Name: Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes (Video) | JoVE Journal
Uploaded: 2012-04-19T13:00:00.000+00:00
Duration: 6 min 47 s
Description: Learn retrograde dye loading for neuroscience using a simple polyethylene suction pipette technique. This low-cost method introduces fluorescent and calcium-sensitive dyes into neurons for studying activity and morphology.

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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.
1. Type-I and Type-II Pipettes
Start by pulling two short sections of polyethylene tubing (PE90, Clay Adams Brand) over the flame of an alcohol lamp7 to produce pipettes with tapered tips. One pipette (Type-I) should be short (3-7 mm) with a small tip that can tightly hold the target root or tract (Outer Diameter: 0.2-0.4 mm; Inner Diameter: 0.1-0.3 mm). Then pull a second pipette (Type-II) with a longer (8-12 cm) thinner, shaft and a very fine tip (Outer Diameter: 0.2-0.3 mm; inner Diameter: 0.1-0.2 mm) that can be inserted into the Type-I pipette as far as its tip. The second thinner pipette will be used to aspirate and introduce artificial cerebrospinal fluid (aCSF) and dye solution respectively from the Type-I pipette.
2. Positioning of Type-I Pipette
Dissect and isolate the spinal cord8,9 and place it in a bath, superfused with chilled aCSF (~16 °C) throughout the loading process. Place the Type-I pipette on an electrode holder (H1/12 electrode holder, Narishige) so that its back opening can be easily connected to a syringe (1ml U-100 Insulin syringe, Becton Dickenson or comparable) via a flexible tubing (PharMed BPT, Cole-Parmer, #AY242002; 10 cm) (Fig.1A-B).
 
3. Dye Application
<ol>
	<li>Using the syringe, first draw aCSF into the Type-I pipette (Fig.1B; 2A) followed by the axonal tract or root to be filled (Fig. 2B). The flexible tubing should then be removed from the back opening. The Type-II pipette is attached to another syringe and inserted into the Type-I pipette holding the axonal tract or root. Using the syringe attached to the type-II pipette, aspirate aCSF so that the residual aCSF just covers the axonal tract (Fig.1C; 2C-D). Then withdraw the type-II pipette from the type-I pipette. Monitor the aCSF level for a few minutes to verify a good seal on the axonal tract or nerves (see Section 3 in Troubleshooting for "good seal").</li>
	<li>Dissolve the dye in aCSF or 0.2% triton X-100 in double distilled water and draw it into the Type-II pipette while paying attention not to include air bubbles. Insert the Type-II pipette containing the dye into the Type-I pipette and into the residual aCSF solution (Fig. 2E). Slowly release the dye into the aCSF using gentle positive pressure (Fig. 2F). Be careful not to introduce any air bubbles or to cause displacement of the target tissue from inside the tip. Withdraw the type-II pipette after sufficient amount of dye has been released (3-5 μL of dye solution).</li>
</ol>
4. Incubation
Incubate the tissue in the dark between 6 to 20 hours depending on the type of experiment. Once sufficient time has been allowed for filling, gently pull away the electrode from the tissue leaving the tissue ready for imaging or histology.
Troubleshooting
<ol>
	<li>If the target tissue does not enter easily into the tip of the Type-I pipette or is loose once inside, then the tip diameter is the wrong size. If this occurs, pull and use a different pipette.</li>
	<li>Before adding the dye to the aCSF in the type-I pipette, make sure that enough aCSF remains so that it can be reached with the Type-II pipette tip (Fig. 2D). Otherwise, an air gap will exist once the dye is added. If this happens, do not add the dye and remove the axonal tract from the pipette and then draw it back in with sufficient aCSF to be reached by the Type-II pipette.</li>
	<li>If the aCSF level in the Type-I pipette increases after it was aspirated with the Type-II pipette, it means that the seal with the tissue is not good. Use a different electrode, otherwise the dye will be diluted during the labeling procedure.</li>
	<li>If an air bubble is introduced into the Type-I pipette while injecting the dye, drain out the bubble by advancing the Type-II pipette tip close to the bubble and gently applying a weak negative pressure using the attached syringe.</li>
</ol>
5. Representative Results
To illustrate an application of the method, we simultaneously loaded motoneurons, sensory afferents and spinal interneurons with three different dextran-conjugated fluorescent dyes (Fig. 3A). As illustrated in Fig. 3B, three classes of neuron are labeled; sensory afferents (red), interneurons projecting into the ventral funiculus (green) and motoneurons (blue).
<img alt="Pipette-based microinjection setup diagram; flexible elastomer tubing; syringe connection." src="/files/ftp_upload/4008/4008fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Schematic showing Pipette Type-I and Type-II assembly. (A) A Type-I pipette (light blue) is placed on an electrode holder so that it's back opening can be easily connected to the flexible elastomer tubing (B) and into which a Type-II pipette can be inserted (C).
<img alt="Spinal nerve drug delivery diagram, elastomer tubing setup, dye application process." src="/files/ftp_upload/4008/4008fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Schematic showing the loading process of motoneurons with fluorescent dyes. (A) A Type-I pipette is placed near the ventral root to be filled. (B) Suction is applied to the pipette to draw aCSF into the pipette followed by the root. (C-D) Disconnect the flexible elastomer tubing from the backend of Type-I pipette and reduce the amount of aCSF by applying suction to the Type-II pipette. (E) Insert the tip of the Type-II pipette containing the dissolved dye into the remaining aCSF in the Type-I pipette. (E-F) Slowly fill the Type-I pipette with the dye and then remove the Type-II pipette.
<img alt="Spinal cord diagram with fluorescent dextran tracers; neural pathway visualization at rostral S4." src="/files/ftp_upload/4008/4008fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Application of the filling procedure to the isolated mouse spinal cord. (A) Schematic showing the three Type-I pipettes used to fill different neuronal classes in the spinal cord. Motoneurons (blue; Cascade Blue dextran) and sacral sensory afferents (red; Texas-Red dextran) were backfilled through the ventral and dorsal roots, respectively. Fluorescein dextran was used to load spinal interneurons (green) whose axons ascend through the ventral funiculus (VF). 1 mg of each dextran dye (10,000MW; Molecular Probes) was dissolved in 6 μL distilled water containing 0.2% Triton X-100 (B). Confocal image of a section from the sacral cord of a spinal cord in which the neurons were back-labeled as described in A. Note that the labeled sensory afferents are primarily ipsilateral with a few contralateral projections. The labeled motoneurons are ipsilateral to the filled ventral root whereas the labeled interneurons are contralateral to the side of the fill (arrow head). Calibration bar 200 μm.

<ol><li>Nance, D. M., Burns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+dextrans+as+sensitive+anterograde+neuroanatomical+tracers%3A+applications+and+pitfalls%5BTitle%5D)+AND+%22Brain+Res.+Bull%22%5BJournal%5D)">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/pubmed?term=((Fluorescent+dextran-amines+used+as+axonal+tracers+in+the+nervous+system+of+the+chicken+embryo%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((Optical+imaging+of+neuronal+activity+in+tissue+labeled+by+retrograde+transport+of+Calcium+Green+Dextran%5BTitle%5D)+AND+%22Brain+Research+Protocols%22%5BJournal%5D)">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/pubmed?term=((Imaging+nervous+system+activity%5BTitle%5D)+AND+%22Curr.+Protoc.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Real-time+imaging+of+neurons+retrogradely+and+anterogradely+labelled+with+calcium-sensitive+dyes%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((Voltage-sensitive+dye+recording+using+retrogradely+transported+dye+in+the+chicken+spinal+cord%3A+staining+and+signal+characteristics%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((Spinal+Cord+Electrophysiology+II%3A+Extracellular+Suction+Electrode+Fabrication%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((In+vitro+brainstem-spinal+cord+preparations+for+study+of+motor+systems+for+mammalian+respiration+and+locomotion%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((Spinal+Cord+Electrophysiology%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((Functionally+reduced+sensorimotor+connections+form+with+normal+specificity+despite+abnormal+muscle+spindle+development%3A+the+role+of+spindle-derived+neurotrophin+3%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Early+functional+impairment+of+sensory-motor+connectivity+in+a+mouse+model+of+spinal+muscular+atrophy%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Studies+of+sacral+neurons+involved+in+activation+of+the+lumbar+central+pattern+generator+for+locomotion+in+the+neonatal+rodent+spinal+cord%5BTitle%5D)+AND+%22Soc.+Neurosci.+Abstr%22%5BJournal%5D)">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/pubmed?term=((Imaging+the+spatiotemporal+organization+of+neural+activity+in+the+developing+spinal+cord%5BTitle%5D)+AND+%22Dev.+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Segmental+patterns+of+vestibular-mediated+synaptic+inputs+to+axial+and+limb+motoneurons+in+the+neonatal+mouse+assessed+by+optical+recording%5BTitle%5D)+AND+%22J.+Physiol.+%28Lond%29%22%5BJournal%5D)">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/pubmed?term=((Organization+of+functional+synaptic+connections+between+medullary+reticulospinal+neurons+and+lumbar+descending+commissural+interneurons+in+the+neonatal+mouse%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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 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,...

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

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.

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. ...

The overall goal of this procedure is to introduce a high concentration of fluorescent or ion sensitive dye into neurons or any neuronal tract using a simple low-cost technique. This is accomplished by first producing two types of pipettes, type one and type two. Then connect a flexible elastomer tubing to the type one pipette and apply positive pressure to draw up the target neuronal tract into the type one pipette, verifying that a good seal is made.Next, insert the type two pipette into the type one pipette and use the type two pipette to aspirate the residual A CSF from the type one pipette. Then introduce the dye, but no air bubbles to the type one pipette. The final step is to incubate the neurons in the dark between six to 20 hours.Ultimately, after the removal of the type one pipette, the preparation is ready for in vitro, electrophysiological, or morphological studies. The main advantage of this technique over existing method like bath application, BOS injection, and electroporation, is that the D is delivered to a defined neuronal population. This method can help to answer key questions in the neuroscience field, such as activity, connectivity, morphology, and distribution of defined neurons.Generally, the technique requires practice, particularly ensuring that the dye does not get diluted within the type one pipette and no air bubbles are introduced into it. Begin this procedure by pulling the polyethylene tubing over the flame of an alcohol lamp to produce two short pipettes with tapered tips. First, pull a short type one pipette with a small tip that can tightly hold the target root or tract.Next, pull a type two pipette with a longer thinner shaft and a very fine tip that can be inserted into the type one pipette as far as its tip. Afterward, connect the type two pipette to EC clipped 19 gauge needle. Then place the isolated spinal cord in a bath.Super fuse it with chilled A CSF throughout the loading process. After that, place the type one pipette on an electrode holder so that its back opening can be easily connected to a syringe via a flexible tubing. In this procedure, first draw a CSF into the type one pipette using a syringe.Next, draw the axonal tract or root to be filled. Then remove the flexible tubing from the back opening. After that, attach the type two pipette to another syringe.Insert it into the type one pipette, which is holding an axonal tract or neuronal root under the microscope. Use the syringe attaching to the type two pipette to aspirate A CSF so that the residual A CSF just covers the axonal tract. Then withdraw the type two pipette from the type one pipette.Monitor the A CSF level for a few minutes to verify a good seal on the axonal tract or nerves. Afterward, dissolve the dye in A CSF or 0.2%Triton X 100 in double distilled water. Draw it into the type two pipette by applying weak negative pressure to the attached syringe while being careful not to draw in any air bubbles.Then insert the type two pipette containing the dye into the residual A CSF solution In the type one pipette, slowly release the dye into the A CSF using gentle positive pressure to the syringe. Be sure not to introduce any air bubbles or to cause displacement of the target tissue from the inside of the tip. If an air bubble is introduced into the type one pipette while injecting the dye, draw the bubble out By advancing the type two pipette tip close to the bubble, then gently applying a weak negative pressure using the attached syringe.After sufficient amount of dye has been released, withdraw the type two pipette. Next, incubate the tissue in the dark for six to 20 hours, depending on the type of experiment. Once sufficient time has been allowed for filling, gently pull away the electrode from the tissue.Then the tissue is ready for imaging or histology. Here is a schematic diagram that shows the loading of three different dextro, conjugated fluorescent dyes simultaneously to motor neurons, sensory afferent and spinal inter neurons, motor neurons and sacral sensory afferent were backfilled with Cascade blue Dextran and Texas red dextran through the ventral and dorsal roots respectively. Green fluorescent dextran has been loaded to spinal inter neurons whose axons ascend through the ventral funiculus.Shown here is a confocal image of a section from the sacral spinal cord in which the neurons have been back labeled. Note that the labeled sensory afferent are primarily ipsilateral. With a few contralateral projections, the labeled motor neurons are ipsilateral to the filled ventral root, whereas the labeled inter neurons are contralateral to it as indicated by the arrowhead.Following this procedure, additional method can be performed in order to answer key questions such as functional connectivity, excitatory inhibitor, sinus type, the distribution, and the neurotransmitter production in the DI field neurons. This technique paved the way for researchers in the field of spinal cord and brainstem neurophysiology to emit specific neuronal population using ion sensitive dyes. After watching this video, you should have good understanding of how to apply fluorescent and or ion sensitive dyes into neurons and sensory fibers in the isolated spinal cord.

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Learn retrograde dye loading for neuroscience using a simple polyethylene suction pipette technique. This low-cost method introduces fluorescent and calcium-sensitive dyes into neurons for studying activity and morphology.

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

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