Fast-scan cyclic voltammetry (FSCV) is an electroanalytical technique commonly used for the detection of neurotransmitters, hormones or metabolites in vitro and in vivo. A carbon microelectrode with a diameter in the lower micrometer range functions as the probe and is positioned very close to the site under investigation. A triangular voltage signal is applied at high scan rates of typically about 400 V/s and repeated with a frequency of 4-10 Hz (Fig. 1a).

The resulting FSCVs can be analyzed by detecting the current peak amplitude on top of a large background. A background subtraction is used to better identify the current peaks. This procedure works well due to the good stability of the background current. The stimulus for the release or uptake of the analyte is synchronized with the recording of the FSCV and is often applied by an external stimulus generator.

The advantages of this technique are its high temporal resolution (millisecond range), its high spatial resolution (lower micrometer range), good specificity (due to distinguishable redox potentials) and sensitivity (nanomolar range) for many analytes.

FSCV_Brain
Scheme 1: A carbon microelectrode is positioned on a brain slice to detect neurotransmitters. The applied potential program is illustrated in red and the resulting current response is illustrated in blue. The background of the FSCV is shown as a dotted line.
Fig. 1a: Voltage program for FSCV.
Fig. 1b: FSCV (blue) with dopamine oxidation and reduction peaks (blue boxes) and background FSCV (black)
Fig. 1c: Background-subtracted FSCV.

The potential program of a FSCV experiment for dopamine detection consists of a fast triangular ramp with a scanrate of typically 400 mV/s. Larger scan rates are not beneficial, because the oxidation and reduction peaks are shifted further towards the potential limits and can be distorted. Additionally, the electrode stability decreases when using scan rates as high as 1000 V/s. The ramp is followed by holding the potential at the lower limit -0.4 V. The total length of the sweep is between 100 ms and 250 ms depending on the frequency. The time of the triangular ramp is only around 9 ms.

Typically a frequency of no larger than 10 Hz is applied. The reaction of dopamine at the carbon microelectrode is adsorption-controlled. Larger frequencies result in reduced peak amplitudes, because the time for adsorption of dopamine is too short and no pre-concentration at the microelectrode can occur.

Since the potential is held at the lower potential limit for about 90% of the experiment time, it has to be chosen carefully. A potential of -0.4 V is applied, because it is negative enough to enhance the adsorption of dopamine during the potential ramps, but not too negative to have an influence from the oxygen reduction reaction which accurs around -0.6 V.

The upper potential limit is typically chosen to be 1.3 V. Although this positive potential is not necessary to detect the peak, it has been demonstrated that the carbon microelectrode is “activated” at these more positive potentials and as a result the peak currents are increased significantly. The “activation” of the microelectrode involves the introduction of more edge planes by breaking carbon bonds. These edge planes are much more active for adsorption of dopamine than basal planes. Choosing even higher upper potential limits will cause etching of the carbon microelectrodes and reduces their life spans.

Because the FSCV experiment can yield more than a thousand FSCVs, the results  are commonly plotted in a false color map. The potential is plotted vs. the time and the currents from the background-subtracted FSCV are displayed in color (Fig. 2). The dopamine oxidation peak is shown in red and the reduction peak is shown in blue.

The graph below shows a line profile along the gray line in the map. It was set to the oxidation peak maximum.

Fig. 2: False color map of the FSCV experiments (top) and line profile along the grey line (bottom)

FSCV with PATCHMASTER and POTMASTER

Whether you are using one of our patch clamp amplifiers with PATCHMASTER or one of our potentiostats with POTMASTER, you can perform FSCV experiments with synchronized triggering of external stimuli. Individual potential ramps and stimulus pattern can be set up and customized. Each FSCV is analyzed and the results are displayed in the Analysis Window. The results can be exported to plot false color maps.

FSCV-annotations
Fig. 3: PATCHMASTER software during a FSCV experiment. The Oscilloscope Window shows the current FSCV. The analysis is not shown but can be configured before and after the experiment.
Automatic background-subtraction

Once a stable FSCV background is achieved it can be saved as the background and directly subtracted from the measurements during the acquisition (Fig. 4). It will be marked as Background in the analysis tree and can be reviewed later. Additionally, the background is displayed as pink trace. This way, the user has full control over the experiment.

FSCV_w_Background
Fig. 4a: Raw FSCV data in the oscilloscope window of PATCHMASTER.

The left image shows the raw FSCV data in black and the background FSCV in pink. Due to the high scan rates the background is very large and obscures the small current peaks from dopamine oxidation. The right image shows the background-subtracted peaks.

PATCHMASTER and POTMASTER allow automatic background subtraction.

Dopamin_4_3
Fig. 4b: Background-subtracted FSCVs.
Automatic peak detection

In the online analysis an automatic peak analyisis is performed. The cursor bounds are set before the experiment, but can also be adjusted afterwards (Fig. 5a). The peak current and peak position are displayed in a separate analysis graph (Fig. 5b).

Cursors
Fig. 5 a: Setting of cursors for the peak detection.
Peak-time
Fig. 5b: The peak current is displayed vs. the experiment time.
Synchronized stimuli

PATCHMASTER and POTMASTER can control external stimulus generators or other external devices for stimulating cells. The external device is triggered by the software to stimulate at predefined times during the experiments. The length, number and frequency of the stimuli are defined. This way, the FSCV experiment is synchronized with the stimulation and you know exactly when the stimulus was applied.

Individual design of potential programs

The software allows the creation of individual potential programs. Fig. 6 shows screenshots of the Pulse Generator File in PATCHMASTER and POTMASTER which illustrate examples of possible potential programs for FSCV experiments.

If you come across a novel FSCV potential program for improving your neurotransmitter detection, you can easily create it in PATCHMASTER and POTMASTER.

a)

b)

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Fig. 6: Screenshots from the Pulse Generator File in PATCHMASTER or POTMASTER: a) symmetrical potential ramps; b) asymmetrical potential program; c) complex potential program.