The aim of this procedure is to measure the reverse transport of dopamine in a neuron that is voltage clamped using simultaneous patch clamp and pyrometry to measure DAT mediated D release. This is accomplished by first testing the integrity of the pyrometric electrode with dopamine. The second step is to place the pyrometric electrode near the cell and achieve a whole cell patch of the neuron with a patch pipette.
Next, the voltage of the cell is controlled with the patch pipette and the DAT mediated dopamine released by the cell is recorded with an pyrometric electrode simultaneously. The final step is to add the DAT blocker and record the whole cell and pyrometry currents again to allow the current specifically mediated by the dopamine transporter to be subtracted out. The significance of this technique is the ability to measure reverse transport of dopamine via the dopamine transporter while simultaneously controlling the voltage of the dopaminergic neuron.
The simultaneous patch clamp and barometry recording system requires an inverted microscope with excellent DIC optics and a long working distance lens to generate a DC light source for the system. With low electrical noise, connect the microscope lighthouse to a car battery. The hydraulic micro manipulators are used because they do not appreciably contribute to the noise levels in our configuration.
A right-handed manipulator is used for the voltage clamp, whole cell recording and the left-handed manipulator for the pyrometry. To prepare the electrode for wholesale recording, pull a quartz pipette with a P 2000 puller. Typically, we use a pipette with a resistance of three to four mega ohms.
Next, fill the electrode with the pipette solution containing one millimolar dopamine. Then mount it on the pipette holder on the right manipulator. Be sure to wrap the container that holds the dopamine containing pipette solution with aluminum foil and keep it on ice when it is not in use.
As dopamine is oxidizable, keeping the solution on ice and protected from light will decrease the oxidation rate. Thread the silver wire into the electrode holder, then thread the am parametric adapter onto the electrode holder. Next, fill the tric electrode with enough mercury to contact the silver wire.
When the electrode is mounted to the adapter, be sure to inspect the pro CFE low noise carbon fiber and parametric electrode under the microscope to ensure that the tip is clean and intact. Be sure to hold the far end of the carbon fiber in order to protect the tip of the carbon fiber from damage. Then mount the pro CFE Tric electrode.
Subsequently mount the assembled electrode on the left hydraulic manipulator to examine the integrity of the tric electrode. Lower it into a glass bottom Petri dish containing the external solution. Record the baseline current in the absence of dopamine.
Then add 10 microliters of a one millimolar dopamine solution to the dish. A good emper metric electrode should record an increase in the oxidative current. Be sure to check the integrity of the emper metric electrode before and after the experiments.
In this procedure, retrieve the dopamine neurons or cells engineered to express dopamine transporter placed in the glass bottom Petri dish from the incubator. Gently wash the cells or dopamine neurons three times with warm external solution. To visualize the cells and perform the experiment, mount the glass bottom Petri dish onto the microscope stage and find the correct focal point to visualize the cells clearly.
Next, apply positive pressure to the patch electrode. Using a one milliliter syringe, gently bring both electrodes down into the solution and closer the cell with the micro manipulators. Then position the emper metric electrode on the left side of the cell and the patch electrode on the right.
When the patch electrode is touching the cell. Apply light suction by mouth or syringe to form a giga seal on the cell. After that, rupture the seal with suction to achieve whole cell configuration, and allow five to eight minutes for the dialysis of the dopamine containing internal solution into the cell.
Then record DAT mediated currents by stepping the cell membrane voltage between minus 60 and plus 100 millivolts from a holding potential of minus 40 millivolts through the pipette. To determine the specific DAT mediated dopamine flx record the emper metric currents in the absence and presence of a DAT antagonist. The specific DAT mediated tric current is then determined by subtracting the traces recorded in the presence of DAT antagonist from the emper traces recorded without the antagonist.
An upward deflection in the tric currents corresponds to an outward flux of dopamine shown here. A DAT mediated currents recorded by stepping the membrane voltage by 20 millivolts between minus 60 and plus 100 millivolts from a holding potential of minus four C millivolts with the pipette containing two millimolar dopamine in the whole cell patching mode. Here is the Emper metric current acquired simultaneously with the whole cell current.
After watching this video, you should have a good understanding of how to measure reverse transport of dopamine via the dopamine transporter in a voltage clamped neuron. Remember to check the integrity of the parametric electrode before and after experiments, and don't forget to subtract the traces recorded in the presence of that antagonist from the tric traces recorded without the antagonist.
