Name: chronic imaging of mouse visual cortex using a thinned-skull preparation
Uploaded: 2010-10-25T17:00:00
Description: Watch this Scientific Journal Video about Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation at JoVE.com

This work was supported by Burroughs Wellcome Career Award in the Biomedical Sciences (A.K.M), The Whitehall Foundation Research Grant (A.K.M), Sloan Foundation Fellowship (A.K.M), NIH EY019277 (A.K.M), and an NEI funded, Vision Training Grant, EY013319 (E.A.K.).

<ol>
 <li>To visualize neurons in the intact brain using two-photon microscopy, a preparation where neurons are labeled with fluorescent markers is used. In the experiments presented here we use the GFP-M transgenic mouse line which labels layer 5 pyramidal cells1. Layer 5 pyramidal cells project dendritic processes into superficial layers, allowing the visualization of dendrites and dendritic spines up to a depth of 300 &mu;m below the level of the pia. An alternate approach is to label cells using viral markers (see Lowery <em...

<ol>
	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-51 (2000).</li><li>Holtmaat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4 (8), 1128-1144 (2009).</li><li>Majewska, A. K., Newton, J. R., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+synaptic+structure+in+sensory+cortical+areas+in+vivo.">Remodeling of synaptic structure in sensory cortical areas in vivo.</a> J Neurosci. 26 (11), 3021-3029 (2006).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat Protocols. 5 (2), 201-208 (2010).</li><li>Xu, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex.">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nat Neurosci. 10 (5), 549-551 (2007).</li></ol>

Chronic imaging using two-photon microscopy is becoming an increasingly popular technique to study morphological changes triggered during plasticity2-5. Here we demonstrate a thinned-skull preparation to follow identified dendritic spines in the intact mouse brain on different imaging days. In this protocol, the skull is left intact, causing minimal damage to the cortex and resulting in very low levels of neuroinflammation which may alter brain function6. This allows the animal to be imaged immedia...

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chronic imaging of mouse visual cortex using a thinned-skull preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

Watch this Scientific Journal Video about Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation at JoVE.com

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

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