The overall goal of this procedure is to identify afferent neurons by retrograde dye labeling and to study the ion channels in these neurons using patch clamp electrophysiology and immunochemistry. This is accomplished by first injecting DAI into the target tissue to label the innervating afferent neurons. The second step is to isolate afferent neurons from dorsal root ganglia.
Next, the labeled neurons are patch clamped to record the gated ion channels, the final step is to identify expressed ion channels in the labeled neurons using immunochemistry. Ultimately using a combination of patch, clamp and immunochemistry, we are able to identify the expression pattern as well as the functional properties of the ion channels in afferent neurons. The main advantage of this method over other existing method is that it's a non-invasive method and the DAI that we use, DAI I is known for its rapid uptake by the neuronal terminals.
Lack of transfer between neurons and long-term retentions within the neurons that we inject. Demonstrating the procedure will be myself and Stephanie, research technician from our lab For dye injection. First, shave the hair over the calf area on both legs of the anesthetized rat using an electric razor.
Next, fill a one CC sterile syringe with 200 microliters of 1.5%Dai solution. Slowly insert the needle parallel to the long axis of each gastro muscle and inject 100 microliters of dai into the muscle prior to withdrawing the needle from the muscle. Pull back on the syringe plunger to ensure the dye no longer flows from the tip.
Wait 10 seconds and then withdraw the needle from the muscle. Four to five days are needed for dye distribution to set up for dissection. Place a beaker with 10 milliliters of HBSS, A 35 millimeter Petri dish and a 100 millimeter Petri dish on ice.
Next, weigh out seven milligrams of collagenase, one milligram of DNA and 10 milligrams of trypsin preheat, a shaking water bath to 37 degrees Celsius and warm up a 15 milliliter centrifuge tube containing 10 milliliters of EBSS in the bath. After euthanizing the rat prior to dissection, quickly cut open the skin on the back of the animal. Then remove the connective and muscle tissue along the spinal column for better exposure.
Subsequently, remove the lumbar section of the vertebral column. Next, place the isolated vertebral column in the 100 millimeter Petri dish. Apply chilled HBSS solution using a sterile pasture pipette to keep the preparation moist and cold.
Cut the muscles from the sides of the vertebral column. Perform a dorsal laminectomy by using rongeurs or scissors to gently remove the bone and closing the recesses containing the dorsal root ganglia.Repeatedly. Apply ice cold HBSS during this procedure.
Under the microscope, isolate the DRG by cutting the peripheral and central nerves, leaving one to two millimeters of nerve to facilitate handling of the ganglia. Then place the isolated DRG into the 35 millimeter Petri dish containing ice cold HBSS buffer. Repeat the procedures until all L four and L five DRG have been isolated.
Remove excess nerves and blood vessels using micro scissors and forceps. Cut open the DRG for better exposure to the enzymes, but do not de sheath the DRG for DRG neuron dissociation. Add the enzymes to the warm EBSS media, then vortex to dissolve and filter sterilize.
Next place all DRG into the enzyme solution with a sterile pasture pipe head. After that bubble the flask with 95%oxygen and 5%CO2 for 60 seconds before tightly capping the flask. Then incubate the flask in the 37 degrees Celsius shaking water bath for 60 minutes at 240 RPM.
At the end of the incubation vigorously. Shake the flask 50 times to dissociate the DRG under sterile conditions. Add five milliliters of MEM plus to the flask to stop the enzymatic activities.
Transfer the flask contents to a sterile 15 milliliters centrifuge tube, and centrifuge at 16 times G for six minutes. Afterward, remove and discard the supernatant. Add five milliliters of MEM plus to the sample tritrate gently with pipette.
To break up the pellet repeat centrifugation step, remove and discard the supernatant and add 500 microliters of MEM plus gently tritrate with the one milliliter pipette tip on a pipette to make a cell suspension. After that, place a polylysine coated glass cover slip into an empty, sterile 35 millimeter dish. Then add a drop of the cell suspension to the center of the cover slip.
Repeat this procedure until the cell solution is exhausted. Place the 35 millimeter dishes into a 37 degree Celsius and 5%CO2 incubator for one hour to allow the neurons to adhere to the cover slips lips after one hour. Add an additional two milliliters of media into each dish and return them to the incubator to perform patch clamp recording.
First, identify the dye labeled DRG neurons using a microscope with epi fluorescence. Next, fill the glass electrode with internal solution. Gently tap to remove any trapped bubbles.
Then place the electrode in the electrode holder. Lower the electrode to the cell using a micro manipulator. Once the electrode touches the cell, apply gentle suction via the suction tube until a tight seal is formed.
Then clamp the membrane voltage at the desired holding potential. Obtain the whole cell patch clamp configuration by applying stronger suction. To remove the membrane patch at the tip of the electrode, once whole cell patch clamp configuration has been established, start the recording.
In this procedure, fix the neurons on the cover slips with 4%para formaldehyde for one hour. Then perme the neurons with 2%tween 20 in PBS for 10 minutes. Next, incubate the cells in blocking solution for one hour.
The test cells are incubated in the blocking solution plus primary antibody overnight. The control set of neurons are left to incubate overnight in blocking solution without any primary antibodies. The next day, wash the cover slips with PBS buffer three times for five minutes each, followed by two washes of five minutes each with 1%tween 20 to remove unbound primary antibody.
Then incubate the test and control cover slips with the appropriate fluorescent tagged secondary antibody for 30 minutes, which binds to the primary antibody to permit visualization. After that, wash the cover slips with PB S3 times for five minutes each to remove excess secondary antibody. Next, mount the cover slips on a clean glass slide using mounting media.
Then apply clear nail polish to the edge of the cover slip to fix it in place. Once dry, visualize the neurons and capture images using the software accompanying the fluorescence microscope. This is a representation of Dai injection into the gastro muscle and retrograde transportation from muscle to the DRG.
The L four and L five DRG are labeled here. The afferent neurons are shown in the bright field image and this fluorescent image shows one dye labeled muscle afferent neuron. This histogram shows the DII fluorescent intensity of sensory neurons.
The large peak is from unlabeled neurons and is fitted with a Gaussian equation to determine the mean intensity and the standard deviation. The dashed vertical line represents the threshold for DAI labeling. The histogram comprises data from 1047 neurons with 167 being labeled with dai.
Here is a representative muscle afferent neuron with good control of membrane voltage. The sodium currents at minus 20, minus 10 and zero millivolts are shown on the left and the current voltage relationship is shown on the right. While attempting this procedure, it's important to remember to quickly but gently dissect out the DRGs and to keep the preparation moist repeatedly at ice code hbss.
Following this procedure, other efferent neurons can be labeled and identified to understand their role in physiology.
