The overall goal of this procedure is to prepare oblique slices of the spinal cord that allow for the stimulation of the ventral roots. The main advantage of this technique is that it allows motoneural identifications for ventral-root stimulation and to study the ventral cells enervated by motoneural axon collaterals. We first had the idea for this method when needing a reliable way to stimulate ventral cells.
Individuals new to this method will struggle because of the difficulty to properly execute the different steps of the dissection. Begin by perfusing a P2 to P11 mouse with low-sodium ACSF. After decapitating the mouse and removing the skin on the back, make two cuts through the shoulders and down the rib cage.
Then, cut the spinal cord as low as possible in the caudal section to isolate the vertebral column and ribs from the lower part of the animal. Flip the vertebral column and remove the viscera still attached to the ribs. Transfer the vertebral column to another smaller, silicon-filled Petri dish.
Add cold ACSF and bubble with carbogen and use four insect pins to hold the tissue dorsal-side up. Then perform a laminectomy of the dorsal side by first inserting the tip of fine scissors between the bone and the spinal cord and cutting the bone from the rostral end, making sure to stay away from the white matter. Alternate the cuts on each side while using tweezers to hold the band of bone already cut away.
Next, use small tweezers to lift the dura and small scissors to cut along the rostral caudal access on both sides. Removal of the dura is a critical step. It is well worth spending a few extra minutes to do this thoroughly.
Once the dura is removed, use a blunt glass tip to gently push cord towards the left side of the groove formed by half-cut spinal column. Then cut the ventral and dorsal roots on the bright side as far as possible from where they enter the cord. Repeat the operation on the left side, always going from rostral to caudal.
After dissection, slip the spinal cord out of the vertebral column. Then use a small insect pin to secure the cord for the dorsal surface-up. Delicately remove any pieces of attached membrane.
Once cleaned, trim both ends of the cord and insert a bent insect pin into the anterior part of the cord to mark its orientation. Then transfer the spinal cord to ice-cold intracellular solution. Next, cool the previously prepared molten agar on a mixture of ice and water.
Stir the agar while measuring the temperature. When the temperature of the agar reaches 38 degrees Celsius, hold the spinal cord by the insect pin and immerse it in the agar, rostral-side down. Make sure the spinal cord is as straight as possible, away from the walls, with the caudal part slightly upward.
Leave the beaker in the mixture of ice and water to allow the agar to solidify around the cord as quickly as possible. After solidification, cut the agar block containing the spinal cord in such a way that the base of the block is at a 35-degree angle with the lumbar part of the cord. The dorsal surface should be facing away from the base.
Glue the block into the chamber of the vibratome, using cyanoacrylate glue. It is critical to mount the block correctly to maintain the continuity of the motor pools with the ventral roots from which they exit. Immerse the block in potassium gluconate solution.
Set the vibratome parameters. Next, cut 350 400 micron-thick slices of the lumbar region, which is identifiable by its curvature and larger diameter. Transfer the suitable slices to ACSF at 34 degrees Celsius.
Typically, there are four to five suitable slices with ventral roots extending two millimeters or more. After approximately 30 minutes at 34 degrees Celsius, cool the slices to room temperature, then begin the recording session. Prepare a box of various pipettes with tip diameters ranging from 40 to 170 microns in advance.
To prepare the section pipettes, select pipettes with a long tapure. Then, using a diamond knife, make cuts at different positions and under a dissecting microscope, break each pipette by hitting the tip with tweezers. Next, remove the chamber from the recording microscope and place it under a dissecting microscope.
Select a slice that contains a ventral root of sufficient length to be mounted on a suction-stimulation electrode. Mount the slice, orienting it with the ventral roots upward, then delicately cut the agar around the ventral roots while leaving the rest of the agar around the slice and place the grid to hold the sample down. Mount the chamber back onto the microscope and continuously perfuse the recording chamber with ACSF at a rate of one to two milliliters per minute at room temperature.
Using a glass pipette filled with ACSF and connected to a syringe, suck one of the ventral roots. In order to achieve good stimulation of the ventral root, the pipette tip needs to be tight around the ventral root. Achieve patch-clamp recording of the desired cell type and record the effect of the ventral root stimulation as described previously.
This image shows the results from voltage-clamp recording. The recordings of the upper panels are taken from different motoneurons than the ones in the bottom panels. The upper panel shows the antidromic response of a motoneuron to a one-volt stimulation in red, and a five-volt stimulation in black.
The lower trace shows the orthodromic action potential response to a 20-volt stimulation in red, and a 30-volt stimulation in black. Black dots indicate stimulus timing. The double arrow heads emphasize the difference in latencies between ortho-and antidromic spikes.
Here, current clamp recordings are shown. The upper panel shows the antidromic response to 10-volt and 15-volt stimulations in red and black, respectively. The lower panel shows the orthodromic response to 25-and 40-volt stimulations in red and black, respectively.
Again, the stimulus timing and differences in latencies are indicated. This image of the Renshaw cell pool was acquired using an oblique condenser and a 20x objective. Note the motoneuron axons merging into the ventral root as shown by the arrow.
This image of the same region was taken using a 40x objective. Putative Renshaw cells are indicated with arrow heads. Shown here is a voltage-clamp recording of a Renshaw cell following stimulation of the ventral root, indicated by the black dot.
QX-314 in the intracellular solution prevented the cell from firing and glycine and gaba responses were blocked by 3 micromolar gabazine and one micromolar strychnine, respectively. The red trace is the average of the gray ones. Once mastered, this technique can be performed in 20 or 30 minutes.
While attempting this procedure, it's important to remember to keep the preparation in cold medium at all time. After watching this video, you should have a good understanding of how to prepare oblique slices of spinal cord suitable for ventral-root stimulation. Ventral-root stimulation can be used to easily confirm the motoneural identity, and more importantly, to reliably stimulate the ventral cells.
