Amperometry approach curve profiling to understand the regulatory mechanisms governing the concentration of intestinal extracellular serotonin

At a Glance

Metadata	Details
Publication Date	2024-05-07
Journal	Scientific Reports
Authors	Mark S. Yeoman, Sara Fidalgo, Gianluca Marcelli, Bhavik Anil Patel
Institutions	University of Brighton, University of Kent
Citations	2
Analysis	Full AI Review Included

Executive Summary

This study introduces a novel electroanalytical technique, Amperometry Approach Curve Profiling (AACP), utilizing Boron-Doped Diamond (BDD) microelectrodes to decouple the regulatory mechanisms governing extracellular serotonin (5-HT) concentration in intestinal tissue.

Core Value Proposition: AACP provides a simple, single-series measurement method to simultaneously quantify 5-HT release (via the exponential intercept) and 5-HT reuptake (via the exponential slope) from steady-state basal current levels.
Sensor Technology: A 76 µm Boron-Doped Diamond (BDD) microelectrode was used, demonstrating high sensitivity and stability (maintaining ~90% signal integrity over 20 minutes) for selective 5-HT detection at +650 mV vs. Ag|AgCl.
Physiological Decoupling: The technique successfully differentiated regulatory activity between the ileum and colon, confirming that the colon exhibits significantly greater 5-HT reuptake activity than the ileum.
Autoreceptor Identification: AACP confirmed the presence of an inhibitory 5-HT₄ autoreceptor in the colonic Enterochromaffin (EC) cells, which reduces 5-HT release when activated.
Pharmacological Quantification: The method was validated by generating dose-response curves for the SERT inhibitor Fluoxetine, yielding precise half maximal inhibitory concentration (IC₅₀) values for the SERT transporter (0.43 ± 0.08 µM in ileum; 0.32 ± 0.04 µM in colon).
Engineering Significance: This approach limits tissue usage and provides a robust methodology for profiling transmitter regulation, applicable across various pathophysiological conditions.

Technical Specifications

Parameter	Value	Unit	Context
Working Electrode Material	Boron-Doped Diamond (BDD)	N/A	Microelectrode diameter: 76 µm
Electrochemical Method	Constant Potential Amperometry	N/A	Selective detection of 5-HT overflow
Applied Potential	+650	mV vs. Ag	AgCl
E-T Distance Range (Actual)	141 to 707	µm	Range used for approach curve profiling
Manipulator Angle Correction	47	°	Correction applied to micromanipulator readings
Perfusion Flow Rate	4	ml min^-1	Continuous flow of Krebs’ buffer
Operating Temperature	37	°C	Warm, oxygenated Krebs’ buffer solution
BDD Signal Stability	~90	%	Signal integrity retained after 20 min recording
SERT IC₅₀ (Fluoxetine, Ileum)	0.43 ± 0.08	µM	Half maximal inhibitory concentration
SERT IC₅₀ (Fluoxetine, Colon)	0.32 ± 0.04	µM	Half maximal inhibitory concentration
5-HT₄ Autoreceptor Sensitivity	40	%	Increase in extracellular 5-HT required for half maximal inhibition
SERT Protein Band Size	~67	kDa	Detected via Western Blot analysis

Key Methodologies

The Amperometry Approach Curve Profiling (AACP) technique was conducted using the following protocol:

Tissue Preparation: Murine ileum and colon segments were excised, bisected along the mesenteric border, and pinned mucosal layer up in a flow bath continuously perfused with warm (37 °C) oxygenated Krebs’ buffer (4 ml min^-1).
Electrode Setup: A three-electrode system was used, featuring a 76 µm BDD microelectrode (working), a “no leak” Ag/AgCl electrode (reference), and a platinum wire (auxiliary).
Electrode Conditioning: BDD electrodes were pre-fouled in 10 µM 5-HT for 5 minutes prior to biological measurements to ensure stable current recordings.
Amperometric Recording: Constant potential amperometry was applied at +650 mV vs. Ag|AgCl to selectively monitor 5-HT overflow.
Approach Curve Profiling: The BDD electrode was sequentially positioned over the tissue surface at five specific, corrected electrode-tissue (E-T) distances (141, 282, 424, 565, and 707 µm). Current was recorded for 40 seconds at each distance.
Pharmacological Intervention: Control measurements were followed by repeating the AACP profile on the same tissue segment after perfusion with various pharmacological agents, including SERT inhibitors (Fluoxetine, Paroxetine, Citalopram) and 5-HT₄ receptor modulators (GR113808, Cisapride).
Data Processing: The natural log of the current difference (Δi, relative to the bulk media current at > 1 mm) was plotted against the E-T distance. A linear regression fit was applied to derive the slope (marker of reuptake) and the intercept (marker of release).
Protein Analysis: Western blot analysis was performed on ileum and colon tissue lysates to quantify SERT protein content (detected at ~67 kDa) for comparison with functional reuptake data.

Commercial Applications

The AACP methodology, coupled with BDD electrochemistry, is highly relevant for industries focused on drug discovery, biosensing, and GI health research:

Drug Discovery and Pharmacology:
- SSRIs and Therapeutics: Rapid, quantitative assessment of the efficacy and potency (IC₅₀ determination) of novel selective serotonin reuptake inhibitors and other drugs targeting neurotransmitter transporters.
- Autoreceptor Modulators: Direct measurement of the sensitivity and regulatory effect of agonists and antagonists on EC cell autoreceptors (e.g., 5-HT₄), crucial for developing GI motility drugs.
Advanced Biosensing and Materials Science:
- BDD Sensor Integration: Utilizing the robust, fouling-resistant properties of BDD microelectrodes for long-term, stable monitoring in complex biological environments, extending beyond ex vivo models.
- Microfluidic/Organ-on-a-Chip Systems: Integrating AACP into microfluidic platforms to study localized neurotransmitter dynamics and gradients under controlled flow conditions.
Gastroenterology and Disease Modeling:
- Functional GI Disorders (FGIDs): Identifying specific alterations in 5-HT release versus reuptake kinetics in tissue models of diseases like Irritable Bowel Syndrome (IBS) or inflammatory conditions, guiding personalized treatment strategies.
- Aging and Disease Profiling: Monitoring how regulatory mechanisms change due to aging, diet, or disease states, providing a mechanistic understanding of GI pathophysiology.

View Original Abstract

Abstract Enterochromaffin (EC) cells located within the intestinal mucosal epithelium release serotonin (5-HT) to regulate motility tones, barrier function and the immune system. Electroanalytical methodologies have been able to monitor steady state basal extracellular 5-HT levels but are unable to provide insight into how these levels are influenced by key regulatory processes such as release and uptake. We established a new measurement approach, amperometry approach curve profiling, which monitors the extracellular 5-HT level at different electrode-tissue (E-T) distances. Analysis of the current profile can provide information on contributions of regulatory components on the observed extracellular 5-HT level. Measurements were conducted from ex vivo murine ileum and colon using a boron-doped diamond (BDD) microelectrode. Amperometry approach curve profiling coupled with classical pharmacology demonstrated that extracellular 5-HT levels were significantly lower in the colon when compared to the ileum. This difference was due to a greater degree of activity of the 5-HT transporter (SERT) and a reduced amount of 5-HT released from colonic EC cells. The presence of an inhibitory 5-HT 4 autoreceptor was observed in the colon, where a 40% increase in extracellular 5-HT was the half maximal inhibitory concentration for activation of the autoreceptor. This novel electroanalytical approach allows estimates of release and re-uptake and their contribution to 5-HT extracellular concentration from intestinal tissue be obtained from a single series of measurements.

Tech Support

Original Source

DOI: https://doi.org/10.1038/s41598-024-61296-9