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.
