Sensitive and Selective Measurement of Serotonin in Vivo Using Fast Cyclic Square-Wave Voltammetry

Abstract

Although N-shaped fast scan cyclic voltammetry (N-FSCV) is well-established as an electroanalytical method to measure extracellular serotonin concentrations in vivo, it is in need of improvement in both sensitivity and selectivity. Based on our previous studies using fast cyclic square-wave voltammetry (FCSWV) for in vivo dopamine measurements, we have modified this technique to optimize the detection of serotonin in vivo. A series of large amplitude square-shaped potentials was superimposed onto an N-shaped waveform to provide cycling through multiple redox reactions within the N-shaped waveform to enhance the sensitivity and selectivity to serotonin measurement when combined with a two-dimensional voltammogram. N-Shaped fast cyclic square-wave voltammetry (N-FCSWV) showed significantly higher sensitivity to serotonin compared to conventional N-FSCV. In addition, N-FCSWV showed better performance than conventional N-shaped FSCV in differentiating serotonin from its major interferents, dopamine and 5-hydroxyindoleascetic acid (5-HIAA). It was also confirmed that the large amplitude of the square waveform did not influence local neuronal activity, and it could monitor electrical stimulation evoked phasic release of serotonin in the rat substantia nigra pars reticulata (SNr) before and after systemic injection of escitalopram (ESCIT, 10 mg/kg i.p.), a serotonin selective reuptake inhibitor.

Figure 1.

Schematic N-FCSWV waveform and voltammogram process. (A) N-FCSWV consists of an N-shaped staircase potential ramp modified with square-shaped potential pulses. At each step, two equal height and oppositely directed potential pulses are imposed. (B) Background subtraction processing in N-FCSWV to remove capacitive background current from the whole current response. The whole voltammogram after serotonin injection was subtracted from the voltammogram before serotonin injection. The new background subtracted voltammogram obtained with multiple redox reactions of serotonin. (C) 2D voltammogram reconstruction. The voltammogram from a biphasic square wave at each step of the ramp is stacked vertically at the ramp potential and displayed with a pseudocolor map.

Figure 2.

Serotonin 1 δM response during waveform parameter optimization. (A) Optimizing E_Staircase (0.0133 V − 0.075 V). Voltammograms for E_Staircase = 0.0133 V and E_Staircase = 0.05 V are shown (inset). As E_Staircase increases, serotonin sensitivity increased significantly, except 0.05 and 0.075 V (n = 5 electrodes, one-way ANOVA with Tukey’s multiple comparison test). (B) Optimizing E_sw. Square pulse amplitude. Voltammograms for E_sw = 0.1 and 0.4 V are shown (inset). As E_sw increases, serotonin sensitivity increased significantly, except between 0.3 and 0.4 V (n = 5 electrodes, one-way ANOVA with Tukey’s multiple comparison test). (C) Optimizing initial voltage. Initial voltage is where the waveform starts (−0.2 V − 0.1 V). Voltammograms indicated a deficit pattern (E_Initial = 0.1 V, inset, left) and intact pattern (E_Initial = −0.1 V, inset, right). There were no significant differences of serotonin sensitivity between various E_Initial (n = 5 electrodes, one-way ANOVA with Tukey’s multiple comparison test). (D) Optimizing switching potential (1.0 V − 1.5 V). Switching potential is a peak potential where the forward sweep changes to a backward sweep. As switching potential increased, serotonin sensitivity decreased except between 1.0 and 1.1 V and did not significantly affect serotonin sensitivity (n = 5 electrodes, one-way ANOVA with Tukey’s multiple comparison test).

Figure 3.

Comparison of sensitivity to serotonin using N-FSCV and N-FCSWV. Conventional N-shaped waveform was used for N-FSCV. N-FCSWV showed significantly higher sensitivity compared to conventional N-FSCV (n = 5 electrodes, paired t test, p = 0.0246 (50 nM), p = 0.0104 (100 nM), p = 0.0163 (200 nM), p = 0.0131 (500 nM)).

Figure 4.

Selectivity of N-FCSWV. 2D pseudo color plot (top panel) and voltammogram (bottom panel) for (A) 100 nM serotonin, (B) 1 δM 5HIAA, and (C) 100 nM dopamine.

Figure 5.

Serotonin selectivity curve. Red squares represent serotonin responses to different concentrations. Blue squares represent the addition of 5-HIAA to the serotonin solution with 5-HIAA being 10-fold higher in concentration compared to serotonin (25 nM, 50 nM, 100 nM, 200 nM, 500 nM for serotonin and 250 nM, 500 nM, 1 δM, 2 δM, 5 δM for 5-HIAA), and there were no significant differences among mixtures (n = 4 electrodes, multiple t test).

Figure 6.

Serotonin release in the rat SNr evoked by electrical stimulation of the rat MFB. (A) Representative 2D voltammogram of evoked serotonin release before (left, control) and 30 min after ESCIT administration (right). (B) (left) MFB stimulation-evoked serotonin concentration changes versus time before and 30 min after ESCIT treatment (n = 4 rats, vertical lines of each point represent ± SEM, black bar on time axis indicates electrical stimulation). (right) Serotonin clearance (t_1/2) of control and 30 min after ESCIT administration. There was significant delay to t_1/2 (p = 0.049, paired t test).