Biomedical subjects

R M Wightman

Publications and source records attributed to R M Wightman.

At least 19 recordsLinked to original sources

PubMed2006

Phasic dopamine signaling during behavior, reward, and disease states.

The neurotransmitter dopamine is important in reward processing, however its precise modulatory role is still being investigated. Carbon-fiber microelectrodes can be used to monitor dopamine on a subsecond time scale in the striatum and nucleus accumbens of rats during behavior, and this approach is providing new insights into the mechanisms that control its extracellular concentration as well as the conditions under which it is released. Three main processes govern the amount of dopamine measured extrasynaptically: exocytotic release, neuronal uptake, and diffusion away from the release site. By monitoring local extracellular dopamine concentrations in the striatum following electrical stimulation of dopamine-containing neurons, release, uptake and diffusion can be individually examined and quantified. Dopaminergic neurons have been shown to fire in two firing modes, tonic and bursts at higher frequency. Electrical stimulation can be designed to mimic either mode to examine their effects on dopamine release. Burst firing causes a transient increase in extracellular dopamine while tonic firing causes a new steady-state level. In behaving primates, dopaminergic neurons display short-latency, phasic firing to primary reward and conditioned cues associated with reward. These bursts code differences between actual and predicted rewards. In rats, transient dopamine release in terminal regions that mimics that seen during burst firing has been demonstrated during reward-related cues. Taken together, these studies indicate that phasic dopamine release is a critical mediator of reward-related processes.

Action Potentials↗

PubMed2003

A role for presynaptic mechanisms in the actions of nomifensine and haloperidol.

Psychomotor stimulants and neuroleptics exert multiple effects on dopaminergic signaling and produce the dopamine (DA)-related behaviors of motor activation and catalepsy, respectively. However, a clear relationship between dopaminergic activity and behavior has been very difficult to demonstrate in the awake animal, thus challenging existing notions about the mechanism of these drugs. The present study examined whether the drug-induced behaviors are linked to a presynaptic site of action, the DA transporter (DAT) for psychomotor stimulants and the DA autoreceptor for neuroleptics. Doses of nomifensine (7 mg/kg i.p.), a DA uptake inhibitor, and haloperidol (0.5 mg/kg i.p.), a dopaminergic antagonist, were selected to examine characteristic behavioral patterns for each drug: stimulant-induced motor activation in the case of nomifensine and neuroleptic-induced catalepsy in the case of haloperidol. Presynaptic mechanisms were quantified in situ from extracellular DA dynamics evoked by electrical stimulation and recorded by voltammetry in the freely moving animal. In the first experiment, the maximal concentration of electrically evoked DA ([DA](max)) measured in the caudate-putamen was found to reflect the local, instantaneous change in presynaptic DAT or DA autoreceptor activity according to the ascribed action of the drug injected. A positive temporal association was found between [DA](max) and motor activation following nomifensine (r=0.99) and a negative correlation was found between [DA](max) and catalepsy following haloperidol (r=-0.96) in the second experiment. Taken together, the results suggest that a dopaminergic presynaptic site is a target of systemically applied psychomotor stimulants and regulates the postsynaptic action of neuroleptics during behavior. This finding was made possible by a voltammetric microprobe with millisecond temporal resolution and its use in the awake animal to assess release and uptake, two key mechanisms of dopaminergic neurotransmission. Moreover, the results indicate that presynaptic mechanisms may play a more important role in DA-behavior relationships than is currently thought.

Animals↗

PubMed2002

Dopamine autoreceptor regulation of release and uptake in mouse brain slices in the absence of D(3) receptors.

The effects of the dopamine D(3) receptor, a putative autoreceptor, have been investigated by comparing behavioral and neurochemical properties of wild-type mice and mice with a genetic deletion of the D(3) receptor. The D(3) knock-out mice were modestly hyper-responsive to a novel environment relative to wild-type mice, and, consistent with this, quantitative in vivo microdialysis revealed elevated striatal dopamine extracellular levels. The dynamic actions of autoreceptors on electrically evoked dopamine release were examined in striatal brain slices from these animals and monitored with fast scan cyclic voltammetry at carbon-fiber microelectrodes. Quinpirole, a dopamine receptor agonist with potency at both D(2) and D(3) receptors, inhibited evoked dopamine in a dose-dependent manner with a slightly higher dose required in the knock-out animals (EC(50) of 60+/-10 nM in wild-type animals and 130+/-40 in D(3) knock-out animals; both curves had a Hill slope near 2). Dopamine synthesis inhibition with alpha-methyl-p-tyrosine caused released dopamine levels to decrease in each genotype. However, regulation of secretion by autoreceptors was still operant. Dose-response curves to quinpirole were unchanged in D(3) knock-out tissue, but secretion-regulated release exhibited a Hill slope decreased to 1 in the wild-type animals. In both genotypes, similar quinpirole-evoked increases in uptake rate were evident following synthesis inhibition. These data are consistent with the D(3) receptor having a small but significant role as a dopamine autoreceptor that partially regulates secretion, but not synthesis, in the caudate-putamen.

Animals↗

PubMed2001

Determination of release and uptake parameters from electrically evoked dopamine dynamics measured by real-time voltammetry.

Quantifying mechanisms underlying extracellular signaling by the neurotransmitter dopamine (DA) is a difficult task, particularly in the complex extracellular microenvironment of the intact brain. In this study, two methods for evaluating release and uptake from DA dynamics monitored by real-time voltammetry are described. Both are based on a neurochemical model characterizing electrically evoked levels of DA as a balance between these opposing mechanisms. The theoretical basis of what is called here nonlinear regression and single curve analyses is given. Fitting simulated data tests the reliability of the methods. The two analyses are also compared with an experimental data set describing the effects of pharmacologically inhibiting the DA transporter in the caudate-putamen (CP) and nucleus accumbens (NAc). The results indicate that nonlinear regression and single curve analyses are suitable for quantifying release and uptake mechanisms underlying DA neurotransmission. Additionally, the most important experimental finding of this technical study was the independent confirmation of high affinity (approximately 0.2 microM) DA uptake in the intact striatum.

Action Potentials↗

PubMed2001

Microscopic imaging with electrogenerated chemiluminescence.

The use of electrogenerated chemiluminescence (ECL) at microelectrodes as a light source for scanning optical microscopy is demonstrated. Cone-shaped microelectrodes were constructed by flame etching carbon fibers to a fine point. ECL generated in solution at such electrodes was forced to the apex of the conducting surface by using high-frequency (20-kHz) potential pulses and high concentrations of ECL reagents in the solution. ECL arose from the reaction of 9,10-diphenylanthracene radical cation with the radical anion of benzonitrile, the solvent. The electrode/light source was raster-scanned a finite distance above the sample surface, and images were generated with standard scanning probe software by collecting the transmitted light with a microscope objective. These images compared favorably to optical images of the same sample. A resolution of approximately 600 nm was achieved with this arrangement even though a feedback loop was not employed to control the tip distance from the sample. The source was sufficiently bright (1.82 pW) that well-defined transmittance spectra could be obtained at individual locations on the sample.

Journal Article↗

PubMed2001

Sub-second changes in accumbal dopamine during sexual behavior in male rats.

Transient (200--900 ms), high concentrations (200--500 nM) of dopamine, measured using fast-scan cyclic voltammetry, occurred in the nucleus accumbens core of male rats at the presentation of a receptive female. Additional dopamine signals were observed during subsequent approach behavior. Background-subtracted cyclic voltammograms of the naturally-evoked signals matched those of electrically-evoked dopamine measured at the same recording sites. Administration of nomifensine amplified natural and evoked dopamine release, and increased the frequency of detectable signals. While gradual changes in dopamine concentration during sexual behavior have been well established, these findings dramatically improve the time resolution. The observed dopamine transients, probably resulting from neuronal burst firing, represent the first direct correlation of dopamine with sexual behavior on a sub-second time scale.

Animals↗

PubMed2001

Terminal effects of ethanol on dopamine dynamics in rat nucleus accumbens: an in vitro voltammetric study.

To assess the direct effects of acute ethanol on dopamine (DA) terminals, evoked DA release and uptake were measured in rat nucleus accumbens slices using fast-scan cyclic voltammetry. Low and moderate concentrations of ethanol (20, 45 and 100 mM) did not alter evoked DA release, while high concentrations (150 and 200 mM) significantly decreased DA release (18 and 36%, respectively) in a calcium-dependent manner. No significant difference was found between the rate of DA disappearance measured before and after the drug. These data indicate that uptake of DA through the dopamine transporter is unaffected by ethanol, even at high concentrations. Therefore, low to moderate concentrations of ethanol have no effect on DA dynamics at the level of the nerve terminal in the nucleus accumbens. This is consistent with the hypothesis that cell body regions of DA neurons are the primary target for the stimulating and reinforcing effects of ethanol. High concentrations of ethanol can locally depress DA release, and this may correlate with the sedative actions of the drug.

Alcohol-Induced Disorders, Nervous System↗

PubMed2001

Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation.

Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.

Action Potentials↗

PubMed2001

Catecholamine release and uptake in the mouse prefrontal cortex.

Monitoring the release and uptake of catecholamines from terminals in weakly innervated brain regions is an important step in understanding their importance in normal brain function. To that end, we have labeled brain slices from transgenic mice that synthesize placental alkaline phosphatase (PLAP) on neurons containing tyrosine hydroxylase with antibody-fluorochrome conjugate, PLAP-Cy5. Excitation of the fluorochrome enables catecholamine neurons to be visualized in living tissue. Immunohistochemical fluorescence with antibodies to tyrosine hydroxylase and dopamine beta-hydroxylase revealed that the PLAP labeling was specific to catecholamine neurons. In the prefrontal cortex (PFC), immunohistochemical fluorescence of the PLAP along with staining for dopamine transporter (DAT) and norepinephrine transporter (NET) revealed that all three exhibit remarkable spatial overlap. Fluorescence from the PLAP antibody was used to position carbon-fiber microelectrodes adjacent to catecholamine neurons in the PFC. Following incubation with L-DOPA, catecholamine release and subsequent uptake was measured and the effect of uptake inhibitors examined. Release and uptake in NET and DAT knockout mice were also monitored. Uptake rates in the cingulate and prelimbic cortex are so slow that catecholamines can exist in the extracellular fluid for sufficient time to travel approximately 100 microm. The results support heterologous uptake of catecholamines and volume transmission in the PFC of mice.

Alkaline Phosphatase↗

PubMed2001

Light-emitting electrochemical processes.

Electrochemical processes leading to light emission are reviewed, with emphasis on aspects of this subject relevant to the understanding and optimization of electrogenerated luminescence (EL) in organic thin-film materials. The basic energetic requirements of light emission from electrochemically initiated solution redox reactions [electrogenerated chemiluminescence (ECL)] are reviewed first. This review is followed by a discussion of light-emitting electrochemical processes that have been observed in hybrids of ionically conducting polymers and electronically conducting polymers. Finally, the features of EL in insulating polymers and molecular thin films are reviewed, along with recent electrochemical and ECL studies of the small-molecule components of certain organic light-emitting diodes. These studies provide a conceptual framework for understanding and optimizing these materials and the EL process.

Journal Article↗

PubMed2001

Effect of acute ethanol on striatal dopamine neurotransmission in ambulatory rats.

The effect of ethanol on evoked dopamine release in the caudate putamen has been measured in behaving animals with in vivo electrochemistry. Dopamine was measured with fast-scan cyclic voltammetry in adult male rats to resolve the competing processes of dopamine uptake and release. Ethanol dose dependently decreased dopamine efflux compared with saline-treated animals: to 89% of controls with 0.5 g/kg, 70% with 1 g/kg, 34% with 2.5 g/kg, and 18% with 5 g/kg. This decrease was not due to a change in uptake, as measured by the rate of dopamine disappearance after stimulation, and therefore can be attributed to decreased dopamine release. Additionally, it was not mediated by a decrease in biosynthesis, as measured by L-DOPA accumulation after NSD 1015 administration. The selective dopamine uptake inhibitor GBR 12909 compensated for the effects of high doses of ethanol on dopamine release. Moreover, GBR 12909 induced faster restoration of the righting reflex in rats sedated with 2.5 g/kg, but not 5 g/kg, ethanol. In brain slices containing the caudate putamen, ethanol suppressed dopamine release only at the highest dose tested (200 mM). The difference in responses between the slice and the intact animal indicates that ethanol exerts its effects in the cell body regions of dopamine neurons as well as in terminals. These neurochemical results, combined with published accounts of microdialysis measures of extracellular dopamine and electrophysiological recordings of dopamine neurons, demonstrate that ethanol has a profound effect on dopamine neurons whose net result is a suppression of dopamine neurotransmission at high doses.

Animals↗

PubMed2000

Subsecond adsorption and desorption of dopamine at carbon-fiber microelectrodes.

High-repetition fast-scan cyclic voltammetry and chronoamperometry were used to quantify and characterize the kinetics of dopamine and dopamine-o-quinone adsorption and desorption at carbon-fiber microelectrodes. A flow injection analysis system was used for the precise introduction and removal of a bolus of electroactive substance on a sub-second time scale to the disk-shaped surface of a microelectrode that was fabricated from a single carbon fiber (Thornel type T650 or P55). Pretreatment of the electrode surfaces consisted of soaking them in purified isopropyl alcohol for a minimum of 10 min, which resulted in S/N increasing by 200-400% for dopamine above that for those that were soaked in reagent grade solvent. Because of adsorption, high scan rates (2,000 V/s) are shown to exhibit equivalent S/N ratios as compared to slower, more traditional scan rates. In addition, the steady-state response to a concentration bolus is shown to occur more rapidly when cyclic voltammetric scans are repeated at short intervals (4 ms). The new methodologies allow for more accurate determinations of the kinetics of neurotransmitter release events (10-500 ms) in biological systems. Brain slice and in vivo experiments using T650 cylinder microelectrodes show that voltammetrically measured uptake kinetics in the caudate are faster using 2,000 V/s and 240 Hz measurements, as compared to 300 V/s and 10 Hz.

Adsorption↗

PubMed2000

Vesicular Ca(2+) participates in the catalysis of exocytosis.

Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracellular Ca(2+) evoked by depolarizing agents. Subcellular studies revealed that reserpine and tetrabenazine at concentrations near their K(i) values not only could increase cytoplasmic catecholamines but also could displace Ca(2+) from vesicles. Furthermore, transient exposure to tetrabenazine and reserpine, but not methyl reserpate and reserpic acid, induced exocytotic release of catecholamines. Reserpine induced a rise in intracellular Ca(2+), as detected by whole-cell measurements with Fura-2. It could induce exocytosis, albeit at a lower frequency, in Ca(2+)-free solutions, supporting an internal Ca(2+) source. Depletion of endoplasmic reticulum and mitochondrial Ca(2+) pools did not eliminate the reserpine-activated release. These results indicate that vesicular Ca(2+) can play an important role in exocytosis and under some conditions may be involved in initiating this process.

Animals↗

PubMed2000

Correlation between behavior and extracellular dopamine levels in rat striatum: comparison of microdialysis and fast-scan cyclic voltammetry.

Recently, fast-scan cyclic voltammetry (FSCV) has been adapted for real-time measurements of evoked dopamine (DA) release and uptake in freely moving rats. Using the advantages of this experimental design in combination with behavioral measures, we examined the effect of GBR 12909 (20 mg/kg, i.p.), a selective DA uptake inhibitor, on striatal extracellular DA dynamics and compared these data to that obtained by microdialysis. These studies established that both techniques report changes in DA that correlate with the kinetics of GBR 12909-induced behavioral effects. However, the time course of changes in evoked DA levels detected by FSCV was more closely linked with the changes in stereotypy than microdialysis measurements.

Animals↗

PubMed2000

Quantitation of in vivo measurements with carbon fiber microelectrodes.

Fast-scan cyclic voltammetry (FSCV) at carbon fiber disk microelectrodes and quantitative microdialysis were used to measure striatal concentration changes of N-acetyl-p-aminophenol (APAP, acetaminophen) following an intraperitoneal injection of 75 mg/kg APAP in rats. The goal of this work was to determine which in vitro calibration procedure, precalibration or postcalibration, gave the most accurate results when using carbon fiber microelectrodes in vivo. Voltammetric detection of APAP in vivo was complicated with normal electrodes by interference from pH changes. An electrode treatment was used to minimize electrode sensitivity to pH and this allowed successful APAP detection. In vitro calibrations of the treated carbon fiber disk microelectrodes before and after the in vivo experiment were used to calculate APAP concentration changes measured in vivo and compared to microdialysis results. The maximal striatal APAP concentration determined by microdialysis, adjusted for in vitro recovery, was 23.1 microM. The electrochemical results were approximately two times greater (postcalibration) or smaller (precalibration) than the microdialysis result.

Acetaminophen↗

PubMed2000

Real-time amperometric measurements of zeptomole quantities of dopamine released from neurons.

Amperometry with carbon-fiber microelectrodes provides a unique way to measure very small chemical concentration changes at the surface of biological cells. In this work, an investigation of dopamine release from individual neurons isolated from the mouse retina is described. The mice were genetically modified so that, in cells that expressed the protein responsible for catecholamine synthesis, tyrosine hydroxylase, the marker protein, placental alkaline phosphatase, was also expressed. This modification allowed for identification of the dopamine-containing cells among the many present in the freshly dissociated retina. Release of dopamine was evoked by chemical secretagogues delivered from micropipets that were calibrated with respect to response time and concentration delivered. Amperometric measurements were recorded with low-noise patch clamp amplifiers, and the primary noise source was found to be the electrode capacitance. Dopamine release occurred in the form of transient concentration spikes, consistent with release from small intracellular vesicles. With optimized filtering of the data, the quantity secreted during each release event could be determined. The average quantity determined at one cell was 52 zmol. However, the spikes were quite variable in size and the amount released per event ranged from 8 to 170 zmol. These measurements allow an estimation of the concentration of released transmitter in a synapse.

Animals↗

PubMed2000

Differential quantal release of histamine and 5-hydroxytryptamine from mast cells of vesicular monoamine transporter 2 knockout mice.

The recent availability of mice lacking the neuronal form of the vesicular monoamine transporter 2 (VMAT2) affords the opportunity to study its roles in storage and release. Carbon fiber microelectrodes were used to measure individual secretory events of histamine and 5-hydroxytryptamine (5-HT) from VMAT2-expressing mast cells as a model system for quantal release. VMAT2 is indispensable for monoamine storage because mast cells from homozygous (VMAT2(-/-)) mice, while undergoing granule-cell fusion, do not release monoamines. Cells from heterozygous animals (VMAT2(+/-)) secrete lower amounts of monoamine per granule than cells from wild-type controls. Investigation of corelease of histamine and 5-HT from granules in VMAT2(+/-) cells revealed 5-HT quantal size was reduced more than that of histamine. Thus, although vesicular transport is the limiting factor determining quantal size of 5-HT and histamine release, intragranular association with the heparin matrix also plays a significant role.

Animals↗

PubMed2000

Interplay between membrane dynamics, diffusion and swelling pressure governs individual vesicular exocytotic events during release of adrenaline by chromaffin cells.

Release of adrenaline by chromaffin cells occurs through a process involving docking and then fusion of a secretory vesicle to the cytoplasmic membrane of the cell. Fusion proceeds in two main stages. The first one leads to the creation of a stable fusion pore passing through the two membranes and which gives a constant release flux of neurotransmitter (pore-release stage). After a few milliseconds, this initial stage which is not investigated here proceeds through a sudden enlargement of the initial pore (full-fusion stage) up to the complete incorporation of the vesicle membrane into that of the cell and total exposure of the initial matrix vesicle core to the extracellular fluid. The precise time-resolved dynamics of the release and of the vesicle membrane during the full-fusion phase can be extracted with a precision never achieved so far by de-convolution of experimental chronoamperometric currents monitored during individual exocytotic secretion events. The peculiar dynamics of the vesicle membrane proves that exocytotic events are powered by the swelling of the matrix polyelectrolyte core of the vesicle, although they are kinetically regulated by diffusion in the matrix and by the dynamics of the vesicle and cell membranes. Two simple theoretical models based on the dynamics of pores are developed to account for these dynamics and are shown to predict behaviors which are essentially identical to the experimental ones. This offers a new view of the kinetic grounds which control the full-fusion stage, and therefore provides a new interpretation of the sudden transition between the pore-release and the full-fusion stages. This transition occurs when the increasing membrane surface tension energy due to the refrained internal swelling pressure overcomes the edge energy of the pore, so that the initial fusion pore becomes unstable and is disrupted. This new view predicts that secretory vesicles which contain matrixes energetically similar to those of the adrenal cells investigated here can be separated into two classes according to their radius and catecholamine content. Small vesicles (less than ca. 25 nm radius, and containing less than ca. 20000 molecules) should always release through pores. Larger vesicles should always end into fusing except if another mechanism closes the pore before ca. 10000 molecules of catecholamines have been released.

Animals↗