whole-cell patch clamp recording in a substantia gelatinosa neuron of a spinal cord slice

Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice

To conduct whole-cell patch-clamp recordings from substantia gelatinosa or SG neurons, use potassium-ion-based intracellular solutions for recording, while applying cesium cation-based solution only for the recording of inhibitory postsynaptic 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 perfuse the slice with bubbled ACSF at room temperature, and set the perfusion rate at 2 to 4 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 micropipette with an appropriate volume of potassium-ion-based or cesium-ion-based intracellular solution. Then, insert the micropipette into the electrode holder, and ensure that the intracellular solution is contacting the silver wire inside the holder. Bring the micropipette 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 micropipette 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 micropipette 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.

whole-cell-patch-clamp-recording-substantia-gelatinosa-neuron-spinal

Universidad de Cartagena UDEC

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Neuroscience

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Electrophysiology Techniques

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preparation of Solutions and Materials
<ol>
	<li>Solutions&#8203;
	<ol>
		<li>Prepare artificial cerebrospinal fluid (ACSF) (in mM): 117 sodium chloride (NaCl), 3.6 potassium chloride (KCl), 1.2 sodium dihydrogen phosphate dihydrate (NaH2PO4&#183;2H2O), 2.5 calcium chloride dihydrate (CaCl2&#183;2H2O), 1.2 magnesium chloride hexahydrate (MgCl2&#183;6H2O), 25 sodium bicarbonate (NaHCO3), 11 D-glucose, 0.4 ascorbic acid, and 2 sodium pyruvate. See Table 1.</li>
		<li>Prepare K+-based intracellular solution (in mM): 130 K-gluconate, 5 KCl, 10 Na2-phosphocreatine, 0.5 ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA), 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 magnesium adenosine triphosphate (Mg-ATP), and 0.3 lithium guanosine triphosphate (Li-GTP). See Table 2.</li>
		<li>Prepare Cesium (Cs+)-based intracellular solution (in mM): 92 cesium methane sulfonate (CsMeSO4), 43 cesium chloride (CsCl), 10 Na2-phosphocreatine, 5 tetraethylammonium (TEA)-Cl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 Li-GTP. See Table 3. 
		Note: All solutions must be prepared using distilled water. ACSF and sucrose-ACSF should be carbogenated (95% O2 and 5% CO2 mixture) prior to use to maintain an optimal pH of approximately 7.4, and the osmolality of these two solutions should be adjusted to 300&#8211;310 milliosmols (mOsm). Because ascorbic acid could affect calcium channels, this agent must be omitted if one would like to record calcium currents. The osmolality and pH of intracellular solutions should be measured and adjusted to 290&#8211;300 mOsm and 7.2&#8211;7.3, respectively. It is recommended to filter the intracellular solutions with 0.2 &#181;m filters and store the solutions as 1 mL aliquots at -20 &#176;C. Cs+ and TEA are applied in Cs+-based intracellular solution to block the potassium channel, which is conducive to using the amplifier to hold the membrane potential steady at 0 mV when recording inhibitory postsynaptic currents (IPSCs).</li>
	</ol>
	</li>
	<li>Instruments
	<ol>
		<li>For a typical electrophysiological system, use an upright microscope equipped with infrared differential interference contrast (IR-DIC) and a high-resolution water-immersion objective, a CCD/CMOS camera, a patch-clamp amplifier, a micropipette holder, and a micromanipulator allowing fine adjustment of the pipette position. An XY stage is also needed to move the microscope.</li>
		<li>Mount all equipment on a vibration isolation table surrounded by a Faraday cage. Connect a video monitor to the video camera to observe the neurons and visualize the micropipettes.</li>
	</ol>
	</li>
	<li>Micropipettes
	<ol>
		<li>Make recording electrodes from borosilicate glass capillaries using a micropipette puller. The typical pipette resistance ranges from 3-6 M&#937; when filled with intracellular solution.</li>
	</ol>
	</li>
</ol>
2. Whole-cell Patch-clamp Recordings
<ol>
	<li>To conduct the whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons, use K+-based intracellular solution for most recording cases while applying Cs+-based solution only for the recording of inhibitory postsynaptic currents.</li>
	<li>Gently move a spinal cord slice to the recording chamber, and then maintain it with a U-shaped platinum wire attached with nylon threads firmly for optimal slice stability. Steadily perfuse the slice with bubbled ACSF at RT through a gravity system and set the perfusion rate at 2&#8211;4 mL/min to achieve sufficient oxygenation.</li>
	<li>Identify the region of SG (a translucent band) using a low-resolution microscope lens, choose a healthy neuron by using the high-resolution objective as the target cell, and adjust it to the center of the video monitor screen.</li>
	<li>Fill a micropipette with an appropriate volume of K+-based or Cs+-based intracellular solution as needed, insert the micropipette into the electrode holder, and ensure that the intracellular solution contacts the silver wire inside the holder.</li>
	<li>Bring the micropipette into focus, immerse it into the ACSF using a micromanipulator, and then apply a mild positive pressure (~1 psi when measured with a manometer) to force the micropipette away from any dirt and debris.</li>
	<li>Move the micropipette towards the targeted neuron gradually. Release the positive pressure once the pipette approaches the neuron, and a very small dimple forms on the neuronal membrane to form a gigaseal.</li>
	<li>Alter the holding potential to -70 mV, which is close to the physiological resting membrane potential (RMP) of a cell. Then, apply a transient and gentle suction to the micropipette to rupture the membrane and create a good whole-cell configuration. 
	Note: After transferring the slice into the recording chamber, ensure steady perfusion for at least 5 min to clear the debris on the slice surface. It is worth noting that the ability to distinguish between healthy and unhealthy/dead neurons is of paramount importance for good sealing and stable recording. An unhealthy/dead neuron has a swollen or shrunken appearance, together with a visible large nucleus, while a healthy neuron is characterized by a 3-dimensional (3D) shape with a bright and smooth membrane, and its nucleus is invisible. To achieve a whole-cell configuration, it is essential to compensate for fast or slow capacitance step-by-step when necessary. At room temperature (RT), the liquid junction potential is calculated to be 15.1&#8211;15.2 millivolts (mV) and 4.3&#8211;4.4 mV in K+-based and Cs+-based intracellular solutions, respectively. In our studies, the recorded data were not corrected for liquid junction potential.</li>
	<li>Recordings of intrinsic membrane properties
	<ol>
		<li>Record firing properties: Test the firing pattern of each neuron in the current clamp with a series of 1 s depolarizing current pulses (25&#8211;150 picoamps (pA) with 25 pA increment) at RMP. Measure the threshold, amplitude, and half-width of a single action potential offline.</li>
	</ol>
	</li>
</ol>
Table 1: Recipe for ACSF
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Molecular Weight</td>
			<td>Concentration (mM)</td>
			<td>g/L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.5</td>
			<td>117</td>
			<td>6.84</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.5</td>
			<td>3.6</td>
			<td>0.27</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>156</td>
			<td>1.2</td>
			<td>0.19</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>147</td>
			<td>2.5</td>
			<td>0.37</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>203</td>
			<td>1.2</td>
			<td>0.24</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84</td>
			<td>25</td>
			<td>2.1</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>180</td>
			<td>11</td>
			<td>1.98</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>198.11</td>
			<td>0.4</td>
			<td>0.08</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>110</td>
			<td>2</td>
			<td>0.22</td>
		</tr>
	</tbody>
</table>
Table 2: Recipe for K+-based intracellular solution
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Molecular Weight</td>
			<td>Concentration (mM)</td>
			<td>mg/100 mL</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>234.2</td>
			<td>130</td>
			<td>3044.6</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.5</td>
			<td>5</td>
			<td>37.28</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>453.38</td>
			<td>10</td>
			<td>453.38</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>380.35</td>
			<td>0.5</td>
			<td>19.02</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>238.31</td>
			<td>10</td>
			<td>238.3</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>507.18</td>
			<td>4</td>
			<td>202.9</td>
		</tr>
		<tr>
			<td>Li-GTP</td>
			<td>523.18</td>
			<td>0.3</td>
			<td>15.7</td>
		</tr>
	</tbody>
</table>
Table 3: Recipe for Cs+-based intracellular solution
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Molecular Weight</td>
			<td>Concentration (mM)</td>
			<td>mg/100 mL</td>
		</tr>
		<tr>
			<td>CsMeSO4</td>
			<td>228</td>
			<td>92</td>
			<td>2097.6</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>168.36</td>
			<td>43</td>
			<td>723.95</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>453.38</td>
			<td>10</td>
			<td>453.38</td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td>165.71</td>
			<td>5</td>
			<td>82.86</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>380.35</td>
			<td>0.5</td>
			<td>19.02</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>238.31</td>
			<td>10</td>
			<td>238.3</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>507.18</td>
			<td>4</td>
			<td>202.9</td>
		</tr>
		<tr>
			<td>Li-GTP</td>
			<td>523.18</td>
			<td>0.3</td>
			<td>15.7</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Used for the preparation of ACSF and PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td>Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Sigma</td>
			<td>71500</td>
			<td>Used for the preparation of ACSF, sucrose-ACSF and PBS</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Used for the preparation of ACSF</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>A7631</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td>Used for the preparation of sucrose-ACSF</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Wako</td>
			<td>169-11835</td>
			<td>Used for the preparation of K+-based intracellular solution</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P1937</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>Li-GTP</td>
			<td>Sigma</td>
			<td>G5884</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>CsMeSO4</td>
			<td>Sigma</td>
			<td>C1426</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Sigma</td>
			<td>C3011</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td>Sigma</td>
			<td>T2265</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Hengxing Chemical Reagents</td>
			<td></td>
			<td>Used for the preparation of PBS</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW150F-4</td>
			<td>1.5 mm OD, 1.12 mm ID</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>Used for the preparation of micropipettes</td>
		</tr>
		<tr>
			<td>Infrared CCD camera</td>
			<td>Dage-MIT</td>
			<td>IR-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier</td>
			<td>HEKA</td>
			<td>EPC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Y stage</td>
			<td>Burleigh</td>
			<td>GIBRALTAR X-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Advanced</td>
			<td>FISKE 210</td>
			<td></td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Mettler Toledo</td>
			<td>FE20</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Zhu, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58479">Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for performing electrophysiological recordings in substantia gelatinosa (SG) neurons of spinal cord slices, using whole-cell patch-clamp techniques to analyze neuron firing patterns. This method allows the study of neural responses to stimuli in SG neurons of the spinal cord.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preparation of Solutions and ...

Place a rat spinal cord slice in a recording chamber and secure it with a tissue anchor.
Perfuse the slice with artificial cerebrospinal fluid.
Under a microscope, identify the translucent substantia gelatinosa or SG region and select a healthy SG neuron.
Position a patch pipette filled with an ionic solution near the neuron.
Apply mild positive pressure to remove any debris from the pipette tip.
Move the pipette towards the neuron.
Then, release the positive pressure to form a dimple on the neuronal membrane, creating a seal.
Alter the holding potential to bring it close to the resting membrane potential, or RMP.
Use brief suction to rupture the membrane, establishing a whole-cell configuration.
At RMP, apply depolarizing electrical pulses.
These pulses open voltage-gated sodium channels, allowing a rapid influx of sodium ions.
This influx of ions triggers an action potential, resulting in neuron firing.
Record the neuron firing patterns.

16 vues. Enregistrement par patch clamp de cellules entières dans un neurone de la substance gélatinosa d’une coupe de moelle épinière

Vidéo: Enregistrement par patch clamp de cellules entières dans un neurone de la substance gélatinosa d’une coupe de moelle épinière - Expérience

Comprendre les réponses des motoneurones à la stimulation de la moelle épinière

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.

Pleins feux sur l’auteur : Faire progresser la stimulation de la moelle épinière - Explorer les réponses cellulaires des motoneurones grâce à l’électrophysiologie par patch-clamp 

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.

Préparation ex vivo d’une tranche de moelle épinière pour l’enregistrement d’un patch-clamp de cellules entières dans les motoneurones lors d’une stimulation de la moelle épinière

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

Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice

Transcription

Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice