Tonic Serotonin Measurements In Vivo Using N-Shaped Multiple Cyclic Square Wave Voltammetry

Abstract

Here, we present the development of a novel voltammetric technique, N-shaped multiple cyclic square wave voltammetry (N-MCSWV) and its application in vivo. It allows quantitative measurements of tonic extracellular levels of serotonin in vivo with mitigated fouling effects. N-MCSWV enriches the electrochemical information by generating high dimensional voltammograms, which enables high sensitivity and selectivity against 5-hydroindoleacetic acid (5-HIAA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), histamine, ascorbic acid, norepinephrine, adenosine, and pH. Using N-MCSWV, in combination with PEDOT:Nafion-coated carbon fiber microelectrodes, a tonic serotonin concentration of 52 ± 5.8 nM (n = 20 rats, ±SEM) was determined in the substantia nigra pars reticulata of urethane-anesthetized rats. Pharmacological challenges with dopaminergic, noradrenergic, and serotonergic synaptic reuptake inhibitors supported the ability of N-MCSWV to selectively detect tonic serotonin levels in vivo. Overall, N-MCSWV is a novel voltammetric technique for analytical quantification of serotonin. It offers continuous monitoring of changes in tonic serotonin concentrations in the brain to further our understanding of the role of serotonin in normal behaviors and psychiatric disorders.

Figure 1.

N-shaped multiple cyclic square wave voltammetry. (A) Schematic design of the N-shaped cyclic square waveform. (B) Design of N-MCSWV (top) and the oxidation–reduction current responses (bottom). (C) Dynamic background subtraction performed within the series of N-shaped CSWs along with capacitive background current simulation. Representative data showing a voltammogram (top) and its 2D color plot (bottom) for 100 nM serotonin in Tris solution and recorded with N-MCSWV at a carbon fiber microelectrode.

Figure 2.

N-MCSWV parameter optimization. (A) Determining the number of CSWs in the single N-MCSWV waveform (n = 6 electrodes with ±SD, Mann–Whitney test, waveform 6 vs 10: p = 0.0260, 7 vs 10: p = 0.240, 8 vs 10: p = 0.818, 9 vs 10: p > 0.999). Optimization of the (B) E_SW (0.1–0.4 V), (C) E_Staircase (0.0125–0.075 V), and (D) gap (0–4 ms) (n = 6 electrodes each with ±SD, non-parametric ANOVA Kruskal–Wallis test).

Figure 3.

N-MCSWV parameter optimization for fouling mitigation. (A) Optimization switching potential (n = 6 electrodes, ±SD). (B) Comparison of the N-MCSWV waveform switching potentials of 1.0 and 1.3 V for 8 h (n = 6 electrodes). The bold line represents the normalized mean charge over time, and thin lines represent SEM. (C) Serotonin response changes in the 24 h of recording (n = 6 electrodes, ±SEM).

Figure 4.

N-MCSWV measurement of serotonin and its sensitivity. (A) Steady-state currents in response to incremental increases in serotonin (10–200 nM) in vitro. (B) Concentration calibration plot of serotonin concentrations (n = 6 electrodes, ±SD, R² = 0.9750).

Figure 5.

N-MCSWV response of various neurochemicals and its selectivity. (A) Serotonin 100 nM, 5-HIAA 10 μM, histamine 1 μM, norepinephrine 1 μM, dopamine 100 nM, DOPAC 2 μM, ascorbic acid 200 μM, adenosine 1 μM, ΔpH +0.2, and ΔpH −0.2. (B) Voltammogram response of serotonin 100 nM and 5-hydroindoleacetic acid (5-HIAA) 10 μM (right). (C) Oxidation current response changes in a single staircase of the N-MCSWV waveform. (D) Principal component analysis of the serotonin 100 nM and 5-HIAA 10 μM. (E) 5-HIAA interference to the serotonin signal, serotonin 100 nM, serotonin 100 nM + 5-HIAA 1 μM, and serotonin 100 nM + 5-HIAA 10 μM (n = 3 electrodes, non-parametric ANOVA Kruskal–Wallis test, p = 0.2458).

Figure 6.

Pharmacological confirmation of tonic serotonin in vivo. Mean ± SEM tonic serotonin concentrations in response to (A) saline (n = 5), (B) escitalopram (10 mg/kg, n = 5), (C) nomifensine (20 mg/kg, n = 5), and (D) desipramine (20 mg/kg, n = 5).