This article details the electrophysiological characterization of genetically engineered interneurons derived from oligodendrocyte precursor cells. The study demonstrates the successful reprogramming and maturation of these neurons through whole-cell patch-clamp techniques.
Take an immobilized genetically engineered mouse coronal slice in a recording chamber perfused with oxygenated buffer.
The slice contains the striatal region, which has interneurons reprogrammed from oligodendrocyte precursor cells marked by an interneuron marker and fluorescent protein expression.
Assemble a recording pipette consisting of an electrode immersed in an intracellular solution.
Microscopically identify a fluorescent interneuron.
Maintain a positive pressure in the pipette and advance it toward the target interneuron, contacting the membrane.
Apply negative pressure to form a high-resistance seal.
Apply negative pressure pulses to break the membrane, establishing a whole-cell configuration.
Maintain the interneuron's membrane potential at a constant negative value.
Apply brief incremental currents, opening the ion channels and changing the membrane potential, triggering an action potential.
Eventually, the membrane returns to the resting potential.
Observe these changes in membrane potential in response to the current injection, indicating neuronal maturation and successful reprogramming.
After transferring one tissue section to a recording chamber for electrophysiology, mount the glass pipette on the recording electrode, and lower it into the solution. Double-check the resistance of the electrode. Then, slowly approach the reprogrammed cell with the pipette, keeping a slight positive pressure in the electrode to avoid plugging the tip, and check that the cell is GFP-positive before patching.
When the cell is patched, maintain the cell in current clamp from minus 60 to minus 70 millivolts, and inject 500-millisecond currents from minus 20 to plus 90 picoamperes with 10-picoampere increments to induce action potentials. This is indicative of a neuronal maturation and successful reprogramming.
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