Biomedical subjects

R M Wightman

Publications and source records attributed to R M Wightman.

At least 91 records · Page 5Linked to original sources

PubMed1994

Heterogeneity of evoked dopamine overflow within the striatal and striatoamygdaloid regions.

The heterogeneity of evoked dopamine overflow in vivo was examined and compared in striatal and striatoamygdaloid regions of the rat. The characteristics of appearance and disappearance rates and the maximum concentration elicited were determined from overflow curves measured by fast-scan cyclic voltammetry. Overall, the characteristics of evoked dopamine overflow were quite variable in the striatum compared to the relative uniformity of overflow in the basolateral amygdaloid nucleus. In addition, there was a significant decrease in the extracellular disappearance rate of evoked dopamine with depth in the striatum. This gradient did not alter with passage from the caudate-putamen to the nucleus accumbens and no change was observed for the appearance rate or maximum concentration. In contrast, differences in evoked dopamine overflow within the striatoamygdaloid region were sharply defined dorsoventrally and appeared to be region-specific. Dopamine terminal fields in the striatum are not clearly demarcated into the caudate-putamen and nucleus accumbens, but may exist as a continuum. The uptake of dopamine appears to be the distinguishing characteristic for the regulation of extracellular dopamine levels in the striatum and the basolateral amygdaloid nucleus.

Amygdala↗

PubMed1994

Extracellular ionic composition alters kinetics of vesicular release of catecholamines and quantal size during exocytosis at adrenal medullary cells.

The temporal resolution of carbon-fiber microelectrodes has been exploited to examine the plasticity of quantal secretory events at individual adrenal medullary cells. The size of individual quantal events, monitored by amperometric oxidation of released catecholamines, was found to be dependent on the extracellular ionic composition, the secretagogue, and the order of depolarization delivery. Release was observed with either exposure to 60 mM K+ in the presence of Ca2+ or exposure to 3 mM Ba2+ in solutions of different pH, with and without external Ca2+. Ba2+ was demonstrated to induce Ca(2+)-independent exocytotic release for an extended period of time (> 4 min) relative to release induced by K+ (approximately 30 s), which is Ca2+ dependent. In all cases, simultaneous changes of intracellular divalent cations, monitored by fura-2 fluorescence, accompanied quantal release and had a similar time course. Exocytosis caused by Ba2+ in Ca(2+)-free medium had a larger mean spike area at pH 8.2 than at pH 7.4. When Ba(2+)-induced spikes measured at pH 7.4 were compared, the spikes in Ca(2+)-free medium were found to be broader and shorter but had the same area. Release induced by K+ after exposure to Ba2+ was comprised of larger quantal events when compared with preceding K+ stimulations. Finally, spikes obtained with Ba2+ exposure at an extracellular pH of 5.5 had a different shape than those obtained in more basic solutions. These changes in spike size and shape are consistent with the interactions between catecholamines and other intravesicular components.

Adrenal Medulla↗

PubMed1994

Efflux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain.

Synaptic release of dopamine in the nucleus accumbens of the intact rat brain elicited by a single electrical impulse applied to ascending dopaminergic fibers results in extracellular concentrations sufficient to bind the known dopamine receptors. The dopamine concentration observed after four rapid, sequential pulses is exactly four times greater and is unaffected by pharmacological antagonism of dopamine uptake and receptor sites at supramaximal concentrations. Thus, dopamine efflux from the synaptic cleft is not restricted by binding to intrasynaptic proteins on the time scale of the measurements (50-100 msec). The extracellular concentration, as a result of a single stimulus pulse, is 0.25 microM and is rapidly removed by extrasynaptic uptake. This maximal, transient concentration of dopamine is 60 times higher than steady-state concentrations reported previously using dialysis techniques, illustrating that dopamine extracellular concentrations are spatially and temporally heterogenous. In contrast to ACh transmission at the neuromuscular junction, the dopamine synapse in the telencephalon is designed for the effective efflux of dopamine from the synaptic cleft to the extrasynaptic compartment during neurotransmission.

Animals↗

PubMed1994

Different kinetics govern dopaminergic transmission in the amygdala, prefrontal cortex, and striatum: an in vivo voltammetric study.

The regulation of extracellular dopamine (DA) concentrations was examined and compared in vivo in four projection fields of mesotelencephalic dopaminergic neurons with fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Transient electrical stimulation of ascending DA fibers in a near physiological range of frequencies (10-20 Hz) elicited similar levels of extracellular DA in the medial prefrontal cortex (MPFC), basal lateral amygdaloid nucleus (BAN), caudate-putamen (CP), and nucleus accumbens (NAc) despite the documented 90-fold disparity in DA tissue levels and terminal density. However, marked differences were observed in the dynamics and overall frequency dependence of the evoked synaptic overflow of DA. These differences are due to the significantly different rates of release and uptake found in each of the four regions. For example, rate constants for the release of the four regions. For example, rate constants for the release and uptake of DA were similar in the MPFC and BAN but approximately 8 and 50 times less, respectively, than that in the CP and NAc. When the parameters were normalized to endogenous DA tissue content, a unique picture emerged: compared to all other regions, relative release was 10-fold greater in the MPFC while relative uptake was at least 10 times less in the BAN. The results further differentiate the functional characteristics of mesotelencephalic dopaminergic systems and demonstrate the regiospecific nature of DA neural transmission in the brain. In addition, the regulation of extracellular DA levels in the MPFC and BAN is suitable for the "long-range" transfer of chemical information in the brain and is consistent with a hypothesis of extrasynaptic neurotransmission.

Amygdala↗

PubMed1993

Temporal characteristics of quantal secretion of catecholamines from adrenal medullary cells.

Exocytotic release of vesicular catecholamine from individual bovine adrenal medullary cells was detected with carbon fiber microelectrodes. Release was elicited from cells permeabilized with 20 microM digitonin in extracellular solutions of pH 5.5, 7.4, or 8.2, and with 100 microM nicotine at pH 7.4. Release detected amperometrically with a 6-microns radius electrode and 1-micron cell-electrode spacing was qualitatively similar for each pH and stimulus. However, amperometric detection with smaller electrodes (radius = 1 micron), cyclic voltammetry, or increased cell-electrode spacing with the larger electrode all resulted in a severe reduction in size and frequency of spikes detected at pH 5.5. Thus, the existence of a steep catecholamine concentration gradient at the cell surface is necessary to cause dissociation of the vesicular matrix at low extracellular pH. At an extracellular pH of 7.4, the distribution of amperometric spike widths measured with a 1-micron cell-electrode spacing was found to be inconsistent with that predicted for diffusional dispersion during transport from the cell surface to the electrode. Both of these results agree with the hypotheses that the chromaffin vesicle matrix normally exists in an aggregated state that can be dissociated by a chemical driving force. Some of the spikes exhibit a pre-spike feature. These were present more often following permeabilization in acidic pH as opposed to more alkaline solutions, and were most prevalent following exposure to nicotine at pH 7.4. The variability in the occurrence of the pre-spike feature suggests it originates from free catecholamine within the vesicle, since the molar fraction bound by the vesicular matrix is regulated by the pH-dependent conformation and Ca(2+)-dependent binding affinity of chromogranin A, a major protein in the vesicle.

Action Potentials↗

PubMed1993

Principles of voltammetry and microelectrode surface states.

In vivo voltammetry is approaching the end of its second decade as a technique to explore extracellular concentrations in the brain. The issues of selectivity and sensitivity, which caused considerable discussion and confusion in the early 1980s, are now resolved. It is clear that in vivo voltammetry and dialysis are complimentary tools to understand neurotransmitter dynamics. The two chief advantages of voltammetry compared to dialysis, improved temporal resolution and reduced tissue damage, make this technique exceptionally well suited for providing information which is complementary to that obtained by single-unit recording and is uniquely capable of providing information on the short-term regulation of extracellular levels of biogenic amines.

Animals↗

PubMed1993

Evoked extracellular dopamine in vivo in the medial prefrontal cortex.

The measurement of evoked extracellular dopamine in the medial prefrontal cortex by using fast-scan cyclic voltammetry with carbon-fiber microelectrodes was established and release characteristics of mesoprefrontal dopamine neurons were examined in vivo in anesthetized rats. Despite the sparse dopaminergic innervation and the presence of more dense noradrenergic and serotonergic innervations overall in the medial prefrontal cortex, the measurement of extracellular dopamine was achieved by selective recording in dopamine-rich terminal fields and selective activation of ascending dopamine neurons. This was confirmed by electrochemical, pharmacological, and anatomical evidence. An increased release capacity for mesoprefrontal dopamine neurons was also demonstrated by the slower decay of the evoked dopamine response after inhibition of catecholamine synthesis and the maintenance of the evoked dopamine response at higher levels in the medial prefrontal cortex compared with the striatum during supraphysiological stimulation.

Animals↗

PubMed1993

Real-time monitoring of electrically stimulated norepinephrine release in rat thalamus: II. Modeling of release and reuptake characteristics of stimulated norepinephrine overflow.

As in the preceding study, electrical stimulation was used to effect release overflow of norepinephrine in the rat thalamus. Using a weak electrochemical pretreatment of a carbon fiber electrode, it was possible to "tune in" the electrochemical response signal for norepinephrine without metabolite interference. This reasonably selective signal was then used to study the degradation of norepinephrine release ability caused by prolonged stimulation. Further, the signals were modeled by the method used successfully for stimulated dopamine overflow, providing hitherto unavailable information on the temporal and spatial characteristics of norepinephrine release overflow. Pertinent comparisons between the release characteristics of the dopamine and norepinephrine systems show that the half-life for norepinephrine in the extracellular fluid space is approximately 1 s in thalamus compared with 33 ms for dopamine in caudate.

Animals↗

PubMed1992

Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis.

Secretion of catecholamines is observed as a series of current spikes when measured at the surface of a bovine adrenal medullary cell in culture with a carbon-fiber microelectrode operated in the amperometric mode. Prior work has shown that these spikes are due to detection of concentrated packets of catecholamines which are released from individual vesicles after their fusion with the cell membrane, a process known as exocytosis. The shape of the individual current spikes, detected with a 5-microns spacing between the hemispherical cell and the electrode, has been compared to the shape generated by a theoretical model. The model consists of an instantaneous point source of material on a surface which subsequently diffuses to a disk that consumes the emitted material. The pertinent diffusion conditions have been evaluated with finite difference and random walk digital simulations. The two methods give identical results when the point source is located on a plane. The more realistic simulation geometry, emission from a hemispherical surface, was evaluated with the random walk method. The simulations allow a set of criteria to be established to evaluate diffusion-controlled broadening of spike shape. The broad range of spike widths observed experimentally and their individual shapes measured with 5-microns cell-electrode spacing are consistent with diffusion from point sources randomly distributed across a hemispherical surface. The data can be described with the diffusion coefficient for catecholamines in free solution. The model enables evaluation of signal-to-noise losses and correction for diffusional losses which are dependent on electrode radius.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Quantal secretion of catecholamines measured from individual bovine adrenal medullary cells permeabilized with digitonin.

Secretion of catecholamines from individual bovine adrenal medullary cells grown in primary culture has been investigated with a carbon-fiber microelectrode placed adjacent to the cells. Oxidation of catecholamines at the electrode surface results in changes in current, which give a real-time measure of catecholamine secretion. Chemical agents are introduced to the individual cells by pressure ejection from micropipettes. When incubated in Ca(2+)-containing buffers, secretion is not observed. However, permeabilization of the cell by exposure to 20 microM digitonin for approximately 15 s results in a Ca(2+)-dependent secretion, and the contents of individual vesicles are detected in the form of sharp spikes. The rate at which spikes occur is a function of the Ca2+ concentration in the external media and reaches a maximum at 19 microM Ca2+. The area of the spikes range from 0.1 to greater than 10 picocoulombs, but the majority are less than 2 picocoulombs, corresponding to less than 6 x 10(6) molecules detected per spike. Histograms of the spike areas are essentially independent of the Ca2+ concentration, indicating that the population of vesicles which undergo exocytosis is the same for all concentrations. Exocytotic secretion can be distinguished from nonexocytotic release by analysis of the shape of the spikes.

Adrenal Medulla↗

PubMed1992

Quantitative determination of catecholamines in individual bovine adrenomedullary cells by reversed-phase microcolumn liquid chromatography with electrochemical detection.

A microcolumn liquid chromatographic method for the determination of norepinephrine and epinephrine in single bovine adrenomedullary cells is described. A single cell is isolated from a culture plate, (3,4-dihydroxybenzyl)amine is added as an internal standard, and the cell is lysed with perchloric acid. After centrifugation, the supernatant is injected directly into a 42 or 50 microns Inner diameter C18 reversed-phase column operated with electrochemical detection. Detection limits for norepinephrine and epinephrine were 46 and 75 amol, respectively. Of the 22 cells examined, 8 contained predominantly norepinephrine, 10 contained predominantly epinephrine, and at least 4 cells contained significant amounts of each. On average, a single cell contained approximately 150 fmol of catecholamine.

Adrenal Medulla↗

PubMed1992

Simultaneous measurement of oxygen and dopamine: coupling of oxygen consumption and neurotransmission.

Fast-scan cyclic voltammetry was used to simultaneously measure increases in dopamine concentration and decreases in O2 concentration evoked by brief electrical stimulation (two pulses at 10 Hz) in slices of rat caudate nucleus. Dopamine concentration began increasing immediately after the first pulse and reached a maximum within 200 ms of stimulation. The O2 concentration began to decrease 300-700 ms after onset of stimulus. Responses for both dopamine and O2 were dependent on external Ca2+ and were Cd2+ and tetrodotoxin sensitive. Only the O2 response was sensitive to CN- (0.15 mM). At short times after exposure to 50 microM ouabain, electrically stimulated dopamine overflow was increased by 150% and electrically stimulated changes in O2 concentration were unaffected. Maximum dopamine concentration was increased 28% by sulpiride (2 microM), 78% by L-DOPA (60 microM), 105% by nomifensine (10 microM) and unaffected by nialamide (10 microM). Maximum decrease in O2 concentration was increased by 25% by sulpiride and unaffected by nialamide, L-DOPA, or nomifensine. The decreases in O2 concentration are indicative of increased O2 consumption and are a measure of oxidative energy production evoked by electrical stimulation. The increase in dopamine is due to the release of dopamine balanced by uptake and serves as an indication of neurotransmitter activity. The results indicate that increases in oxidative energy production following electrical stimulation are dependent on external Ca2+ entry through Cd(2+)-sensitive channels. Possible mechanisms for this coupling are discussed.

Animals↗

PubMed1992

Regulation of transient dopamine concentration gradients in the microenvironment surrounding nerve terminals in the rat striatum.

Synaptic overflow of dopamine in the striatum has been investigated during electrical stimulation of the medial forebrain bundle in anesthetized rats. Dopamine has been detected with Nafion-coated, carbon-fiber electrodes used with fast-scan voltammetry. In accordance with previous results, dopamine synaptic overflow is a function of the stimulation frequency and the anatomical position of the carbon-fiber electrode. In some positions the concentration of dopamine is found to respond instantaneously to the stimulus when the time-delay for diffusion through the Nafion film is accounted for. In these locations the measured rates of change of dopamine are sufficiently rapid such that extracellular diffusion is not apparent. The rate of dopamine overflow can be described by a model in which each stimulus pulse causes instantaneous release, and cellular uptake decreases the concentration between stimulus pulses. Uptake is found to be described by a constant set of Michaelis-Menten kinetics at each location for concentrations of dopamine from 100 nM to 15 microM. The concentration of dopamine released per stimulus pulse is found to be greatest at low frequency (< or = 10 Hz) with stimulus trains, and with single-pulse stimulations in nomifensine-treated animals. The frequency dependence of release is not an effect of dopamine receptor activation; haloperidol (2.5 mg/kg) causes a uniform increase in release at all frequencies. The absence of diffusional effects in the measurement locations means that the constants determined with the electrode are those operant inside intact striatal tissue during stimulated overflow. These values are then extrapolated to the case where a single neuron fires alone. The extrapolation shows that while the transient concentration of dopamine may be high (200 nM) at the interface of the synapse and the extrasynaptic region, it is normally very low (< 6 nM) in the bulk of extracellular fluid.

Animals↗

PubMed1992

Evoked neuronal activity accompanied by transmitter release increases oxygen concentration in rat striatum in vivo but not in vitro.

Dopamine and oxygen (O2) were measured in the caudate nucleus of anesthetized rats and in striatal slices during electrical stimulation. Simultaneous electrochemical detection of dopamine and O2 was accomplished with fast-scan cyclic voltammetry at a Nafion-coated carbon-fiber microelectrode. Stimulation of the medial forebrain bundle resulted in synaptic overflow of dopamine in the caudate nucleus. At the same time, O2 concentration increased in the extracellular fluid with two separate phases. The amplitude of the initial increase directly correlated with the frequency of the stimulus, with the time of maximum concentration reproducible across a range of frequencies. The second increase occurred at later times with a more random amplitude and with a broad, variable shape. Agents which blocked vasodilation affected both phases: atropine attenuated the initial increase, while the second feature was nearly absent after theophylline. Yohimbine and alpha-methyl-p-tyrosine did not affect the O2 responses. Local electrical stimulation of the slice preparation also resulted in dopamine overflow, but a prolonged decrease in O2 concentration accompanied this event. Striatal field stimulation in vivo produced changes in O2 concentration dependent on the relative position of the stimulating and working electrodes, but none of the responses resembled that seen in the caudate slice. Thus, while measurements in brain slices show O2 consumption as a result of stimulated neuronal activity, an apparent elevation of local cerebral blood flow during and after stimulation dominate the in vivo response.

Animals↗

PubMed1992

Dynamic observation of dopamine autoreceptor effects in rat striatal slices.

Fast-scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one-, two-, and 50-pulse, 10-Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 microM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 microM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 microM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 microM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one-pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half-time of approximately 17 s was found. The addition of picrotoxin (100 microM) and glutamate (10 microM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 microM) attenuated overflow for all the trains tested.

Acetylcholine↗

PubMed1992

Effect of chronic haloperidol treatment on stimulated synaptic overflow of dopamine in the rat striatum.

In vivo voltammetry was used to assess the change in stimulated striatal dopamine overflow in response to various treatments with the dopamine receptor antagonist haloperidol. Dopamine overflow was induced with stimulating electrodes implanted in the medial forebrain bundle of anesthetized rats while dopamine concentrations were monitored with Nafion-coated, carbon-fiber microelectrodes implanted in the striatum. An acute challenge of haloperidol (0.5 mg kg-1, i.p.) given to naive animals caused stimulated overflow to increase at all stimulation frequencies (10-60 Hz), with the greatest change, 5-fold, occurring at 30 Hz. These results have been compared to those obtained in a different group of rats given daily injections of haloperidol (0.5 mg kg-1, s.c.) for 30 consecutive days. On the 30th day, dopamine striatal tissue levels and uptake kinetics were not altered by this treatment, but 3,4-dihydroxyphenylacetic acid tissue levels were elevated almost 2-fold. A challenge dose of haloperidol (0.5 mg kg-1, i.p.) administered to the animals treated with chronic haloperidol did not elicit a change in stimulated dopamine overflow. In two other groups, rats were withdrawn from 30-day haloperidol treatment for 3 days or 14 days before experimentation. Stimulated dopamine overflow concentrations in both groups were not significantly different from naive animals. When the withdrawn animals were given a haloperidol challenge (0.5 mg kg-1, i.p.), 15- and 12-fold increases in overflow for 3-day and 14-day withdrawal groups, respectively, were observed at a stimulation frequency of 30 Hz. Thus, chronic treatment with haloperidol induces long-lasting effects on the capacity of dopamine receptors to modulate dopamine release.

Animals↗

PubMed1991

Enzyme-modified organic conducting salt microelectrode.

A miniaturized enzyme-modified electrode has been constructed and evaluated. The tip of a capillary-encased, carbon-fiber electrode is recessed, and tetrathiafulvalene-tetracyanoquinodimethane crystals are electrochemically deposited in the recessed tip. Flavoenzymes are placed in the recess by cross-linking with glutaraldehyde. The specific enzymes used are glucose oxidase to form a microbiosensor for glucose, and a combination of acetylcholine esterase and choline oxidase to form a microbiosensor for acetylcholine. The sensor is operated in an amperometric mode with Eapp = 150 mV versus a sodium saturated calomel electrode, and the response appears to be limited by the kinetics of the enzyme reaction. The effective maximum current density for the glucose electrode is greater than 600 microA/cm2. At low concentrations of glucose, oxygen provides a significant interference by attenuating the signal. The device is simple to prepare and has a rapid response time. Interference from ascorbate has been significantly reduced by the design and by addition of a layer of ascorbate oxidase. Although not yet suitable for use in tissue, the biosensors are suitable for detection in situations where oxygen concentrations do not frequently change.

Acetylcholine↗

PubMed1991

Strategies for low detection limit measurements with cyclic voltammetry.

Cyclic voltammetry of Nafion-coated, carbon-fiber electrodes is used to detect trace concentrations of dopamine, both in a flow injection apparatus and in the brain of an anaesthetized rat. To improve signal-to-noise ratios, the sources of noise during cyclic voltammetry have been determined and strategies have been developed to decrease the noise. With the potentiostat employed, the measured noise is comparable to that expected for Johnson noise from the feedback resistor of the current transducer. Additional noise arises from the waveform generator employed and, in some cases, line noise. Line noise is discriminated against by starting each cyclic voltammogram either in phase or 180 degrees out of phase with the line frequency. When used in vivo, additional noise also arises from the physiological activity of the animal. Detection limits are found to closely correspond to those predicted on the basis of simulation of the voltammetric shape and the measured noise. Detection limits are improved by the use of appropriate analog and digital filtering, ensemble averaging, and appropriate timing of repetitive cyclic voltammograms. The combined use of these techniques enables the in vivo detection of approximately 100 nM of dopamine with a signal-to-noise ratio of 25.

Animals↗