This work was supported by the intramural program of the National Institutes of Neurological Disorders and Stroke at the National Institutes of Health. We would also like to thank Dr. George Mentis for his earlier contributions to the method and to the data in Figure 3.

Fluorescent dextrans have been used as anatomical tools and for imaging neuronal activity1-4. Fields et al., (2009)4 published a protocol for applying ion- and voltage-sensitive dyes to axonal tracts with a focus on the spinal cord as a model system. Here we describe a more detailed procedure for applying fluorescent and/or ion-sensitive dye to the cut ventral roots, dorsal roots or any neuronal tract of the spinal cord for in-vitro optophysiological and morphological studies.
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We have nothing to disclose.

We describe here a simple and cost-effective protocol for introducing dyes into neurons, nerves and spinal tracts. This method involves exposing identified anatomical pathways to a highly concentrated dye solution for the duration of the loading process. This results in retrograde labeling of the target site with little background compared to microinjections, bath application and electroporation techniques. However, the method is limited to sites in the nervous system where a tract of axons can be isolated. Furthermore,...

<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Material Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td rowspan="2">Tubing</td>
 <td>PE90 (IDxOD:0.034"x0.050"; Wall Thikness:0.008")</td>
 <td>Clay Adams Brand Intermedic</td>
 <td>427421</td>
 <td>For Type I &amp; Type II pipettes</td>
 </tr>
 <tr>
 <td>NSF-51(IDxOD:1/16x1/8; Wall Thikness:1/32)</td>
 <td>PharMed BPT, Cole-Parmer</td>
 <td>AY242002</td>
 <td>For Flexible tubing</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>1ml insulin Syringe U-100</td>
 <td>Becton Dickenson</td>
 <td>329650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>19G x1-1/2" Metal Hub Needle</td>
 <td>MONOJECT</td>
 <td>200136</td>
 <td>Connecting Type II pipette to syringe</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Alcohol Lamp</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Stopcock</td>
 <td>Three-way Stopcock with Male Luer Slip Adapter</td>
 <td>Baxter Healthcare Corp.</td>
 <td>2C6241</td>
 <td>Use with syringe</td>
 </tr>
 <tr>
 <td rowspan="3">Holder</td>
 <td>H-1 electrode holder</td>
 <td>Narishige</td>
 <td>H-1/12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnet stand</td>
 <td>Narishige</td>
 <td>GJ-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Narishige</td>
 <td>M-3333</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tweezers</td>
 <td>S&amp;T DUMONT Swiss 00632-11</td>
 <td>DUMONT</td>
 <td>JF-5 TC</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
 </table>
*Those are suggested materials. Can be replaced with any compatible products

retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

retrograde-loading-nerves-tracts-spinal-roots-with-fluorescent

Research

JoVE Journal

Neuroscience

12.2K visualizações.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Nós descrevemos uma técnica simples e de baixo custo para a introdução de elevada concentração de corantes fluorescentes e de cálcio sensíveis em neurónios ou qualquer tracto neuronal, utilizando uma pipeta de sucção de polietileno.