The Institutional Animal Care and Use Committee approved all animal experiments and the studies were conducted in accordance with relevant animal welfare regulations.
1. Animals preparation
2. Patch-clamp recording with SCS (Figure 2)
Thanks to the rigorous low-temperature maintenance during the fine operation (Supplementary Figure 1, Supplementary Figure 2, and Figure 1), the cell viability was good enough to perform subsequent electrophysiological recordings. To simulate the clinical scenario as much as possible, we used micromanipulation to place the SCS cathode and anode near the dorsal midline and DREZ, respectively (Figure 2), which could initiate neural signal in the dorsal horn to propagate to the motor neurons in the dorsolateral region of the motor column. In this study, we used FG to locate the motor neurons with a diameter of 20-50 µm. As shown in Figure 2D,G, we confidently confirmed a healthy FG+ neuron as a motor neuron-the terminal of the spinal circuit. This labeling method paved the way to study how SCS affects the firing pattern. The electrophysiological properties of delayed and immediate firing motor neurons are listed in Supplementary Table 1 and Supplementary Table 2. The methods for calculating the active and passive properties are provided in Supplementary File 1.
When SCS delivered a pulsed alternating electric field to the spinal slice, we first used current-clamp mode to record the response of membrane potential (Figure 4). As we gradually increased the stimulation amplitude with a step of 1/3 motor threshold (Figure 4B,C), the membrane potential also rose with it, but only 1x motor threshold could elicit Aps (Figure 4D). Figure 4D shows that about every 10-20 pulses could elicit an AP, which indicates that a definite AP response regularity to SCS amplitude might exist.
After the SCS was turned off, we continued to record membrane potential. Figure 5 showed that the membrane potential slightly increased to -60 mV, and the neuron fired a series of spontaneous APs. These spontaneous APs last for a short period of time (30-40 s), then the membrane potential returned to -65 mV, indicating that the SCS can temporarily increase the cell excitability.
Then, we used voltage-clamp recordings for EPSC when SCS was on and off (Figure 6). After each SCS pulse, an evoked EPSC could be detected. The latency between the stimulus artifact and EPSC was 2.64 ± 0.38 ms (Supplementary Figure 4). The amplitude of evoked EPSCs was 35.14 ± 12.73 pA (Supplementary Figure 4). The frequency of evoked EPSCs is consistent with the SCS frequency. After subtracting the passive charge balancing, the evoked EPSC can be observed in a viable motor neuron following 1x motor threshold stimulation (Supplementary Figure 5A). The stimulation polarity was reversed after stopping the perfusion for 2 h in the same cell. The stimulation did not induce any EPSC, confirming that the evoked EPSC was not an artifact (Supplementary Figure 5B).
Figure 1: Spinal cord dissection and slicing. (A) Cut the spine at the anterior superior iliac spine (about L4 vertebral level) and the curvature shifting point of the thoracic column (about T6 vertebral level), respectively. (B) Immediately transfer the isolated spine to the anatomical tray with the dorsal side up and the rostral end close to the operator. Fill the anatomical tray with the continuously oxygenated ice-cold cutting solution. (C) Perform laminectomy on both sides from the rostral end. (D) Cut the dorsal dura mater, which is conducive to nutrient uptake between cells and oxygenated artificial cerebrospinal fluid (ACSF). (E) Carefully cut the nerve root. (F) Cut the ventral dura mater.(G)Separate the lumbar enlargement to a length of 6-7 mm. (H) Place the lumbar enlargement on the 35 °slope with the dorsal side up and caudal end down. Use an absorbent filter paper to remove abundant water on the tissue surface. (I) Slowly pour the molten agarose gel into the petri dish. (J) Trim the gel into a cube and mount it on the specimen disc with super glue.(K)Place the specimen disc into the cutting tray, then pour the ice-cold cutting solution.(L)An example of a spinal slice at lumbar enlargement. Please click here to view a larger version of this figure.
Figure 2: Spinal cord stimulation (SCS) configuration and motor neuron imaging. (A) The pulse generator with custom-made electrodes can separately adjust the amplitude, pulse width, and frequency. Inlet showed that the dimensional specification of an electrode contact is 800 µm x 500 µm x 300 µm. (B) Place the anode and cathode near the dorsal midline and dorsal root entry zone (DREZ) via the micromanipulation system, respectively. Place the recording pipette at the dorsolateral region of the motor column to clamp the Fluoro-Gold positive (FG+) motor neurons. (C) A healthy immediate firing motor neuron with infrared differential interference contrast microscope (IR-DIC) imaging (60x). (D) The same neuron with only fluorescence imaging (60x), shining Fluoro-Gold (FG) particles represent that this neuron is a motor neuron.(E)Merged image of IR-DIC and fluorescence in FG+ motor neuron. (F-H)A healthy delayed firing motor neuron with IR-DIC imaging (60x). Please click here to view a larger version of this figure.
Figure 3: Distinguish delayed and immediate firing motor neurons using a 5 s depolarizing current injection. (A) Immediate firing motor neurons:Low rheobase can induce immediate and repetitive firing with stable firing frequency; (B) Delayed firing motor neurons: High rheobase can induce a delayed onset for repetitive firing with an accelerating firing rate. Please click here to view a larger version of this figure.
Figure 4: Spinal cord stimulation (SCS) elicited action potentials (APs) firing in motor neurons. (A) When no stimulation was applied, the resting membrane potential (RMP) was -65 mV. (B) 1/3x motor threshold stimulus cannot elicit APs. (C) 2/3x motor threshold stimulus cannot elicit APs.(D) 1x motor threshold stimulus can elicit APs, and every 10-20 pulses can elicit an AP. Please click here to view a larger version of this figure.
Figure 5: Spontaneous action potentials (APs) firing after spinal cord stimulation (SCS). After the SCS was turned off, the neuron fired a series of spontaneous APs for a short period of time (30-40 s), then the resting membrane potential (RMP) returned to -65 mV. Please click here to view a larger version of this figure.
Figure 6: Illustration of spinal cord stimulation (SCS) parameter and evoked excitatory postsynaptic current (EPSC). (A) Illustration of SCS parameter; (B,C) Following a single stimulation pulse (1x motor threshold stimulation), the evoked EPSC can be observed; (D) After subtracting the passive charge balancing, the latency and amplitude of the EPSC can be calculated. Please click here to view a larger version of this figure.
Supplementary Figure 1: Instrument preparation. (A) Perfusion tray: at 10 min before the perfusion, place crushed ice on the 10-cm petri dish filled with silica gel. (B) Anatomical tray: in the night before sacrifice, place the self-made anatomical tray filled with silica gel in the -80 °C refrigerator. (C) Cutting tray: in the night before sacrifice, place the cutting tray with water band in the -80 °C refrigerator. (D) Agarose casting slope: at 30 min before the perfusion, place a 35° agarose slope with base plates in the center of a 35-mm petri dish. Please click here to download this File.
Supplementary Figure 2: Intracardial perfusion. (A) Lift the xiphoid process. (B) Cut the sternum along both sides of the xiphoid process to open the chest and expose the heart. Be careful not to damage the internal thoracic vessels, or massive bleeding will fill the entire operating field and hamper the operator from identifying the ventricle apex or right atrium. (C) Insert a 22 G needle at the left ventricle apex for perfusion and cut the right atrium for fluid exit. Please click here to download this File.
Supplementary Figure 3: Confirming the dorsal root activity. Use suction electrodes to apply 1x motor threshold stimulation to the dorsal root. If an evoked action potential is detected in the motor neurons, we can confirm that the activity of the dorsal root is intact. Please click here to download this File.
Supplementary Figure 4: Latency and amplitude of evoked EPSCs when SCS was on. Please click here to download this File.
Supplementary Figure 5: Demonstration of the validity of the evoked EPSC. (A) Following 1x motor threshold stimulation after subtracting the passive charge balancing, the evoked EPSC can be observed in a viable motor neuron; (B) After stopping the perfusion for 2 h in the same cell, the stimulation polarity was reversed, 1x motor threshold stimulation did not induce any EPSC, which confirmed that the evoked EPSC was not an artifact. Please click here to download this File.
Supplementary File 1: Calculation methods of the active and passive properties. Please click here to download this File.
Supplementary Table 1: Passive properties of motor neurons. Please click here to download this File.
Supplementary Table 2: Active properties of motor neurons Please click here to download this File.
Adenosine 5’-triphosphate magnesium salt | Sigma | A9187 | |
Ascorbic Acid | Sigma | A4034 | |
CaCl2·2H2O | Sigma | C5080 | |
Choline Chloride | Sigma | C7527 | |
Cover slide tweezers | VETUS | 36A-SA | Clip a slice |
D-Glucose | Sigma | G8270 | |
EGTA | Sigma | E4378 | |
Fine scissors | RWD Life Science | S12006-10 | Cut the diaphragm |
Fluorescence Light Source | Olympus | U-HGLGPS | |
Fluoro-Gold | Fluorochrome | Fluorochrome | Label the motor neuron |
Guanosine 5′-triphosphate sodium salt hydrate | Sigma | G8877 | |
HEPES | Sigma | H3375 | |
infrared CCD camera | Dage-MTI | IR-1000E | |
KCl | Sigma | P5405 | |
K-gluconate | Sigma | P1847 | |
Low melting point agarose | Sigma | A9414 | |
MgSO4·7H2O | Sigma | M2773 | |
Micromanipulator | Sutter Instrument | MP-200 | |
Micropipette puller | Sutter instrument | P1000 | |
Micro-scissors | Jinzhong | wa1020 | Laminectomy |
Microscope for anatomy | Olympus | SZX10 | |
Microscope for ecletrophysiology | Olympus | BX51WI | |
Micro-toothed tweezers | RWD Life Science | F11008-09 | Lift the cut vertebral body |
NaCl | Sigma | S5886 | |
NaH2PO4 | Sigma | S8282 | |
NaHCO3 | Sigma | V900182 | |
Na-Phosphocreatine | Sigma | P7936 | |
Objective lens for ecletrophysiology | Olympus | LUMPLFLN60XW | working distance 2 mm |
Osmometer | Advanced | FISKE 210 | |
Patch-clamp amplifier | Axon | Multiclamp 700B | |
Patch-clamp digitizer | Axon | Digidata 1550B | |
pH meter | Mettler Toledo | FE28 | |
Slice Anchor | Multichannel system | SHD-27H | |
Spinal cord stimulatior | PINS | T901 | |
Toothed tweezer | RWD Life Science | F13030-10 | Lift the xiphoid |
Vibratome | Leica | VT1200S | |
Wide band ultraviolet excitation filter | Olympus | U-MF2 |
Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.
Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.
Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.