preparation of spinal cord slice for the whole-cell patch-clamp recording in motor neurons

脊髄刺激に対する運動ニューロン応答の理解

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Andreas Rowald et al. reported that SCS enables lower limb locomotor and trunk function within a single day1. Exploring the biological mechanism of SCS for locomotor recovery is a critical and trending research field for developing a more precise SCS strategy. For example, Gr&#233;goire Courtine&#39;s team demonstrated that excitatory Vsx2 interneuron and Hoxa10 neurons in the spinal cord are the key neurons to response to SCS, and cell-specific neuromodulation is feasible to restore the rat walking ability after SCI2. However, few studies focus on the electrical mechanism of SCS at a single-cell scale. Although it is well-known that the suprathreshold direct current stimulus can elicit the action potentials (APs) in the classic squid experiment3,4,5, how the pulsed alternating electrical stimulation, such as SCS, affects the motor signal generation is still unclear.
Given the complexity of intraspinal neural circuits, appropriate selection for cell population is important for investigating the electrical mechanism of SCS. Although SCS restores motor function by activating the proprioceptive pathway6, the motor neurons are the final unit to execute the motor command, derived from integrating proprioception information afferent input7. Therefore, directly studying the electrical characteristics of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation.
As we know, patch-clamp is the golden-standard method for cellularly electrophysiological recording with extremely high spatiotemporal resolution8. Therefore, this study describes a method using a patch clamp to study the electrical responses of motor neurons to SCS. Compared with the brain patch-clamp9, the spinal cord patch-clamp is more difficult due to the following reasons: (1) The spinal cord is protected by the vertebral canal with tiny volume, which requires very fine micromanipulation and rigorous ice-cold maintenance to obtain better cell viability. (2) Because the spinal cord is too slender to be secured on the cutting tray, it should be immersed in low-melting point agarose and trimmed after solidification.
Hence, this method provides technical details in dissecting the spinal cord and maintaining the cell viability at the same time so as to smoothly study the electrical mechanism of SCS on motor neurons and avoid unnecessary trials and mistakes.

著者スポットライト:脊髄刺激の進歩 - パッチクランプ電気生理学による運動ニューロンの細胞応答の探索 

脊髄刺激時の運動ニューロンにおける全細胞パッチクランプ記録のための脊髄スライスの ex vivo 調製

Spinal cord stimulation can help patients resolve most motion functions after injury. Most neurons are to execute sensory mode behaviors. Studying the electric response of most neurons can aid in understanding the underlying logic of spinal load neuromodulation.A patch clamp is the golden standard method for cellular electrophysiological recordings with the extremely high spatial temporal resolution. Therefore, this study describes a method using a patch clamp to simultaneously record the aortas stimulus characteristics and cellular responses of motor neurons at a single-cell scale. Compared with the brain patch clamp, the spinal cord patch clamp is more difficult, because the spinal cord is well protected by the vertebral canal with tiny volume.Therefore, this protocol provides technical details in quickly micro dissecting the spinal cord and reverse maintenance to obtain better cell viability. Patch clamp allows a unique understanding of the synaptic transmissions and the understanding of action potentials, coding pattern activities by the spinal cord stimulation.

Watch this Scientific Journal Video about Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons at JoVE.com

Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons  

運動ニューロンの全細胞パッチクランプ記録のための脊髄スライスの作製

脊髄を解剖し、腰椎肥大部を分離した後、背側を上にして尾側を下にして腰椎肥大部を35度の傾斜に置きます。吸収性濾紙を使用して、組織表面の余分な水分を吸収します。溶融したアガロースゲルを腰椎肥大の入ったペトリ皿に慎重に注ぎます。ペトリ皿を氷水混合物に置き、ゲルが急速に冷却されるようにします。ゲルを15 x 10 x 10ミリメートルの立方体に成形し、瞬間接着剤を使用して試料ディスクに取り付けます。ビブラトームの設定を、厚さ350マイクロメートル、速度0.14〜0.16ミリメートル/秒、振幅1ミリメートル、振動周波数85ヘルツに調整します。次に、カバースライドピンセットを使用してスライスをクリップし、連続的に酸素化された人工脳脊髄液で満たされたインキュベーションチャンバーに入れます。

preparation-spinal-cord-slice-for-whole-cell-patch-clamp-recording

Research

JoVE Journal

Neuroscience

Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons

274 ビュー.  Tsinghua University. 脊髄を解剖し、腰椎肥大部を分離した後、背側を上にして尾側を下にして腰椎肥大部を35度の傾斜に置きます。吸収性濾紙を使用して、組織表面の余分な水分を吸収します。溶融したアガロースゲルを腰椎肥大の入ったペトリ皿に慎重に注ぎます。ペトリ皿を氷水混合物に置き、ゲルが急速に冷却されるようにします。ゲルを15 x 10 x 10ミリメートルの立方体に成形?...

ビデオ: Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons  

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

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.

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

神経科学

Spinal Cord Dissection for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After exposing the heart of an anesthetized rat, cut the right atrium using fine scissors and inject 100 milliliters of chilled perfusing fluid at approximately two milliliters per second within one minute. Position the rat in the prone position. Cut the spine at the anterior superior iliac spine and the curvature shifting point of the thoracic column.Immediately place the isolated spine into the oxygenated ice cold perfusing solution to wash off residual blood and fat tissue. Then transfer the isolated spine to the anatomical tray with its dorsal side up and the rostral end near the operator. Fill the tray with 50 milliliters of continuously oxygenated ice cold cutting solution.Under a dissection microscope, cut the vertebral pedicles on both sides from the rostral end using micro scissors. Next, delicately sever the dura mater along its dorsal midline with micro scissors. Lift the rostral part of the spinal cord carefully and cut the nerve root, leaving about one millimeter reserved.After the spinal cord has been separated from the vertebral canal, secure it with the ventral side facing up using two insect pins. Trim any excessive nerve roots, leaving about one millimeter intact. Then separate the lumbar enlargement to a length of six to seven millimeters using a micro scissor.

運動ニューロンにおける全細胞パッチクランプ記録のための脊髄解剖

After dissecting the spinal cord and separating the lumbar enlargement, place the lumbar enlargement on a 35 degree slope with the dorsal side up and the caudal side down. Absorb any excess water on the tissue surface using an absorbent filter paper. Carefully pour the molten agarose gel into the Petri dish containing the lumbar enlargement.Position the Petri dish in the ice water mixture to ensure rapid gel cooling. Shape the gel into a cube of 15 by 10 by 10 millimeters, and mount it onto the specimen disc using super glue. Adjust the vibratome settings to a thickness of 350 micrometers, a speed of 0.14 to 0.16 millimeters per second, an amplitude of one millimeter, and a vibration frequency of 85 hertz.Now, use the cover slide tweezers to clip a slice and place it into the incubation chamber, filled with continuously oxygenated artificial cerebrospinal fluid.

Patch-Clamp Recording to Study the Electrical Responses of Motor Neurons to Spinal Cord Stimulation

To begin, place the prepared spinal cord slice in the recording chamber. Then place the anode of the micro-manipulation system near the dorsal midline in the cathode close to the dorsal root entry zone for proper spinal cord stimulator positioning. Now switch to a 60X objective lens to identify a healthy neuron having a smooth and bright surface without visible nuclei.Slightly decrease the infrared intensity, turn on the fluorescent light source, and adjust the light filter to the wideband ultraviolet excitation option. Now, select an appropriate Fluoro-Gold-positive motor neuron and gradually lower the electrode until it is near the neuron. As the pipette contacts its surface, look for a small membrane indentation at the tip level and release the previously-applied positive pressure.Then apply negative pressure to the pipette using a syringe and wait until the resistance value rises to gigaohms on the software interface. Clamp the membrane potential at minus 70 millivolts. Press the fast capacitance compensation button on the amplifier's software interface, then apply a brief negative pressure to rupture the cell membrane and press the slow capacitance compensation button on the amplifier's software interface.Apply the spinal cord stimulation for one to two seconds keeping the amplitude between one and 10 milliamperes. Determine the motor threshold by gradually increasing the stimulation amplitude until the first action potential is observed. Distinguish delayed and immediate firing motor neurons using a five-second depolarizing current injection around rheobase in the current clamp mode.After turning off the spinal cord stimulation, continue recording the membrane potentials to capture the spontaneous action potentials firing. When the amplitude of spinal cord stimulation was raised, the membrane potential was increased, and the action potentials were triggered every 10 to 20 pulses. After the spinal cord stimulation was turned off, the neuron fired a series of spontaneous action potentials for a short period of time.Then, the resting membrane potential returned to minus 65 millivolts.