Eric W. Kristensen

Real-time characterization of dopamine overflow and uptake in the rat striatum

The rate of overflow and disappearance of dopamine from the extracellular fluid of the rat striatum has been measured during neuronal stimulation. Overflow of dopamine was induced by electrical stimulation of the medial forebrain bundle with biphasic pulse trains. The instantaneous concentration of dopamine was measured with a Nafion-coated, carbon fiber microelectrode implanted in the brain. The measurement technique, fast-scan cyclic voltammetry, samples the concentration of dopamine in less than 10 ms at 100 ms intervals. Identification of dopamine is made with cyclic voltammetry. Stimulated overflow was measured as a function of electrode position, stimulation duration, stimulation frequency, and after administration of L-DOPA and nomifensine. The observed concentration during a 2-s, 60-Hz stimulation was found to alter with position of the carbon fiber electrode. For stimuli of 3 s or less the amount of overflow was found to be a linear function of stimulus duration at a fixed electrode position. The observed overflow was found to be steady-state at a frequency of 30 Hz, suggesting a balance between uptake and synaptic overflow under these conditions. The experimental data was found to be successfully modelled when the balance of uptake and stimulated overflow was considered. It was assumed that each stimulus pulse releases a constant amount of dopamine (125 nM), and that uptake follows a Michaelis-Menten model for a single uptake site with Km = 200 nM and Vmax = 5 microM/s. The increase in stimulated overflow observed after L-DOPA (250 mg/kg) could be modelled by a 1.6-fold increase in the amount of dopamine release with no alteration of the uptake parameters. The increase in modelled by an increase in Km. In addition, the fit of the modelled data to the experimental data was improved when diffusion from the release and uptake sites was considered.

Effects of restricted diffusion at ultramicroelectrodes in brain tissue: The pool model: theory and experiment for chronoamperometry

Abstract The theory for chronoamperometry has been considered for the case where diffusion involves transport from media with lower diffusion coefficient and reduced volume fractions to a medium of unrestricted diffusion adjacent to the electrode. An analytical expression is developed in spherical coordinates for the special case of slow charge transfer kinetics and conditions where steady state behavior is approached, that is, for the experimental case of interest here. It is shown that the form of the resulting expression is similar to that obtained for slow charge transfer in homogeneous media. However, the expression obtained in restricted media is dependent on the radius of the media adjacent to the electrode and the volume fraction of the outer media. The chronoamperometric response of disk-shaped voltammetric electrodes in rat brain tissue is found to agree with the model predicted by these expressions. Evaluation of the data with the model enables an estimate of the size of the region of free diffusion next to the electrode to be made.

Sinusoidal voltammetry: a frequency based electrochemical detection technique

Abstract Sinusoidal voltammetry is an electrochemical technique, which uses a large amplitude sinusoid as the potential waveform and performs data analysis in the frequency domain. When the amplitude of the applied potential waveform is large (i.e. >50 mV) the current–potential behavior of the electrochemical interface is extremely non-linear. As a result the faradaic response exhibits signal intensity at higher order harmonics of the fundamental excitation frequency. In contrast, the major source of noise, due to the capacitive charging current, is primarily linear and the vast majority of its intensity remains at the fundamental frequency. The dramatic difference in the frequency response between these signals can be exploited in many ways to enhance both the signal-to-noise ratio and selectivity of an electrochemical measurement. The extent of faradaic signal distribution to the harmonics of the fundamental excitation frequency is dependant on many standard voltammetric parameters. In addition to enhanced sensitivity at the higher harmonics, redox species with different electrochemical properties (e.g. E °, number of electrons, electron transfer rate constant, etc.) can be detected selectively based on their unique ‘fingerprint’ frequency response. The experimental parameters (e.g. E switch , scan rate, excitation potential window amplitude, etc.) can be optimized, as well as the frequency and phase angle to achieve the best selectivity for the analyte of interest.

Time resolved dopamine overflow from synaptosomes and chopped striatal tissue with rapid superfusion

Overflow of dopamine has been measured with a rapid superfusion apparatus in an attempt to obtain a system in which overflow is a measure of the primary release process. The tissue samples employed, chopped tissue and synaptosomes, were prepared from rat striatum. The superfusion system employed an on-line amperometric detector to provide temporal information. In addition, liquid chromatography with electrochemical detection was used for identification and quantification of dopamine. Dopamine release could be induced from both samples by exposure to K+ in the presence of Ca2+. The presence of pargyline (0.1 mM) did not significantly affect overflow from either sample. In addition, dopamine stores could be replenished in both samples by exposure to 0.5 microM DA, an effect blocked by amphetamine, nomifensine, and amfonelic acid. However, overflow from synaptosomes showed considerably less distortion from interactions of released substances with the tissue than from chopped tissue. The temporal profile of overflow was more rapid and uptake inhibitors did not affect overflow during depolarization. Since overflow from synaptosomes appears to be more closely related to release, the temporal response of this preparation to K+ stimulations was examined in more detail. A linear relation between dopamine overflow and log (K+) was obtained with 3-s exposures to K+. In contrast, a sigmoidal relationship was obtained with 30-s exposures. Thus, the data support the concept that depolarization of nerve terminals by K+ is a biphasic process that can be temporally resolved.

Background-subtraction of fast-scan cyclic staircase voltammetry at protein-modified carbon-fiber electrodes

Background-subtraction techniques were applied to the voltammetry of nicotinamide adenine dinucleotide (NADH) at protein-modified carbon-fiber microelectrodes. The background currents at carbon-fiber electrodes were stable and voltammetric scans immediately before or after the analyte were effectively used for background subtraction. Digital step-potential waveforms were used to excite these carbon-fiber electrodes, where the resulting voltammetric analysis assessed the optimal switching and initial potentials and the electrochemical response time was determined. The initial potential was 0.0 V and the switching potential 1.1 V (versus Ag/AgCl) and the response time was approximately 300 ms. Some sensitivity to NADH was lost and voltammetric prescans were required at protein-modified electrodes to obtain a stable baseline. Current versus time was assessed by the average current of the faradaic region from each voltammogram and by differential current; the average current minus the current from a non-faradaic potential range. Differential current assessments discriminated against artifacts caused by pH (as high as 1.0 pH unit) and ionic strength flux (100 mM). These background-subtraction techniques allowed the faradaic information to be obtained quickly and conveniently while maximizing sensitivity and maintaining selectivity.