Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

This method can help clarify the spinal mechanisms underlying sensory transmission, nociceptive regulation and chronic pain or ache development The main advantage of this technique is that it can permit ideal neuronal preservation and it can mimic in-vivo conditions to a certain extent. Start by pulling recording electrodes from borosilicate glass microcapillaries using a capillary puller. Then, fill a mold with molten agar to prepare a 1.2 centimeter by 1.5 centimeter by 2.0 centimeter agar block.

Trim the block to have a central channel if planning transverse sections. Or a channel flesh with the edge of the block for parasagittal sections. Prior to transcardial profusion and spinal cord extraction, prepare 500 milliliters of sucrose based ACSF.

Cool the solution to ice cold and equilibrate with 95%O2 and 5%CO2. Cool all the dissection tools on ice. Make a longitudinal five centimeter incision on the back skin from caudal to rostral.

Then, cut through the rib cage between the spine and ribs on either side. Use scissors to made a cut at the caudal end of the spine. Then cut away the surrounding tissues and quickly isolate the lumbosacral segment of the spine.

Transfer the lumbosacral segment to a glass dish containing ice cold sucrose based ACSF. With the ventral side upward, use fine scissors to cut through the vertebral pedicle bilaterally and expose the spinal cord carefully. Isolate a two centimeter long section of spinal cord with the lumbosacral enlargement and transfer the spinal section into another glass dish filled with cold sucrose ACSF.

Work under a dissection microscope to remove the meninges and the pia arachnoid membrane. Cut all the ventral and dorsal roots away as quickly as possible. Take care to avoid the injuring to the spinal cord, especially the dorsal horn when removing the meninges and the spinal roots.

Place the spinal cord on the previously trimmed agar block. To prepare transverse slices, attach the ventral side of the spinal cord to the agar with the dorsal side toward the blade. To prepare parasagittal slices, attach the ventral side with superglue to the agar in a vertical direction.

Then, mount the agar block to a platform of a vibratome with superglue. And prepare 300 to 500 micron transverse or parasagittal slices with an advanced speed of 0.025 millimeters per second and a vibration frequency of 80 Hertz. The spinal cord should be sliced dorso-ventrally.

For the best results, the slice should be prepared within 15 to 20 minutes. The thickness of the spinal slice should be no more than 600 microns to satisfy cell visibility. Use a plastic trimmed pipette to transfer the slices onto nylon mesh in a storage chamber containing continuously oxygenated ACSF at 32 degrees Celsius.

To conduct whole-cell patch clamp recordings from substantia gelatinosa or SG neurons, use potassium ion based intracellular solutions for recording while applying caesium cation based solution only for the recording of inhibitory post-synaptic currents. Gently move the spinal cord slice to the recording chamber. And then stabilize it firmly with a U shaped platinum wire with nylon threads for optimal slice stability.

Steadily profuse the slice with bubbled ACSF at room temperature and set the profusion rate at two to four milliliters per minute to achieve sufficient oxygenation. Then, using a low resolution microscope lens, identify the region of SG neurons as a translucent band. Choose a healthy neuron, characterized by a three dimensional shape with a bright and smooth membrane as the target cell using the high resolution objective and adjust it to the center of the video monitor screen.

Fill a micro pipette with an appropriate volume of potassium ion based or caesium ion based intracellular solution. Then insert the micro-pipette into the electrode holder and ensure that the intracellular solution is contacting the silver wire inside the holder. Bring the micro pipette into focus and use the micromanipulator to immerse it into the ACSF.

Apply a mild positive pressure to force away any dirt and debris. Gradually move the micro pipette towards the targeted neuron. Once the pipette approaches the neuron and a very small dimple forms on the neuronal membrane, release the positive pressure to form a gigaseal.

Alter the holding potential to minus 70 millivolts, which is close to the physiological resting membrane potential of a cell. Then, apply a transient and gentle suction to the micro pipette to rupture the membrane and create a good whole-cell configuration. Record firing properties by testing the firing pattern of each neuron in current clamp with a series of one second depolarizing current pulses of 25 to 150 picoamps with 25 picoamp increments at resting membrane potential.

To record somatic subthreshold currents, hold the membrane potential at minus 50 millivolts in voltage clamp mode then apply a series of hyperpolarising voltage pulses of one second duration from minus 60 millivolts to minus 120 millivolts with a 10 millivolt decrement. Record excitatory post-synaptic currents with the potassium ion based intracellular solution in voltage clamp mode at a holding potential of minus 70 millivolts. Record IPSC's using caesium ion based intracellular solution for recording IPSC's.

After holding the membrane potential at minus 70 millivolts for approximately five minutes, gradually change the holding potential to zero millivolts. Wait a few minutes for stabilization and then start to record the IPSC events. Firing patterns determined by injecting a series of one second depolarizing current pulses into an SG neuron at resting membrane potential may be classified as tonic-firing, delayed-firing, gap-firing, initial-burst, phasic-bursting, single-spike and reluctant-firing.

This image shows the evoking protocol for subthreshold currents in voltage clamp. These representative traces show the response to hyperpolarizing current injection classified as IH, IA and IT.This is a representative trace of SEPSC's recorded from SG neurons at minus 70 millivolts in the absence and presence of 50 micromolar APV and 20 micromolar CNQX. This trace shows the recorded SEPC's before the addition of APV and CNQX on an expanded timescale.

Following this procedure are the methods like paired patch clamp recordings or optogenetics can be performed to answer additional questions like characterizing specific neuronal microcircuits in substantia gelatinosa.