This article details a method for whole-cell patch-clamp recording from rat dorsal root ganglia (DRG) neurons. The procedure includes steps for establishing a tight seal and measuring neuronal excitability.
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's excitability, 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.
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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