Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Podsumowanie

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Streszczenie

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets¹. The dopamine signal is terminated by diffusion^2-3, extracellular enzymes⁴, and membrane transporters⁵. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine⁵. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)⁵. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis⁶. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume⁷. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

Protokół

1. Equipment and Supplies

1. Mount a Faraday cage on top of the anti vibration table (TMI) to decrease the background noise.
2. The simultaneous patch clamp amperometry recording system requires an inverted microscope with excellent DIC optics and a long working distance lens. Connect the microscope lighthouse to a car battery. This DC light source for the system will further decrease the electrical noise.
3. Hydraulic micromanipulators (Siskiyou) further decrease noise. In our configuration, we use a right handed manipulator for the whole cell recording, and the left handed for amperometry.

2. Prepare Electrodes for Recording

1. Pull patch electrodes using quartz pipettes on a P-2000 puller (Sutter). Our pull lasts approximately 5 sec, with two heat cycles. This heat time has resulted in consistent resistance (3-4 MΩ) in our whole cell patch pipettes.
2. Fill the electrode with the pipette solution containing 2 mM dopamine and mount it on the right manipulator. Wrap the container holding the pipette solution containing DA with aluminum foil. Keep on ice. Dopamine is oxidizable. Keeping the solution on ice, protected from light will decrease the oxidation rate of dopamine.
3. Gently remove a ProCFE (Dagan) low noise carbon fiber amperometric electrode from the storage box, fill with mercury, mount onto the amperometric adaptor (as depicted in Figure 1), and then mount on the right maniupulator. Protect the tip of the carbon fiber from damage by holding the far end of the carbon fiber. Inspect the electrode with a lab microscope to ensure the tip is clean and intact.
4. Examine the integrity of the amperometric electrode by putting the electrode in a glass bottom Petri dish containing external solution. Record a baseline current in the absence of dopamine. Add 10 μl of a 1mM DA solution to the dish. A good amperometric electrode records an increase in the oxidative current. Repeat this step at the start and end of each experiment to make certain the amperometric electrode works properly.

3. Prepare the Primary Neuronal Culture of Dopamine Neurons or Cells Engineered to Express Dopamine Transporter in Glass Bottom Petri Dishes

1. Gently wash the cells or dopamine neurons three times with warm external solution.
2. Mount the glass bottom Petri dish onto the microscope stage.

4. Visualize Cell and Perform Experiment

1. Find the correct focal point to clearly visualize the cells. Place positive pressure on the patch electrode. Then, gently bring both electrodes down into the solution, and close to the cell.
2. Position the amperometric electrode next to the cell (on left side), and the patch electrode on the right.
3. Attain a gigaohm seal on the cell with the patch electrode. Rupture the seal with suction to achieve whole cell configuration.
4. Allow 5-8 min for dialysis of internal solution containing dopamine into the cell.
5. Use desired voltage step or ramp protocol. Simultaneously acquire data from both the patch pipette and the amperometric electrode to measure whole cell currents and reverse transport of dopamine through the dopamine transporter while the membrane potential is controlled via the patch pipette.

Wyniki

Combined patch clamp with amperometry can measure voltage-dependent DAT-mediated DA efflux. Figure 2A shows a representative experimental configuration and recording of DAT-mediated DA efflux when the intracellular milieu and the membrane potential are clamped by a whole-cell patch pipette. Using this technique, cells expressing YFP-DAT proteins are voltage-clamped with a whole cell patch pipette while an amperometric electrode is placed onto the plasma membrane (Figure 2A). The whole ce...

Dyskusje

Simultaneous voltage-clamp and amperometry has the following benefits. All cell types are accessible and can be used for recording. The identification of the cells or neuron where the recordings are done is simple and straightforward. In particular, if the cell is fluorescently labeled by adding a fluorescent tag to the protein of interest the experimenter can easily select the target cell or neuron. The experimental configuration allows uniform and controlled delivery of pharmacological agents either via the patch pipet...

Materiały

Name	Company	Catalog Number	Comments
Equipment
Anti-vibration table w/faraday cage	Technical Manufacturing Corporation	63-500 series	we use model 63-543
Inverted microscope Nikon TE-2000	Nikon	discontinued	now Eclipse Ti
Two low noise amplifiers axopatch 200b	Molecular Devices		800-635-5577
1-CV 203 BU headstage	Molecular Devices		800-635-5578
1-HL-U pipette holder	Molecular Devices		800-635-5579
Digidata 1440A A/D converter	Molecular Devices		800-635-5580
Two manipulators Siskyou, left and right handed	Siskiyou	MX6600R MX6600L	877-313-6418
Laser pipette puller	Sutter Instruments	P-2000	888-883-0128
Low noise carbon fiber amperometric electrode	ProCFE		www.dagan.com
Low noise quartz pipette	Sutter Instruments	QF100-70-7.5	888-883-0128
12-volt car battery			widely available
Car battery charger			widely available
Reagent
Sodium chloride (NaCl)	Sigma	S7653
HEPES	Sigma	H3375
Dextrose	Sigma	G7528
Magnesium sulfate (MgSO4)	Sigma	M2643
Potassium phosphate monobasic (KH2PO4)	Sigma	P5655
Potassium chloride (KCl)	Sigma	P9333
Calcium chloride dihydrate (CaCl2∙2H20)	Sigma	223506
Magnesium chloride hexahydrate (MgCl2∙6H20)	Sigma	M2670
EGTA	Sigma	E0396