Name: in vivo 2-photon calcium imaging in layer 2/3 of mice
Uploaded: 2008-03-13T12:00:00
Description: Watch this Scientific Journal Video about In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice at JoVE.com

<p class="jove_content">We would like to acknowledge Olga Garaschuk for helpful discussions on optimizing the bulk loading of calcium indicators.</p>

<ol>
<li>Prepare the fluorescent calcium indicator solution. We use acetoxymethyl ester dyes, or AM dyes for short. These are dyes that can be taken up by cells when injected extracellularly. We typically use Oregon-Green BAPTA-1 AM or Fluo-4 AM at a concentration of 1 mM. AM dyes can be obtained from Invitrogen in 50 microgram vials. We first prepare a 20% solution of Pluronic F-127 in DMSO. We warm the solution prior to use every day until its clear. We dissolve the calcium indicator the 20% Pluronic F-127/DMSO for 15-20 minutes to a concentr...

<ol>
	<li>Garaschuk, O., Milos, R. I., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+bulk-loading+of+fluorescent+indicators+for+two-photon+brain+imaging+in+vivo.">Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.</a> <em style='font-style: italic'>Nat Protoc</em>. <b>1</b>, (2006).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+two-photon+calcium+imaging+of+neuronal+networks.">In-vivo two-photon calcium imaging of neuronal networks.</a> <em style='font-style: italic'>Proc Natl Acad Sci USA</em>. <b>100</b>, 7319-7324 (2003).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.&amp;nbsp;&amp;nbsp;+Biomed+Eng+Online+2:13.">ScanImage: flexible software for operating laser scanning microscopes.&amp;nbsp;&amp;nbsp; Biomed Eng Online 2:13.</a> <em style='font-style: italic'>Biomed Eng Online</em>. <b>2</b>, (2003).</li></ol>

<p class="jove_content">The advantage of this method over electrophysiological recordings is that it is less invasive and allows the recordings of activity in dense neuronal neuronal networks.   When doing this procedure it s important to remember to take extreme care when performing the craniotomy not to damage the dura.  Even a small amount of subdural bleeding with greatly obscure the imaging.</p>


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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Oregon Green BAPTA1-AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>O-6807</td>
<td>&nbsp;</td>
<tr>
<td>Fluo 4- AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14201</td>
<td>&nbsp;</td>
<tr>
<td>Sulforhodamine 101</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>S-359</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic F-127 in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Centrifugal Filter (0.45 micron)</td>
<td>Reagent</td>
<td>Millipore</td>
<td>UFC3 0HV 0S</td>
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in vivo 2-photon calcium imaging in layer 2/3 of mice

<p class="jove_content">To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye  that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope.  Co-injection of astrocyte-specific dyes allows one to differentiate  neurons from astrocytes.  The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.</p>

Watch this Scientific Journal Video about In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice at JoVE.com

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

<p class="jove_content">To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .</p>

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Research

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