Optimizing the Temporal Resolution of Fast-Scan Cyclic Voltammetry

Abstract

Electrochemical detection with carbon-fiber microelectrodes has become an established method to monitor directly the release of dopamine from neurons and its uptake by the dopamine transporter. With constant potential amperometry (CPA) the measured current provides a real time view of the rapid concentration changes, but the method lacks chemical identification of the monitored species and markedly increases the difficulty of signal calibration. Monitoring with fast-scan cyclic voltammetry (FSCV) allows species identification and concentration measurements, but often exhibits a delayed response time due to the time-dependent adsorption/desorption of electroactive species at the electrode. We sought to improve the temporal resolution of FSCV to make it more comparable to CPA by increasing the waveform repetition rate from 10 to 60 Hz with uncoated carbon-fiber electrodes. The faster acquisition led to diminished time delays of the recordings that tracked more closely with CPA measurements. The measurements reveal that FSCV at 10 Hz underestimates the normal rate of dopamine uptake by about 18%. However, FSCV collection at 10 Hz and 60 Hz provide identical results when a dopamine transporter (DAT) blocker such as cocaine is bath applied. To verify further the utility of this method, we used transgenic mice that over-express DAT. After accounting for the slight adsorption delay time, FSCV at 60 Hz adequately monitored the increased uptake rate that arose from overexpression of DAT and, again, was similar to CPA results. Furthermore, the utility of collecting data at 60 Hz was verified in an anesthetized rat by using a higher scan rate (2400 V/s) to increase sensitivity and the overall signal.

Figure 1

Dopamine release and uptake responses are dependent on the repetition rate of the cyclic voltammogram waveform. (A,B) Color plots of stimulated dopamine release recorded in the same location in a striatal brain slice with a waveform application frequency of (A) 10 and (B) 60 Hz. Stimulation occurred at t = 0 s. (C) Concentration traces for stimulated dopamine release extracted from the peak oxidation potential for dopamine at 10 and 60 Hz. The dopamine concentration recorded at 10 Hz rises more slowly after stimulation and takes longer to return to baseline. Nonlinear regression accounting for the different adsorption times at the two different repletion rates reveals that the 10 Hz rate yields an underestimate of V_max by about 18% ± 1%. (D,E) Color plots of stimulated dopamine release recorded in the presence of 10 μM cocaine collected at (D) 10 and (E) 60 Hz. (F) Concentration traces for stimulated dopamine release extracted from the peak oxidation potential for dopamine at 10 and 60 Hz in the presence of 10 μM cocaine. Cocaine lowers the uptake rate and renders responses measured with 10 and 60 Hz repetition rates more similar. However, the signal recorded shows a 13% ± 2% greater maximal dopamine concentration at 10 Hz. (G) Increasing concentrations of cocaine (1–20 μM) were used to generate a curve showing the change in K_m caused by cocaine. The K_i of cocaine was calculated from this curve and found to be the same within experimental error regardless of the waveform application frequency.

Figure 2

Addition of ascorbate to striatal brain slices increases amperometric signals. The addition of 500 μM ascorbate to the aCSF perfusing the slice increases the dopamine signal measured by amperometry due to recycling of dopamine-o-quinone caused by the catalytic oxidation of ascorbate by dopamine-o-quinone.

Figure 3

Stimulated dopamine release monitored with fast-scan cyclic voltammetry repeated at 60 Hz and amperometry. Both measurements were in the presence of 500 μM ascorbate. A time delay due to adsorption can be seen in the fast-scan cyclic voltammetric response indicated by a longer time to reach a maximum and a slower decay to baseline. The adsorptive delay with cyclic voltammetry was accounted for using the convolute-and-compare method to obtain rate constants.

Figure 4

Stimulated dopamine release can be monitored in an anesthetized rat at 60 Hz. (A,B) Color plots of stimulated dopamine release collected using 400 V/s as the scan rate collected at (A) 10 and (B) 60 Hz. (C) Concentration versus time traces for the data collected at 10 and 60 Hz from the peak oxidation potential for dopamine using a 400 V/s scan rate. The concentrations calculated at 10 and 60 Hz are not different from each other. (D,E) Color plots of stimulated dopamine release collected using a scan rate of 2400 V/s collected at (A) 10 and (B) 60 Hz. The peak current for dopamine oxidation is about 5.5 times greater than obtained at 400 V/s. (F) Concentration versus time traces for the data collected at 10 and 60 Hz from the peak oxidation potential for dopamine using a 2400 V/s scan rate. Switching from 10 to 60 Hz at this higher scan rate does not dramatically decrease the signal-to-noise ratio as it does at 400 V/s, while maintaining the same calculated peak concentration of dopamine.

Figure 5

Transgenic mice overexpressing DAT have faster striatal dopamine uptake. (A) Representative dopamine release and uptake measured in WT and DAT-Tg mice with amperometry. The dopamine release profile from DAT-Tg animals is smaller in magnitude and has a faster decay than the profile from WT animals. The ratio of V_max values taken from both genotypes gives a DAT-Tg/WT ratio of 1.66 ± 0.36, a significant enhancement (p < 0.01, n = 4 each genotype). (B) Representative release profiles from WT and DAT-Tg animals measured with fast-scan cyclic voltammetry are qualitatively similar to their amperometric counterparts. Deconvolution and simplex modeling (solid lines) of these traces gave V_max values of 5.0 μM/s for the WT animal and 7.2 μM/s for the DAT-Tg animal.