appendix
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Literature
1.
Denk W, Strickler JH, Webb WW: Two-photon laser scanning fluorescence microscopy. Science 1990, 248: 73–76. 10.1126/science.2321027PubMedCrossRef
2.
Lichtman JW, Fraser SE: The neuronal naturalist: watching neurons in their native habitat. Nat Neurosci 2001,4(Suppl):1215–1220.PubMedCrossRef
3.
Misgeld T, Kerschensteiner M: In vivo imaging of the diseased nervous system. Nat Rev Neurosci 2006, 7: 449–463.PubMedCrossRef
4.
Sigler A, Murphy TH: In vivo 2-photon imaging of fine structure in the rodent brain: before, during, and after stroke. Stroke 2010, 41: S117-S123. 10.1161/STROKEAHA.110.594648PubMedCrossRef
5.
Svoboda K, Yasuda R: Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 2006, 50: 823–839. 10.1016/j.neuron.2006.05.019PubMedCrossRef
6.
Tian GF, Takano T, Lin JH, Wang X, Bekar L, Nedergaard M: Imaging of cortical astrocytes using 2-photon laser scanning microscopy in the intact mouse brain. Adv Drug Deliv Rev 2006, 58: 773–787. 10.1016/j.addr.2006.07.001PubMedCrossRef
7.
Scheibe S, Dorostkar MM, Seebacher C, Uhl R, Lison F, Herms J: 4D in in vivo 2-photon laser scanning fluorescence microscopy with sample motion in 6 degrees of freedom. J Neurosci Methods 2011, 200: 47–53. 10.1016/j.jneumeth.2011.06.013PubMedCrossRef
8.
Jung CK, Herms J: Structural Dynamics of Dendritic Spines are Influenced by an Environmental Enrichment: An In Vivo Imaging Study. Cereb Cortex 2012. first published online: October 18, 2012. 10.1093/cercor/bhs317
9.
Niesner R, Andresen V, Neumann J, Spiecker H, Gunzer M: The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging. Biophys J 2007, 93: 2519–2529. 10.1529/biophysj.106.102459PubMedCentralPubMedCrossRef
10.
Schwarzmaier SM, Zimmermann R, McGarry NB, Trabold R, Kim SW, Plesnila N: In vivo temporal and spatial profile of leukocyte adhesion and migration after experimental traumatic brain injury in mice. J Neuroinflammation 2013, 10: 32. 10.1186/1742-2094-10-32PubMedCentralPubMedCrossRef
11.
Jung JC, Mehta AD, Aksay E, Stepnoski R, Schnitzer MJ: In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J Neurophysiol 2004, 92: 3121–3133. 10.1152/jn.00234.2004PubMedCentralPubMedCrossRef
12.
Levene MJ, Dombeck DA, Kasischke KA, Molloy RP, Webb WW: In vivo multiphoton microscopy of deep brain tissue. J Neurophysiol 2004, 91: 1908–1912. 10.1152/jn.01007.2003PubMedCrossRef
13.
Mizrahi A, Crowley JC, Shtoyerman E, Katz LC: High-resolution in vivo imaging of hippocampal dendrites and spines. J Neurosci 2004, 24: 3147–3151. 10.1523/JNEUROSCI.5218-03.2004PubMedCrossRef
14.
Chen X, Leischner U, Varga Z, Jia H, Deca D, Rochefort NL, Konnerth A: LOTOS-based two-photon calcium imaging of dendritic spines in vivo. Nat Protoc 2012, 7: 1818–1829. 10.1038/nprot.2012.106PubMedCrossRef
15.
Margolis DJ, Lutcke H, Schulz K, Haiss F, Weber B, Kugler S, Hasan MT, Helmchen F: Reorganization of cortical population activity imaged throughout long-term sensory deprivation. Nat Neurosci 2012, 15: 1539–1546. 10.1038/nn.3240PubMedCrossRef
16.
De Paola V, Holtmaat A, Knott G, Song S, Wilbrecht L, Caroni P, Svoboda K: Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 2006, 49: 861–875. 10.1016/j.neuron.2006.02.017PubMedCrossRef
17.
Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR: Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 2000, 28: 41–51. 10.1016/S0896-6273(00)00084-2PubMedCrossRef
18.
Hechler D, Nitsch R, Hendrix S: Green-fluorescent-protein-expressing mice as models for the study of axonal growth and regeneration in vitro. Brain Res Rev 2006, 52: 160–169. 10.1016/j.brainresrev.2006.01.005PubMedCrossRef
19.
Lam CK, Yoo T, Hiner B, Liu Z, Grutzendler J: Embolus extravasation is an alternative mechanism for cerebral microvascular recanalization. Nature 2010, 465: 478–482. 10.1038/nature09001PubMedCentralPubMedCrossRef
20.
Zuo Y, Lubischer JL, Kang H, Tian L, Mikesh M, Marks A, Scofield VL, Maika S, Newman C, Krieg P, Thompson WJ: Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination. J Neurosci 2004, 24: 10999–11009. 10.1523/JNEUROSCI.3934-04.2004PubMedCrossRef
21.
Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E, Svoboda K: Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 2002, 420: 788–794. 10.1038/nature01273PubMedCrossRef
22.
Xu HT, Pan F, Yang G, Gan WB: Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat Neurosci 2007, 10: 549–551. 10.1038/nn1883PubMedCrossRef
23.
Piston DW: Imaging living cells and tissues by two-photon excitation microscopy. Trends Cell Biol 1999, 9: 66–69. 10.1016/S0962-8924(98)01432-9PubMedCrossRef
Metadata
Title
An experimental protocol for in vivo imaging of neuronal structural plasticity with 2-photon microscopy in mice
Authors
Christian Stetter
Markus Hirschberg
Bernhard Nieswandt
Ralf-Ingo Ernestus
Manfred Heckmann
Anna-Leena Sirén
Publication date
01-12-2013
Publisher
BioMed Central
Published in
Experimental & Translational Stroke Medicine / Issue 1/2013
Electronic ISSN: 2040-7378
DOI
https://doi.org/10.1186/2040-7378-5-9

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