In vitro electrochemical measurement of serotonin release in the human jejunum mucosa using a diamond microelectrode

Abstract

We report herein on the use of a boron-doped diamond microelectrode (DME) to record oxidation currents in vitro associated with the release of serotonin from enterochromaffin cells in the epithelium of the human intestinal mucosa. Continuous amperometric measurements were made as a function of distance (ln current vs. distance) from the tissue surface in human jejunum specimens. The results demonstrate the capabilities of the DME for the stable and reproducible detection of serotonin in the complex environment of the human tissue. Serotonin release was evoked by the shearing force of a continuously flowing Krebs buffer solution at 36 °C with the tissue pinned down in a flow bath. Reproducible currents with distance were recorded for serotonin oxidation. Increased oxidation currents were observed in the presence of the selective serotonin reuptake inhibitor, fluoxetine, indicating that a significant fraction of the amperometric current recorded is attributable to serotonin oxidation. The nominal reciprocal slope, |slope^-1|, of the ln current vs. distance plots increased from 270 ± 25 μm^-1 in Krebs buffer (N = 3) to 471 ± 65 μm^-1 during fluoxetine addition (N = 3), reflective of a reduced rate of reuptake in the presence of the SERT antagonist. The paper reports on the characterization of the diamond microelectrodes and the in vitro electrochemical measurement data.

Fig. 1

Illustration of the insulated diamond microelectrode design with a conically shaped electrode tip exposed outside of the polypropylene insulation.

Fig. 2

SEM micrographs of (a) an electrochemically-sharpened 40 μm diam. Pt wire, (b) a polycrystalline boron-doped diamond film coated 40 μm Pt wire, (c) the tip of a 40 μm Pt wire covered with polycrystalline diamond film, (d) the surface of polycrystalline diamond film deposited on the Pt wire, and (e) a damaged boron-doped diamond-coated 40 μm diam. Pt wire.

Fig. 3

Visible Raman spectrum recorded from the surface of a diamond-coated Pt wire microelectrode. Excitation wavelength = 532 nm. Laser power at sample = 10 mW. Integration time per spectrum = 5 s.

Fig. 4

Cyclic voltammetric i–E curves for the background in Krebs buffer (pH 7.2) and 10 μmol L⁻¹ serotonin (5-HT) + Krebs buffer at a typical diamond thin film deposited on a sharpened 76 μm diam. Pt wire. The microelectrode was insulated with polypropylene, as illustrated in Fig. 1. Scan rate = 0.1 V s⁻¹.

Fig. 5

Plan-view optical micrographs displaying the microvilli of the human jejunum at two slightly different magnifications. (A) was collected at a slightly higher magnification than (B). EC cells that release serotonin and enterocytes that express the serotonin uptake transporter (SERT) reside within the epithelial layer along the surface of the microvilli.

Fig. 6

Current versus tissue-electrode distance plots in which the current magnitude for 5-HT oxidation plotted against a specific tissue-electrode distance was calculated from the difference in (A) current magnitude measured after 20–30 s at tissue-electrode distances from 2000–50 μm. (B) Natural log (ln) of the current magnitude in (A) plotted against increasing tissue-electrode distance. Data are mean ± S.E.M. N = 3 tissue specimens. Detection potential = 0.8 V vs. Ag/AgCl. The slope of the curve is −0.0036 μm⁻¹ and the y-axis intercept is 8.63 (R² = 0.9691).

Fig. 7

Continuous recordings of serotonin (5-HT) oxidation currents at varying electrode distances (2000–50 μm) of the DME with respect to the mucosa surface. Measurements were made before (Krebs buffer; pH = 7.4), during application, and after (washout) of the selective serotonin uptake inhibitor (SSRI), fluoxetine (1 μmol L⁻¹; 5 min), to determine if increases in current magnitude at each electrode distance were fluoxetine-dependent. Fluoxetine produced a reversible increase in serotonin oxidation currents. Detection potential = 0.8 V vs. Ag/AgCl.

Fig. 8

(A) Ln current (pA) versus distance plots of the serotonin oxidation current with and without fluoxetine (1 μmol L⁻¹). (B) Bar graphs of the aggregated data of curve slope with and without fluoxetine. Decreased serotonin clearance by SERT as shown by a decrease in the slope of the ln (current) versus tissue-electrode distance plot compared to Krebs buffer alone (control). Data are presented for mean ± S.E.M. for N = 3 tissue specimens. Detection potential = 0.8 V vs. Ag/AgCl.

Fig. 9

(A) Continuous amperometric i–t recording of the stable serotonin oxidation current at a distance of 250 μm over a 5 min period. Detection potential = 0.8 V vs. Ag/AgCl. The shearing force of the buffer flow stimulates mechanosensitive receptors expressed by mucosal EC cells; a process that evokes serotonin release. (B) Cyclic voltammetric i–E curves were made in a 10 μmol L⁻¹ serotonin in Krebs buffer before and after extended tissue exposure (~3 months).