Characterization of a Multiple-Scan-Rate Voltammetric Waveform for Real-Time Detection of Met-Enkephalin

Abstract

Opioid peptides are critically involved in a variety of physiological functions necessary for adaptation and survival, and as such, understanding the precise actions of endogenous opioid peptides will aid in identification of potential therapeutic strategies to treat a variety of disorders. However, few analytical tools are currently available that offer both the sensitivity and spatial resolution required to monitor peptidergic concentration fluctuations in situ on a time scale commensurate with that of neuronal communication. Our group has developed a multiple-scan-rate waveform to enable real-time voltammetric detection of tyrosine containing neuropeptides. Herein, we have evaluated the waveform parameters to increase sensitivity to methionine-enkephalin (M-ENK), an endogenous opioid neuropeptide implicated in pain, stress, and reward circuits. M-ENK dynamics were monitored in adrenal gland tissue, as well as in the dorsal striatum of anesthetized and freely behaving animals. The data reveal cofluctuations of catecholamine and M-ENK in both locations and provide measurements of M-ENK dynamics in the brain with subsecond temporal resolution. Importantly, this work also demonstrates how voltammetric waveforms can be customized to enhance detection of specific target analytes, broadly speaking.

Keywords: FSCV; carbon fiber; chromaffin cell; cyclic voltammetry; dopamine; microelectrode; opioid.

Figure 1

Introduction to the MSW. (a) A schematic of the MSW with the parameters of interest labeled. M-ENK, the target analyte, was used for waveform characterization. (b) Representative CVs (top) and mCVs (bottom) for M-ENK and L-ENK (normalized). Current collected during the amperometric period is plotted with respect to time (shaded region). M-ENK and L-ENK differ only at the C-terminus, with either a methionine or a leucine group, respectively. Both pentapeptides share the same first peak, but the presence of the second peak allows M-ENK to be visually distinguished.

Figure 2

Waveform application frequency impacts sensitivity to M-ENK. (a) (top) Accumulation time as the MSW parameter under investigation. (bottom) Representative mCVs for bolus injections of 1 μM M-ENK. (b) Accumulation time (or application frequency) and current (tyrosine peak ∼0.95 V; dotted line) plotted on the abscissa and ordinate, respectively (n = 3 electrodes). Exponential line included as a guide for the eye.

Figure 3

Characterization of the voltammetric signal for M-ENK when varying accumulation potential (a) and scan rate (b). (top) Electrochemical waveforms investigated. The inset is an enlarged view of the region of interest. (middle) Representative mCVs collected in response to 1 μM M-ENK. (bottom) Peak anodic current generated in M-ENK oxidation (∼0.95 V) increased as the accumulation potential decreased and as the scan rate in the second segment of the forward scan increased (n = 5 electrodes per parameter).

Figure 4

Potentials selected at both nodes of the second segment of the forward scan influence the voltammetric response of M-ENK. (top) Schematic of the applied waveforms used to investigate amperometric potentials of +1.2 and +1.3 V (a) and transition potentials of +0.6, +0.65, and +0.7 V (b). (middle) Representative mCVs collected for 1 μM M-ENK. (Bottom) Increasing the amperometric potential from +1.2 to +1.3 V increased the peak signal. Increasing the transition potential significantly decreased signal amplitude. *p < 0.05, **p < 0.01, ***p < 0.001; n = 5 electrodes.

Figure 5

Transition potential that distinguishes the first segment of the forward scan from the second can be tailored to maximize sensitivity or selectivity. Representative mCVs collected with transition potentials of +0.6 V (a) or +0.7 V (b) for detection of 1 μM M-ENK, 500 nM DA, and a mixture of both species containing the same concentrations.

Figure 6

Improved detection of M-ENK with MSW 2.0. (a) A graphic comparison of the two waveforms. (b) Representative mCVs collected for 1 μM M-ENK using MSW 1.0 and MSW 2.0. (c) A direct comparison of calibration plots for M-ENK using these waveforms. ***p < 0.001; n = 5 electrodes.

Figure 7

Simultaneous monitoring of M-ENK and CA dynamics in an adrenal slice preparation with MSW 2.0. (a) Representative data for standards of 750 nM DA and 500 nM M-ENK, and (b) M-ENK and CA released following electrical stimulation (administered at the time indicated by the red dashed line). (top) Color plots of raw voltammetric data. (bottom) mCVs extracted from the color plots at the time indicated by the white dashed line. There is good agreement between the normalized mCVs in the potential range where M-ENK is evident (to the right of the dashed line, 0.7–1.3 V, R² = 0.83).

Figure 8

M-ENK recorded in the dorsal striatum of an anesthetized rat. (top) Representative color plots collected during microinfusion of M-ENK (a), PBS (b), or a cocktail of enkephalinase inhibitors (c). Infusion is marked with the orange bar on the concentration vs time plots (bottom). Inset mCVs were extracted at the time point indicated by the corresponding white dashed lines. Microinfusion of PBS did not result in any observable neurochemical changes, but local infusion of the protease inhibitor cocktail resulted in voltammograms that correlated with those collected after infusion of exogenous M-ENK (R = 0.88).

Figure 9

Neurochemical fluctuations recorded in the dorsomedial striatum of awake, freely behaving rats. Representative color plots are shown, with concentration vs time traces below. Inset mCVs were extracted at the time point indicated by the white dashed lines. (a) Voltammograms that correlate with those collected after infusion of exogenous M-ENK into striatal tissue were evident in the intact striatum (left, R = 0.85), but no CA signal was evident in the 6-OHDA lesioned animal (right). These voltammograms correlate with the M-ENK standard (R = 0.84). (b) A voltammetric signal that correlates with M-ENK fluctuations was recorded in response to the presentation (left) and consumption (middle and right) of unexpected food reward (R = 0.80–0.88).