This protocol uses intracellular recordings of spinal motoneurons, to directly investigate the influence of trans-spinal direct current stimulation on the function of spinal networks. The major advantage of this technique is that it allows intracellular recordings to be performed on fully mature nervous system. Facilitating translation of the experimental observations to practical applications.
After confirming a lack of response to pedal reflex, an anesthetized six month old male Wistar rat, use a dissecting microscope and a number 21 blade, to make a longitudinal skin incision from the sternum to the chin. And use blunt dissection to expose the right jugular vein. Place two, 4-0 ligatures beneath the section of the vein without branching points.
And make one loose knot on the proximal end of the vein segment. And one loose knot on the distal end of the segment. Clamp the vein proximal to the heart, and ligate the distal end of the vein.
Use iris scissors to make an incision between the clamp and the distal ligature. And, holding a flap of the vein, introduce a pre-filled catheter to the point where the vein is blocked by the clamp. Then remove the clamp and push the catheter several millimeters into the vein.
For a tracheal tube placement, use blunt forceps to separate the two mandibular glands covering the sternohyoid muscles. And separate the sternohyoid muscles at the midline, to expose the trachea. Place three, 4-0 ligatures beneath the trachea, and make two knots below the tracheal tube insertion point and one knot above.
Then insert a tracheal tube into the trachea below the third tracheal cartilage. And secure the tube in place with the pre-prepared ligatures. To dissect the hind limb nerves, use a number 21 blade to make a longitudinal cut on the posterior side of the left hind limb, from the Achilles tendon to the hip.
And use scissors to make a cut between the anterior and posterior regions of the popliteal fossa, at the back of the knee joint. Cut two heads of the biceps femoris, to expose the sciatic nerve. And separate the lateral head from the medial head of the gastrocnemius muscle, to expose the tibial nerve and its branches.
Then use number 55 forceps, to carefully dissect the medial gastrocnemius and the lateral gastrocnemius and soleus nerves. Disconnecting them from the surrounding tissues while maintaining their connection to the respective of muscles. To perform the laminectomy, use a number 21 blade to make a longitudinal incision from the sacrum up to the thoracic vertebrae.
And identify the Th13 vertebra, as a lowest thoracic segment with a rib insertion. Then use fine rongeurs to remove the spinous processes and laminae from the Th13 to the L2 vertebrae, to expose the lumbar segments of the spinal cord. To fix the vertebral column, place the rat into a custom made frame on a 37 degrees Celsius heating pad, connected to a closed loop heating system.
And use the skin flaps to form a deep pool over the exposed spinal cord. Place metal clamps below the Th12 transverse processes, and at the L3 spinous processes, and fill the pool with 37 degrees Celsius mineral oil. Use the skin flaps to make a deep pool of mineral oil over the exposed tibial, medial gastrocnemius and lateral gastrocnemius and soleus nerves.
And place the nerves on bipolar silver wire stimulating electrodes. Then connect the electrodes to a square pulse stimulator using separate stimulation channels for each nerve. For a surface electrode placement, under the dissecting microscope, place a silver ball electrode on the left caudal side of the exposed spinal cord.
With a reference electrode inserted in the back muscles. And connect both electrodes to the differential DC amplifier. Use a constant current stimulator to stimulate the medial gastrocnemius and lateral gastrocnemius and soleus nerves, with square pulses of a 0.1 millisecond duration repeated at a three hertz frequency and observe the afferent volleys.
At the end of the simulation, move the surface electrode rostrally, and repeat the stimulation to identify the spinal segments at which the amplitudes of the volleys are the highest for each nerve. After determining the location of the maximum volley, deliver a neuromuscular blocker intravenously to paralyze the rat. While an assistant connects the tracheal tube to an external ventilator in line with a rodent compatible cap nominator.
To open the dura and pia mater, use number 55 forceps to gently lift the dura mater, and cut the tissue caudally from the L5 segment, rostrally up to the L4 segment. Then use a pair of ultra thin 5SF forceps, to make a small patch in the pia, covering the dorsal column between the blood vessels. Exactly at the level of the maximum afferent volley from the medial gastrocnemius and lateral gastrocnemius and soleus nerve.
To place the trans-spinal direct current stimulation electrodes, place a saline soaked sponge on the dorsal side of the Th12 vertebrae. And use fine manipulation to press the sponge with an active trans-spinal direct current stimulation electrode. Then mount a custom pooled microelectrode, onto the micro manipulator, allowing a one to two micron stepping movement and stereotaxic calibration.
And drive a micropipette tip into a selected patch in the pia, at a 15 to 20 degree medial lateral angle. To record the motoneuron membrane and firing properties, in the bridge mode of the intracellular amplifier, stimulate the respective nerve branches to identify the motoneuron on the basis of the all or nothing appearance, of the antidromic action potential. In the discontinuous current clamp mode of the intracellular amplifier with a current switch rate mode four to eight kilohertz, use a 0.5 millisecond intracellular depolarizing current pulses, to evoke an orthodontic action potential, in the motoneuron.
To calculate the cell input resistance, stimulate a motoneuron, with 40 short 100 millisecond pulses of hyperpolarizing one nanogram current. To determine the real base value as the minimum amplitude of the depolarizing current required to elicit a single spike, stimulate a motoneuron with 50 millisecond square wave pulses at increasing amplitudes. Then inject 500 millisecond square wave pulses of depolarizing current, at increasing amplitudes.
In 0.1 to two nano amp steps, to evoke rhythmic discharges of motoneurons. For a trans-spinal direct current stimulation, start the polarization procedures by trans-spinal application of direct current, while maintaining a stable penetration of the motoneuron. Here, a typical orthotropic action potential, evoked by intracellular stimulation that meets all of the criteria for data inclusion is shown.
In this analysis, a cell response to a 100 millisecond hyperpolarizing current pulse of one nanogram. From which both the peak and plateau input resistance of a motoneuron, can be determined from the voltage deflection, can be observed. This expanded voltage trace of a real basic spike, exhibits a clearly marked voltage threshold of the spike.
Indicating the level of membrane depolarization at which voltage gated sodium channels are activated to initiate the action potential. These graphs provide examples of intracellular voltage traces. From two motoneurons, stimulated intracellularly with 500 millisecond square pulses of depolarizing current.
Before, during, and after a trans-spinal direct current stimulation application. Anodal trans-spinal direct current stimulation was found to act toward an increased motoneuron excitability and higher frequencies of rhythmic firing. While cathodal trans-spinal direct current stimulation, acted to towards firing inhibition.
Moreover, the effects of both types of trans-spinal direct current stimulation, outlasted the period of polarization. Inaccurate data can be acquired, if the data inclusion criteria are compromised due to imperfect cell penetration. A failure to compensate the microelectrode resistance and capacitance or a spinal cord instability.
It is imperative that no damage is done to the spinal cord during the dissection as injury can result in spinal shock, making further recordings impossible. It is possible to harvest tissue samples for further histological or immuno cyto chemical analysis. For example, the measurement of c-fos expression can be used as a proxy of neuronal activity.
