We would like to thank Dr. Jeffrey Rothstein for providing BAC GLT1 eGFP mice and GLT1 antibody; Tufts Center for Neuroscience Research (NIH P30 NS047243; PI, Rob Jackson) for providing valuable core facilities; New faculty recruitment grant (NIH P30 5P30NS069254-02; PI, Phil Haydon) in Tufts Neuroscience Department.

1. Assembly of the Microfluidic Culture Chamber (MCP)
<ol>
 <li>MCP (Figure 1) are open chambers designed for compartmented cultures of different types of cells 4. It typically has two compartments that are connected through the central channels (3 &mu;m in diameter). Assembly of MCP with glass-bottomed dishes is necessary for preparing cultures and subsequent imaging analysis.</li>
 <li>First, coat sterile glass-bottomed dishes with Polyornithine (Sigma-Aldrich, 1 mg/ml) dissolved in sodium...

<ol>
	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. D., Stevens-Graham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+neuron-glia+communication.">New insights into neuron-glia communication.</a> Science. 298, 556-562 (2002).</li><li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature. 1, 2128-2136 (2006).</li><li>Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuromuscular+synapse+development+by+glial+cell+line-derived+neurotrophic+factor+and+neurturin.">Regulation of neuromuscular synapse development by glial cell line-derived neurotrophic factor and neurturin.</a> The Journal of biological chemistry. 277, 10614-10625 (2002).</li><li>Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+astroglial+excitatory+neurotransmitter+transporter+GLT1.">Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1.</a> Neuron. 61, 880-894 (2009).</li><li>Regan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+promoter+activity+reveal+a+differential+expression+and+physiology+of+glutamate+transporters+by+glia+in+the+developing+and+mature+CNS.">Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS.</a> The Journal of neuroscience. 27, 6607-6619 (2007).</li><li>Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+glutamate+transporter+subtype+expression+in+astrocytes.">Neuronal regulation of glutamate transporter subtype expression in astrocytes.</a> The Journal of neuroscience. 17, 932-940 (1997).</li><li>Schlag, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+glial+Na+-dependent+glutamate+transporters+by+cyclic+AMP+analogs+and+neurons.">Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.</a> Molecular pharmacology. 53, 355-369 (1998).</li><li>Ponomarev, E. D., Novikova, M., Maresz, K., Shriver, L. P., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+culture+system+that+supports+adult+microglial+cell+proliferation+and+maintenance+in+the+resting+state.">Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state.</a> Journal of immunological. 300, 32-46 (2005).</li><li>Espinosa-Jeffrey, A., Wakeman, D. R., Kim, S. U., Snyder, E. Y., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+system+for+rodent+and+human+oligodendrocyte+specification,+lineage+progression,+and+maturation.">Culture system for rodent and human oligodendrocyte specification, lineage progression, and maturation.</a> Current protocols in stem cell biology. Chapter 2, (2009).</li><li>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=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Debanne, D., Rama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+shape+axonal+signaling.">Astrocytes shape axonal signaling.</a> Science signaling. 4, pe11 (2011).</li></ol>

No conflicts of interest declared.

The MCP based neuron and astrocytes co-culture system allows dissection of detailed neuron to astroglia signaling pathways by allowing only the axons pass the central channels and interacting with the astroglial cells. This co-culture system can be conveniently set up with conventional neuron and astrocyte culture procedures. We also described a practical application of this co-culture system by employing an eGFP based reporter for demonstrating axon-dependent GLT1 promoter activation in astrocytes.
	<p class="...

<table border="1">
 <tbody>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Hyclone</td>
 <td>SH30070.03</td>
 <td>for plating neuron for neuron cutlure medium</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Sigma-Aldrich</td>
 <td>F4135</td>
 <td>for astrocyte culture medium</td>
 </tr>
 <tr>
 <td>Glial derived nerve factor</td>
 <td>R&amp;D systems</td>
 <td>212-GD</td>
 <td>Apply 10-20 ng/ml to neuron side of chamber</td>
 </tr>
 <tr>
 <td>Dulbecco modified eagle medium high glucose</td>
 <td>Sigma-Aldrich</td>
 <td>11995</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>70 mm cell strainer</td>
 <td>BD Falcon</td>
 <td>352350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile glass bottom dish</td>
 <td>MatTek Corporation</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microfluidic culture platforms</td>
 <td>Xona Microfluidics LLC</td>
 <td>SND150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6 wells of the culture plate</td>
 <td>Cellstar</td>
 <td>657 160</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Neuron culture medium
 <ul>
 <li>Neurobasal medium</li>
 <li>2% B27 Neurobasal supplement</li>
 <li>2 mM glutamate by adding 1% 100x GlutaMAX</li>
 <li>1% Penicillin-streptomysin</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 Neuron culture medium for plating cell
 <ul>
 <li>Neurobasal medium</li>
 <li>2% B27 Neurobasal supplement</li>
 <li>2 mM glutamate by adding 1% 100x GlutaMAX</li>
 <li>1% Penicillin-streptomysin</li>
 <li>5% Fetal bovine serum SH30070.03</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 Astrocyte culture medium
 <ul>
 <li>Dulbecco modified eagle medium high glucose</li>
 <li>10% Fetal bovine serum F4135</li>
 <li>1% Penicillin-streptomysin</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 Table 1. Materials used in the microfluidic culture platform-based neuronal axon and glia co-culture system.</td>
 </tr>
 </tbody>
</table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Time-lapse imaging analysis of axon-induced GLT1 promoter activation in astrocytes
The compartmented neuron and astrocyte co-culture system allows only the neuronal processes, especially the axons, to selectively interact with astrocytes. Following the successful establishment of axon and astrocyte (or other glial cells) co-culture in the assembled MCP, different types of axon-glia interactions can be studied such as; axon-induced activation of astroglial gene promoter activation, astrocyte-induc...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Research

JoVE Journal

Neuroscience

14.9K vues.  Tufts University. Cette étude décrit les procédures de mise en place d'un axone des neurones et roman (astro) glie plate-forme de co-culture. Dans ce système de co-culture, la manipulation de l'interaction directe entre un axone unique (et unique cellule gliale) devient possible, ce qui permet une analyse mécaniste du neurone mutuelle à la signalisation gliales.