Name: quantifying synapses: an immunocytochemistry-based assay to quantify synapse number
Uploaded: 2010-11-16T17:30:00
Description: Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Puncta Analyzer Plug-in for Image J was written by Barry Wark (current address: Physion Consulting) in the lab of Ben A. Barres (Stanford University).
Funding;
<ul>
<li>Alfred P. Sloan Foundation</li>
<li>The Esther A. and Joseph Klingenstein Fund, Inc.</li>
<li>Broad Biomedical Research Foundation</li>
<li>Cure for Huntington s Disease Initiative</li>
 </ul>

Solutions to Prepare:
<ol>
 <li>Antibody Buffer:
 <ul>
 <li>150 mM NaCl</li>
 <li>50 mM Tris-Base (Fisher, Cat. No: BP152-5, 50 mM) - 1.21 g</li>
 <li>1% BSA (Sigma, Cat. No: A2153, 1%) - 2.0 g</li>
 <li>100 mM L-lysine (Sigma, Cat. No: L-1137, 100 mM) - 3.65 g</li>
 <li>Adjust pH to 7.4</li>
 <li>0.04% Azide</li>
 <li>Adjust volume to 200 ml with distilled H2O.</li>
 ...

<ol>
	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

The synapse assay described above is based in the context of our experimental goals, wherein we focus largely on excitatory projections of RGCs, either in purified culture or in brain section. We have provided a reference table listing antibodies that work well for labeling excitatory synapses (Table 1).
This synapse assay can be adapted to quantify synapse number of any neuronal population or any other synaptic subtype for which there is a selective pre- and postsynaptic marker. For example,...

<table border="1" cellpadding="0" cellspacing="0">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>Antigen</td>
 <td>Species</td>
 <td>Monoclonal/Polyclonal</td>
 <td>Vendor</td>
 <td>Catalog Number</td>
 <td>Dilution</td>
 <td>Works in Culture</td>
 <td>Works in Sections</td>
 </tr>
 <tr>
 <td rowspan="10">Presynaptic</td>
 <td>Synapsin</td>
 <td>Rabbit</td>
 <td>Polyclonal</td>
 <td>Synaptic Systems</td>
 <td>106004</td>
 <td>1:750</td>
 <td>Y</td>
 <td>N.D.</td>
 </tr>
 <tr>
 <td>Synapsin</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Synaptic Systems</td>
 <td>106001</td>
 <td>1:500</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>Bassoon</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Assay Designs</td>
 <td>VAM-PS003F</td>
 <td>1:500</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>Bassoon</td>
 <td>Guinea Pig</td>
 <td>Polyclonal</td>
 <td>Synaptic Systems</td>
 <td>141004</td>
 <td>1:1000</td>
 <td>Y</td>
 <td>N.D.</td>
 </tr>
 <tr>
 <td>Synaptotagmin 1</td>
 <td>Rabbit</td>
 <td>Polyclonal</td>
 <td>Synaptic Systems</td>
 <td>105002</td>
 <td>1:750</td>
 <td>Y</td>
 <td>N</td>
 </tr>
 <tr>
 <td>Synaptobrevin 2 
 (C1.69.1)</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Synaptic Systems</td>
 <td>104211</td>
 <td>1:500</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>Synaptophysin 
 (C1.7.2)</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Synaptic Systems</td>
 <td>101011</td>
 <td>1:500</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>VGlut1</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Millipore</td>
 <td>MAB5502</td>
 <td>1:2500</td>
 <td>N</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>VGlut1</td>
 <td>Guinea pig</td>
 <td>Polyclonal</td>
 <td>Millipore</td>
 <td>AB5905</td>
 <td>1:2500</td>
 <td>N</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>VGlut2</td>
 <td>Guinea pig</td>
 <td>Polyclonal</td>
 <td>Millipore</td>
 <td>AB2251</td>
 <td>1:2500</td>
 <td>N</td>
 <td>Y</td>
 </tr>
 <tr>
 <td rowspan="6">Postsynaptic</td>
 <td>PSD-95 
 (6G6-1C9 clone)</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Affinity Bio Reagents</td>
 <td>MA1-045</td>
 <td>1:750</td>
 <td>Y</td>
 <td>N</td>
 </tr>
 <tr>
 <td>PSD-95</td>
 <td>Rabbit</td>
 <td>Polyclonal</td>
 <td>Zymed</td>
 <td>51-6900</td>
 <td>1:500</td>
 <td>N</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>Homer</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Synaptic Systems</td>
 <td>160011</td>
 <td>1:500</td>
 <td>Y</td>
 <td>N.D.</td>
 </tr>
 <tr>
 <td>Homer</td>
 <td>Rat</td>
 <td>Polyclonal</td>
 <td>Millipore</td>
 <td>AB5875</td>
 <td>1:500</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>Gephyrin</td>
 <td>Rabbit</td>
 <td>Polyclonal</td>
 <td>Synaptic Systems</td>
 <td>147003</td>
 <td>1:500</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 <tr>
 <td>Gephyrin</td>
 <td>Mouse</td>
 <td>Monoclonal</td>
 <td>Synaptic Systems</td>
 <td>147</td>
 <td>1:200</td>
 <td>Y</td>
 <td>Y</td>
 </tr>
 </tbody>
</table>
Table 1: Lists examples of good pre- and postsynaptic markers that we have successfully utilized in our synapse assay. Be aware that this is not an exhaustive list of all available markers. Y = Yes, N = No, N.D. = Not Determined.
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Cat. No.</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td>20012-027</td>
 </tr>
 <tr>
 <td>poly-d-lysine</td>
 <td>Sigma</td>
 <td>P6407</td>
 </tr>
 <tr>
 <td>Laminin</td>
 <td>Cultrex</td>
 <td>3400-010-01</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Roche Diagnostics Gmbh</td>
 <td>9002-93-1</td>
 </tr>
 <tr>
 <td>Normal Goat Serum</td>
 <td>Gibco</td>
 <td>16210</td>
 </tr>
 <tr>
 <td>VectaShield with DAPI</td>
 <td>Vector Laboratories</td>
 <td>H-1200</td>
 </tr>
 <tr>
 <td>OCT</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 </tr>
 <tr>
 <td>Tris-Base (50 mM)</td>
 <td>Fisher</td>
 <td>BP152-5</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>A2153</td>
 </tr>
 <tr>
 <td>l-lysine</td>
 <td>Sigma</td>
 <td>L-1137</td>
 </tr>
 <tr>
 <td>16% PFA solution</td>
 <td>Electron Microscopy Sciences</td>
 <td>15711</td>
 </tr>
 <tr>
 <td>Granular PFA</td>
 <td>Electron Microscopy Sciences</td>
 <td>19210</td>
 </tr>
 <tr>
 <td>24-well culture plate</td>
 <td>Falcon</td>
 <td>35-3047</td>
 </tr>
 <tr>
 <td>Goat anti-mouse Alexa conjugated antibodies</td>
 <td>Invitrogen</td>
 <td>---</td>
 </tr>
 <tr>
 <td>Supply</td>
 <td>Company</td>
 <td>Cat. No.</td>
 </tr>
 <tr>
 <td>12mm, No. 0 glass coverslips</td>
 <td>Karl Hecht Gmbh</td>
 <td>1105209</td>
 </tr>
 <tr>
 <td>No. 1.5 glass coverslip 
 (for slices)</td>
 <td>VWR Scientific</td>
 <td>48393241</td>
 </tr>
 <tr>
 <td>Glass slides</td>
 <td>VWR Scientific</td>
 <td>48311-703</td>
 </tr>
</tbody>
</table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

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