Search PubMed⌕ Search

Biomedical subjects

R M Wightman

Publications and source records attributed to R M Wightman.

At least 109 records · Page 6Linked to original sources
PubMed1991

Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.

Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

Adrenal Medulla↗
PubMed1991

Etched carbon-fiber electrodes as amperometric detectors of catecholamine secretion from isolated biological cells.

Voltammetric electrodes with micrometer dimensions have been fabricated from carbon fibers etched to a conical shape and insulated with poly(oxyphenylene) following literature procedures. The resultant electrode has a tip radius in the micron range. The response of this electrode is compared to a carbon-fiber electrode prepared by sealing a carbon-fiber electrode in a glass pipet (electrode radius greater than 3.5 microns). While the etched electrodes did not follow electrochemical theory as well as the glass-encased electrode, the etched electrode was found to be suitable for the amperometric measurement of the secretion of catecholamines from isolated bovine adrenal cells. Comparable results are only obtained when the two different electrodes are placed 1 micron from the cell surface. When the etched electrode is placed further away, less secretion is observed, because of diffusion and accompanying dilution.

Animals↗
PubMed1991

Simultaneous electrochemical measurements of oxygen and dopamine in vivo.

Fast-scan cyclic voltammetry, a demonstrated analytical method for the in vivo detection of catecholamine neurotransmitters, is extended to the simultaneous determination of molecular oxygen (O2). Cyclic voltammograms were recorded at a scan rate of 400 V/s at carbon-fiber disk electrodes coated with a perfluorinated ion-exchange material. The peak current for O2 occurs near -1.2 V under these conditions. In flow-injection experiments, these electrodes respond to step changes in dopamine and O2 with a half-rise time of less than 200 ms. The voltammetric peak current is independent of flow rate, indicating a diffusion-limited response unaffected by convection. Several compounds present in the in vivo matrix (adenosine, glutathione, and NAD and glutamic, lactic, and uric acids) were tested and shown not to interfere with the voltammetric signal for O2. These electrodes maintain a stable response in vivo for at least 6 h. They have been used to measure transient increases in both dopamine and O2 in the extracellular fluid of the caudate nucleus of an anesthetized rat in response to an electrical stimulus.

Animals↗
PubMed1991

Secretion of catecholamines from individual adrenal medullary chromaffin cells.

Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cation-exchange polymer. Catecholamine secretion is induced by nicotine (10-500 microM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotine-induced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.

Acetylcholine↗
PubMed1990

Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis.

Nicotinic receptor-mediated secretion of catecholamines from individual cultured bovine adrenal medullary chromaffin cells was measured and characterized with a voltametric microelectrode placed adjacent to the cells. Nicotine-induced secretion is associated with a large increase in chemical spikes that is temporally resolved into the apparent secretion of discrete packets of attomole quantities of easily oxidized molecules. These data are consistent with direct chemical measurement of single exocytotic events.

Adrenal Medulla↗
PubMed1990

Interference by DOPAC and ascorbate during attempts to measure drug-induced changes in neostriatal dopamine with Nafion-coated, carbon-fiber electrodes.

The selectivity of Nafion-coated, carbon-fiber electrodes was evaluated for the voltammetric detection of dopamine in the presence of 3,4-dihydroxyphenylacetic acid (DOPAC) and ascorbate, which are interferant anions in the brain. Nafion coating was applied to both polished carbon-disk electrodes and electrochemically modified electrodes. During in vitro testing, polished disk electrodes showed the greatest selectivity for dopamine at the fastest scan rate tested (300 V s-1), but the drift in signal made slow changes in dopamine difficult to determine. Electrochemically modified electrodes already provide an ascorbate wave distinct from that for dopamine, but Nafion coating actually decreased dopamine selectivity with respect to DOPAC. In vivo testing was carried out in the neostriatum of urethane-anesthetized rats in response to drug- or stimulation-induced increases in dopamine transmission. Administration of haloperidol (0.5 mg kg-1) followed by GBR 12909 (20 mg kg-1), which is known to cause a 10-fold increase in extracellular dopamine, failed to produce a selective signal for dopamine when measured voltammetrically. Comparable results were obtained following administration of amphetamine (2.5 mg kg-1), which also increases dopamine overflow. Voltammetric detection of dopamine was possible, however, during electrical stimulation of dopaminergic afferents in the medial forebrain bundle. Thus, voltammetry with Nafion-coated electrodes is best suited to the measurement of transient changes in extracellular dopamine rather than the relatively prolonged changes in dopamine overflow produced by various drugs.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1989

Microcylinder electrodes as sensitive detectors for high-efficiency, high-speed liquid chromatography.

A carbon fiber microcylinder electrode (r = 3.5 microns) is used as a detector for high-efficiency and high-speed liquid chromatography. The microcylinder has a detection volume of a few picoliters, and can be placed directly at the outlet frit of the column. With proper positioning equipment, the electrode can be placed at the region of the outlet frit where the separation efficiency is highest. Such selective sampling results in greatly increased measured efficiency over a conventional electrochemical detector when short (4 cm) columns of conventional diameter (3.2 mm) are used. The microcylinder detector is sensitive and subpicomole detection limits are obtained in less than 30 s for norepinephrine. The need for expensive positioning equipment is eliminated by mounting the electrode into a fitting which can be mated directly with the column.

Chromatography, Liquid↗
PubMed1989

Heterogeneity of stimulated dopamine overflow within rat striatum as observed with in vivo voltammetry.

Overflow of dopamine has been measured in the striatum of anesthetized rats with 60-Hz, 300-microA electrical stimulations of the medial forebrain bundle. Electrodes were placed in the region of the caudate-putamen or the nucleus accumbens. The elliptical electrodes were fabricated from carbon fibers and had a major radius of 35 microns. Overflow was detected with fast-scan voltammetry repeated at 100-ms intervals. In both brain regions the detected substance had the voltammetric properties of dopamine. Measurements were made at different vertical electrode positions, varied by 100-microns intervals. Significant differences in stimulated overflow were observed between each position in both brain regions. The dimensions over which overflow was observed compare to that of previous descriptions of the patch and matrix topology of the striatum. When the electrode was moved in 10-microns intervals the variance was considerably less. The observed rate of overflow during stimulation exactly correlated with the maximal amount of dopamine observed during a stimulation. In addition, the overflow rate and the disappearance rate also correlated. This suggests that in both brain regions uptake and release sites are anatomically close to one another. The major difference between overflow curves measured in the two different regions was the appearance of an apparent mass transfer barrier to the electrode in the caudate-putamen.

Animals↗
PubMed1989

Effects of D-2 antagonists on frequency-dependent stimulated dopamine overflow in nucleus accumbens and caudate-putamen.

Stimulated dopamine overflow has been measured with in vivo voltammetry in the caudate-putamen and nucleus accumbens. Overflow was induced by electrical stimulation of the medial forebrain bundle with 120 1-ms, 300-microA, biphasic pulses at frequencies between 10 and 60 Hz. Overflow was measured with a Nafion-coated, carbon-fiber electrode used with fast-scan voltammetry (300 V s-1). Quantification and identification of dopamine concentrations down to 100 nM in vivo is possible with this technique. The overflow curves were fit to a kinetic model that describes the measured response as a function of uptake (characterized by a Vmax and Km) and release (characterized by the concentration of dopamine released per stimulus pulse). Overflow curves in both regions could be described with similar kinetic parameters except for the Vmax, which in the nucleus accumbens was only 60% of that measured in the caudate-putamen. Uptake inhibition by nomifensine (20 mg kg-1) caused an apparent 15-fold change in the value of Km in the nucleus accumbens, similar to results previously reported in the caudate-putamen. In contrast, metoclopramide (10 mg kg-1) and sulpiride (100 mg kg-1) altered the apparent amount of dopamine released per stimulus pulse without a change in the uptake kinetics.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

The effect of L-dopa on in vivo dopamine release from nigrostriatal bundle neurons.

We have examined the impact of L-dihydroxyphenylalanine (L-DOPA) on the in vivo release of dopamine (DA) in rat striatum using carbon fiber voltammetry. L-DOPA caused a large increase in the DA released by nigrostriatal bundle stimulation. This was reduced by 94% in animals pretreated with intraventricular 6-hydroxydopamine. We conclude that L-DOPA increases depolarization-induced DA release from DA neurons.

Animals↗
PubMed1988

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.

Algorithms↗
PubMed1988

Differentiation of dopamine overflow and uptake processes in the extracellular fluid of the rat caudate nucleus with fast-scan in vivo voltammetry.

Stimulated overflow of dopamine (DA) into the extracellular fluid of the rat caudate nucleus was measured with fast-scan cyclic voltammetry. DA concentrations were sampled in less than 10 ms at 100-ms intervals with a Nafion-coated, carbon-fiber microelectrode. Overflow of DA was induced by electrical stimulation of the medial forebrain bundle with 300-microA pulses of various duration and frequency. Stimulated overflow was measured as a function of stimulus duration before and after administration of benztropine, bupropion, and amphetamine. These results were correlated with simulated curves based on a simple uptake/overflow model. The observed overflow was assumed to be a function of [DA]p, the concentration of DA which overflows per stimulus pulse, and the kinetics of cellular uptake of DA. Correlation of experimental with stimulated results was obtained at the 95% confidence limit for the duration studies; however, it was not possible to distinguish between the effects of pharmacological agents on uptake and overflow. In contrast, modulation of stimulus frequency did permit such distinction. Simulations of an increase in [DA]p fit results following dihydroxyphenylalanine methyl ester at 95% confidence limits, whereas an equivalent change in the apparent Km did not fit. An increase in the apparent value of Km correlated with results obtained at different frequencies following nomifensine and bupropion administration at the 95% confidence limit, whereas an equivalent increase in [DA]p did not fit. The effects of GBR 12909 best correlated with an increase in the DA available for overflow.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1988

Comparison of uptake of dopamine in rat striatal chopped tissue and synaptosomes.

Uptake of dopamine (DA) by chopped tissue prepared from rat corpus striatum has been examined to determine whether one or two kinetically distinct uptake sites exist. Two methods were used: direct measurement of accumulated [3H]DA and determination of the rate of formation of 3,4-dihydroxyphenylacetic acid (DOPAC) after exposure to DA. The rate of formation of DOPAC in the latter experiments is a direct function of the rate of DA uptake. The rates of [3H]DA uptake and DOPAC formation are both linear with time in the presence of 10 microM substrate. Studies of [3H]DA accumulation into chopped tissue reveal two apparent components with Km values of 160 nM and 3.8 microM, whereas similar experiments with striatal homogenate or synaptosomes yield a single uptake component with a Km equivalent to the lower value found in chopped tissue. Evaluation of DA uptake via the rate of DOPAC formation gave a Km value of 2.3 microM. (High substrate values were used, so a lower value for Km is not apparent in the data.) The high Km-value component was absent in animals with a lesioned striatum induced by prior nigral injections of 6-hydroxydopamine. Several pharmacologic agents (benztropine, amfonelic acid, bupropion, nomifensine and ouabain) were evaluated. All reduced the uptake of DA in chopped tissue, but with reduced potencies compared with the effect of these agents in synaptosomes. The high Km activity in chopped tissue, as well as the apparent reduced potency of uptake inhibitor, appears to arise from the diffusional barrier present inside more intact tissue. This barrier is not present in homogenates or synaptosomes, and, thus, a single uptake process is seen.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1987

Detection of dopamine overflow and diffusion with voltammetry in slices of rat brain.

Voltammetric electrodes have been used to monitor extracellular dopamine in rat brain slices. The electrode tips are small enough to be immersed inside the slice. Specificity for dopamine is increased through the use of voltammetry and a cation exchange membrane at the electrode tip. Dopamine overflow is observed in the caudate nucleus following electrical stimulation (60 Hz, 1 s, 3 V) with an adjacent bipolar electrode. The amount of overflow observed is increased when the tissue is perfused with 10 microM cocaine or nomifensine, both recognized inhibitors of dopamine uptake. The ability of dopamine in the perfusion buffer to permeate the slice was monitored with two voltammetric electrodes, one in the cerebral cortex and the other in the caudate nucleus. At a high concentration (100 microM), dopamine rapidly appeared (2.7 +/- 0.4 min) in the interior of the cortex, but dopamine was not observed in the caudate until a significantly later time (8.9 +/- 1.0 min). To examine whether this difference is a reflection of the presence of different uptake systems in the two regions, pressure ejection was employed. In this experiment a double-barrelled pipette was used to eject dopamine or DOPAC at a fixed distance (approximately 70 micron) from the voltammetric electrode. Ejection of small amounts of both substances could be detected in the cortex. When the ejector-detector assembly was moved to the caudate, dopamine could only be observed following pressure ejection after perfusion of the slice with 10 microM nomifensine. Detection of DOPAC was unaffected. All of these experiments indicate that uptake systems in the caudate keep dopamine concentrations very low in the extracellular fluid of the slice.

Animals↗