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