This article details a method for recording action currents from dopaminergic neurons using an electrophysiology setup. The procedure involves patch-clamping dendrites of neurons in a brain slice to study ion movements and neuronal activity.
Begin with an electrophysiology setup with an immobilized brain slice submerged in an artificial cerebrospinal fluid or aCSF containing different ions.
The setup includes a patch pipette filled with a potassium-rich solution connected to a recording electrode and an amplifier.
Apply positive air pressure to the pipette to prevent its clogging. Then, position it near a dendrite, the signal-receiving extension of a dopaminergic neuron.
The resulting membrane dimpling confirms proximity to the dendrite.
Release the positive pressure to create suction, forming a tight seal between the pipette and the dendritic membrane.
Record the initial ion movement as an action current at the baseline voltage.
Flow-in aCSF with sodium and calcium channel blockers to block the respective channels, preventing ion movement through them.
Apply a negative voltage to open a voltage-gated cation channel.
This channel allows potassium ions to enter the neuron, increasing the action current and suggesting stimulation of the dendrite in a dopaminergic neuron.
In this procedure, transfer a brain slice to the recording chamber. Anchor the slice at the bottom of the recording chamber with a platinum ring, and ensure that the slice is of good quality with a smooth, even surface. Then, select a neuron with a dendrite extending over a long distance in the same plane, and ensure that the dendrite of interest can be followed to a well-defined soma. Next, fill the patch pipette with electrode solution.
To patch the dendrite in cell-attached mode, identify and focus on a portion of the dendrite. Apply positive pressure to the pipette, and lower it using the micromanipulator. Then, position the pipette close to the membrane, and adjust the pressure in order to create a small dimple. Subsequently, release the pressure on the pipette tip and patch the dendrite while controlling the pipette resistance. Try to obtain a seal resistance larger than 1 giga ohm, at best between 3 and 10 giga ohms for cell-attached recordings.
Afterward, retract the pipette away from the membrane by a couple of micrometers to avoid deformation of the dendrite. The action currents seen here represent the spontaneous action potentials of nigral neurons. To suppress the action currents, apply calcium and sodium channel blockers to ACSF. Then, apply a voltage step of negative 90 millivolts from a holding potential of 0 millivolts to evoke the hyperpolarization-activated cation current.
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