Name: retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes
Uploaded: 2012-04-19T13:00:00
Description: Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

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.

وقد استخدمت dextrans فلوري كأدوات التشريحية وللتصوير نشاط الخلايا العصبية 1-4. الحقول وآخرون، (2009) 4 نشر بروتوكول لتطبيق ايون والجهد حساسة للأصباغ إلى مساحات محواري مع التركيز على الحبل الشوكي ونظام نموذجي. نحن هنا وصفا لإجراءات أكثر تفصيلا لتطبيق صبغة ال...

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	<li>Nance, D. M., Burns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextrans+as+sensitive+anterograde+neuroanatomical+tracers:+applications+and+pitfalls.">Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls.</a> Brain Res. Bull. 25, 139-145 (1990).</li><li>Glover, J. C., Petursdottir, G., Jansen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextran-amines+used+as+axonal+tracers+in+the+nervous+system+of+the+chicken+embryo.">Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo.</a> J. Neurosci. Methods. 18, 243-254 (1986).</li><li>McPherson, D. R., McClellan, A. D., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+neuronal+activity+in+tissue+labeled+by+retrograde+transport+of+Calcium+Green+Dextran.">Optical imaging of neuronal activity in tissue labeled by retrograde transport of Calcium Green Dextran.</a> Brain Research Protocols. 1, 157-164 (1997).</li><li>Fields, D. R., Shneider, N., Mentis, G. Z., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+nervous+system+activity.">Imaging nervous system activity.</a> Curr. Protoc. Neurosci. Chapter 2, Unit 2.3 (2009).</li><li>O'Donovan, M. J., Ho, S., Sholomenko, G., Yee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+neurons+retrogradely+and+anterogradely+labelled+with+calcium-sensitive+dyes.">Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes.</a> J. Neurosci. Methods. 46, 91-106 (1993).</li><li>Wenner, P., Tsau, Y., Cohen, L. B., O'Donovan, M. J., Dan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dye+recording+using+retrogradely+transported+dye+in+the+chicken+spinal+cord:+staining+and+signal+characteristics.">Voltage-sensitive dye recording using retrogradely transported dye in the chicken spinal cord: staining and signal characteristics.</a> J. Neurosci. Methods. 70, 111-120 (1996).</li><li>Garudadri, S., Gallarda, B., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+Cord+Electrophysiology+II:+Extracellular+Suction+Electrode+Fabrication.">Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication.</a> J. Vis. Exp. (48), e2580 (2011).</li><li>Smith, J. C., Feldman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+brainstem-spinal+cord+preparations+for+study+of+motor+systems+for+mammalian+respiration+and+locomotion.">In vitro brainstem-spinal cord preparations for study of motor systems for mammalian respiration and locomotion.</a> J. Neurosci. Methods. 21, 321-333 (1987).</li><li>Meyer, A., Gallarda, B., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+Cord+Electrophysiology.">Spinal Cord Electrophysiology.</a> J. Vis. Exp. (35), e1660 (2010).</li><li>Shneider, N. A., Mentis, G. Z., Schustak, J., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+reduced+sensorimotor+connections+form+with+normal+specificity+despite+abnormal+muscle+spindle+development:+the+role+of+spindle-derived+neurotrophin+3.">Functionally reduced sensorimotor connections form with normal specificity despite abnormal muscle spindle development: the role of spindle-derived neurotrophin 3.</a> J. Neurosci. 29, 4719-4735 (2009).</li><li>Mentis, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+functional+impairment+of+sensory-motor+connectivity+in+a+mouse+model+of+spinal+muscular+atrophy.">Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy.</a> Neuron. 69, 453-467 (2011).</li><li>Blivis, D., Mentis, Z., O'Donovan, J. M. G., Lev-Tov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+sacral+neurons+involved+in+activation+of+the+lumbar+central+pattern+generator+for+locomotion+in+the+neonatal+rodent+spinal+cord.">Studies of sacral neurons involved in activation of the lumbar central pattern generator for locomotion in the neonatal rodent spinal cord.</a> Soc. Neurosci. Abstr. 564.8, (2009).</li><li>O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+spatiotemporal+organization+of+neural+activity+in+the+developing+spinal+cord.">Imaging the spatiotemporal organization of neural activity in the developing spinal cord.</a> Dev. Neurobiol. 68, 788-803 (2008).</li><li>Kasumacic, N., Glover, J. C., Perreault, M. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+patterns+of+vestibular-mediated+synaptic+inputs+to+axial+and+limb+motoneurons+in+the+neonatal+mouse+assessed+by+optical+recording.">Segmental patterns of vestibular-mediated synaptic inputs to axial and limb motoneurons in the neonatal mouse assessed by optical recording.</a> J. Physiol. (Lond). 588, 4905-4925 (2010).</li><li>Szokol, K., Glover, J. C., Perreault, M. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+functional+synaptic+connections+between+medullary+reticulospinal+neurons+and+lumbar+descending+commissural+interneurons+in+the+neonatal+mouse.">Organization of functional synaptic connections between medullary reticulospinal neurons and lumbar descending commissural interneurons in the neonatal mouse.</a> J. Neurosci. 31, 4731-4742 (2011).</li></ol>

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

وضع العلامات رجعي من الخلايا العصبية هي طريقة التشريحية مستوى 1،2 التي قد استخدمت أيضا لتحميل الكالسيوم والأصباغ الجهد حساسة إلى الخلايا العصبية 3-6. عموما، يتم تطبيق الأصباغ والبلورات الصلبة أو عن طريق الحقن الضغط المحلية باستخدام ماصات زجاجية. ومع ذلك، وهذا يمكن أن يؤدي إلى التخفيف من شدة العلامات صبغ وانخفاض، وخاصة عندما يطلب من عدة ساعات لنشر صبغ. هنا علينا أن نبرهن تقنية بسيطة ومنخفضة التكلفة لإدخال الأصباغ الفلورية وايون تراعي في الخلايا العصبية باستخدام ماصة شفط البولي ايثيلين مليئة الحل صبغ. هذا الأسلوب يوفر وسيلة يمكن الاعتماد عليها للحفاظ على نسبة عالية من الصبغة في اتصال مع محاور عصبية طوال الإجراء التحميل.

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.

إلى الوراء تحميل من الأعصاب، ومساحات، وجذور العمود الفقري مع الأصباغ الفلورية

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. ...

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Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

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