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

Protokół

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.  

1. Patch Clamp Recordings

1. 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 µm) 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.
2. Choose "V-clamp" mode by turning the mode knob on the amplifier, then open the "Membrane test" 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.
3. 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 "dimple" 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.
4. 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%.
5. Obtain whole-cell recording configuration as described previously.
   1. Briefly penetrate the neuron cell membrane via a short but strong suction. Alternatively, use the amplifier's "zap" function while suction is applied.
   2. 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 MΩ.
   3. Abandon the cell if Rs is initially greater than 30 MΩ 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.
6. Close the "Membrane test" window. Choose "I-clamp normal" mode by turning the mode knob on the amplifier.
7. Click "open protocol" in the software, select and load the protocol for measuring rheobase. Click "record" 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.
   1. Click "open protocol" in the software, select and load the protocol for measuring rheobase. Click "record" 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.
   2. Use data acquisition and analysis software (e.g., Clampfit) to analyze the recorded traces according to the manufacturer'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.
8. Record the Ligand Induced Currents.
   1. Fill a pipette with the specific agonists.
      Note: In the current report, we used 100 µM glutamate and 100 µM AITC to induce the currents mediated by glutamate receptors and TRPA1 receptors respectively.
   2. 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.
   3. Use the manipulator to move the pipette within 50 µm 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.

Table 1

100 ml of intracellular solution
Concentration (mM)	Component	MW	g
130	KGluconate	234.24
10	KCl	74.55
10	HEPES	238.3
10	EGTA
2	MgCl2*6H2O	203.3
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.

Materiały

Name	Company	Catalog Number	Comments
Glass pipette	Sutter	BF150-110-7.5
Anchor	Havard apparatus	64-0250	stabilize the DRG to avoid drift.
Peristaltic pump	WPI
Pipette puller	Sutter	P97
Amplifier	Molecular devices	Axopatch 200B
Digitizer	Molecular devices	1440D
Microscope	NIKON	FN600
Micro-manipulator	Sutter	MPC200
Carbogen (95% O2, 5% CO2)	Local Medical Gas supplier