whole-cell patch clamp recordings on intact rat dorsal root ganglia

Whole-Cell Patch Clamp Recordings on Intact Rat Dorsal Root Ganglia

In this procedure, place the electrode solution on ice to prevent degradation. Next, fill the glass patch pipette with filtered intracellular solution. Then, place the pipette in the head stage pipette holder.
Apply gentle positive pressure to the pipette through a 5-milliliter syringe by displacing the plunger about 1 milliliter before lowering the pipette into the bath solution. Subsequently, choose V-clamp mode on the amplifier, and open the membrane test interface in the software.
Under the microscope, move the pipette close to the target neuron. Next, apply positive pressure from the pipette to traverse the satellite glial cell layer until a sudden enlargement of space between the neuron and the surrounding layer of satellite glial cells is observed. Then, keep moving the pipette towards the neuron until a dimple is observed on the neuron.
In our preparation, the neurons are surrounded by satellite glial cells. Therefore, to penetrate the glial cell layers and reach individual neurons, the recording pipette is maintained at a high positive pressure.
After that, reduce the positive pressure. Achieve a gigaohm seal with gentle suction.
To obtain whole-cell recording configuration, penetrate the neuron cell membrane via short but strong suction.
Alternatively, use the zap function on the amplifier while suction is applied.
Once a whole-cell mode is established, compensate the whole-cell capacitance and series resistance by turning the capacitance and resistance compensation knobs on the amplifier. Then, close the membrane test window. Choose I-clamp normal mode by turning the mode knob on the amplifier.
Afterward, click Open protocol in the software. Select and load the protocol for measuring rheobase, and click Record to start recording. To examine the neuronal excitability, measure the input resistance and rheobase by injecting a graded series of depolarizing currents in steps of 100 picoamps.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. &#160;
1. Patch Clamp Recordings
<ol>
	<li>Place the electrode solution (see Table 1) on ice to prevent degradation. Fill the glass patch pipette with filtered intracellular solution (syringe filter, pore size: 0.2 &#181;m) and place the pipette in the head stage pipette holder. Apply gentle, positive pressure to the pipette through a 5 ml syringe by displacing the plunger about 1 ml before lowering the pipette into the bath solution. 
	Note: This prevents the pipette from becoming blocked.</li>
	<li>Choose &#34;V-clamp&#34; mode by turning the mode knob on the amplifier, then open the &#34;Membrane test&#34; interface in the software. Move the pipette close to the target neuron under microscope inspection. 
	Note: In the intact DRG preparation, a thin layer of satellite glial cells encapsulates each neuron.</li>
	<li>Use positive pressure from the pipette to traverse the satellite glial cell layer until a sudden enlargement of space between the neuron and the surrounding layer of satellite glial cells is observed. Keep moving the pipette towards the neuron until a &#34;dimple&#34; is observed on the neuron. 
	Note: Compared with recordings from disassociated neurons, the positive pressure needs to be slightly larger, which will help penetrate the satellite glial cell layer and increase the chances of successful patching.</li>
	<li>Reduce the positive pressure. Next, achieve a giga ohm seal with gentle suction. 
	Note: With this method, the recording success rate is at least 70%.</li>
	<li>Obtain whole-cell recording configuration as described previously.
	<ol>
		<li>Briefly penetrate the neuron cell membrane via a short but strong suction. Alternatively, use the amplifier&#39;s &#34;zap&#34; function while suction is applied.</li>
		<li>Once a whole-cell mode is established, compensate whole-cell capacitance and series resistance (Rs) by turning the capacitance and resistance compensation knobs on the amplifier. 
		Note: Rs is normally 5-20 M&#937;.</li>
		<li>Abandon the cell if Rs is initially greater than 30 M&#937; or changes by more than 20% during the recording. Also, measure the resting membrane potential; abandon a cell if it is more than -50 mV.</li>
	</ol>
	</li>
	<li>Close the &#34;Membrane test&#34; window. Choose &#34;I-clamp normal&#34; mode by turning the mode knob on the amplifier.</li>
	<li>Click &#34;open protocol&#34; in the software, select and load the protocol for measuring rheobase. Click &#34;record&#34; to start recording. Measure input resistance and rheobase to examine the neuronal excitability by injecting a graded series of depolarizing currents in steps of 100 pA.
	<ol>
		<li>Click &#34;open protocol&#34; in the software, select and load the protocol for measuring rheobase. Click &#34;record&#34; to start recording. Measure input resistance and rheobase to examine the neuronal excitability by injecting a graded series of depolarizing currents in steps of 100 pA.</li>
		<li>Use data acquisition and analysis software (e.g., Clampfit) to analyze the recorded traces according to the manufacturer&#39;s instructions. The software calculates the input resistance (Rin) based on the steady-state I-V relationship during the hyperpolarizing currents delivered. Move the cursor to measure the value for rheobase and membrane threshold. Note: The amplitude of current required to induce the AP is defined as the rheobase, and the lowest voltage for inducing AP is defined as the membrane threshold.</li>
	</ol>
	</li>
	<li>Record the Ligand Induced Currents.
	<ol>
		<li>Fill a pipette with the specific agonists. 
		Note: In the current report, we used 100 &#181;M glutamate and 100 &#181;M AITC to induce the currents mediated by glutamate receptors and TRPA1 receptors respectively.</li>
		<li>Check the pipette to make sure there are no air bubbles inside. Place the pipette in the pipette holder. Connect the pipette holder with a tube connected to a drug dispensing system.</li>
		<li>Use the manipulator to move the pipette within 50 &#181;m of the neuron. Set the drug dispensing system pressure to 1 psi and the duration to 1 sec. Switch the recording mode to voltage clamp, and clamp at -70 mV. Briefly apply the pressure via the drug dispensing system to record a drug-induced current.</li>
	</ol>
	</li>
</ol>
Table 1
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">100 ml of intracellular solution</td>
		</tr>
		<tr>
			<td>Concentration (mM)</td>
			<td>Component</td>
			<td>MW</td>
			<td>g</td>
		</tr>
		<tr>
			<td>130</td>
			<td>KGluconate</td>
			<td>234.24</td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>KCl</td>
			<td>74.55</td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>HEPES</td>
			<td>238.3</td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>EGTA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>MgCl2*6H2O</td>
			<td>203.3</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Notes: Adjust pH to 7.4 with KOH, and osmolarity to 260 - 280 mOsm with sucrose and distilled water. Add 2 mM MgATP, 0.5 mM Na2GTP and filter before immediate use.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass pipette</td>
			<td>Sutter</td>
			<td>BF150-110-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anchor</td>
			<td>Havard apparatus</td>
			<td>64-0250</td>
			<td>stabilize the DRG to avoid drift.</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Molecular devices</td>
			<td>1440D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>FN600</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-manipulator</td>
			<td>Sutter</td>
			<td>MPC200</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen (95% O2, 5% CO2)</td>
			<td>Local Medical Gas supplier</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Gong, K.,et al. <a href="https://app.jove.com/v/54287">Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats.</a> J. Vis. Exp. (2016)
This video demonstrates the examination of sensory neuron excitability in the dorsal root ganglia using the patch clamp technique to measure the rheobase and membrane potential, analyzing the neurons&#39; responses to incremental current injections.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. &#160;
1. Patch Clamp ...

Take immobilized rat dorsal root ganglia, or DRG, in a recording chamber perfused with aCSF.
DRG contains sensory neurons surrounded by satellite glial cells.
Apply positive pressure to a patch pipette filled with intracellular solution and connected to an amplifier, then advance it toward the target neuron.
The positive pressure helps traverse the surrounding satellite glial cell layer and contact the neuron, forming an indentation on its membrane.
Apply negative pressure to form a tight seal with the membrane.
Next, apply negative pressure pulses to establish the whole-cell configuration. Calibrate the amplifier parameters for accurate signal measurements.
To determine the neuron&#39;s excitability,&#160; apply brief incremental currents into the neuron, opening the voltage-gated ion channels and triggering an action potential.
Measure the rheobase, the minimum current needed to trigger an action potential.
Further, apply a ramp current to measure the minimum membrane potential at which the action potential occurs.&#160;

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Whole-Cell Patch Clamp Recordings on Intact Rat Dorsal Root Ganglia

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Whole-Cell Patch Clamp Recordings on Intact Rat Dorsal Root Ganglia