Expanding neurochemical investigations with multi-modal recording: simultaneous fast-scan cyclic voltammetry, iontophoresis, and patch clamp measurements.

Multi-modal recording describes the simultaneous collection of information across distinct domains. Compared to isolated measurements, such studies can more easily determine relationships between varieties of phenomena. This is useful for neurochemical investigations which examine cellular activity in response to changes in the local chemical environment. In this study, we demonstrate a method to perform simultaneous patch clamp measurements with fast-scan cyclic voltammetry (FSCV) using optically isolated instrumentation. A model circuit simulating concurrent measurements was used to predict the electrical interference between instruments. No significant impact was anticipated between methods, and predictions were largely confirmed experimentally. One exception was due to capacitive coupling of the FSCV potential waveform into the patch clamp amplifier. However, capacitive transients measured in whole-cell current clamp recordings were well below the level of biological signals, which allowed the activity of cells to be easily determined. Next, the activity of medium spiny neurons (MSNs) was examined in the presence of an FSCV electrode to determine how the exogenous potential impacted nearby cells. The activities of both resting and active MSNs were unaffected by the FSCV waveform. Additionally, application of an iontophoretic current, used to locally deliver drugs and other neurochemicals, did not affect neighboring cells. Finally, MSN activity was monitored during iontophoretic delivery of glutamate, an excitatory neurotransmitter. Membrane depolarization and cell firing were observed concurrently with chemical changes around the cell resulting from delivery. In all, we show how combined electrophysiological and electrochemical measurements can relate information between domains and increase the power of neurochemical investigations.

Quantitative analysis of iontophoretic drug delivery from micropipettes.

Microiontophoresis is a drug delivery method in which an electric current is used to eject molecular species from a micropipette. It has been primarily utilized for neurochemical investigations, but is limited due to difficulty controlling and determining the ejected quantity. Consequently the concentration of an ejected species and the extent of the affected region are relegated to various methods of approximation. To address this, we investigated the principles underlying ejection rates and examined the concentration distribution in microiontophoresis using a combination of electrochemical, chromatographic, and fluorescence-based approaches. This involved a principal focus on how the iontophoretic barrel solution affects ejection characteristics. The ion ejection rate displayed a direct correspondence to the ionic mole fraction, regardless of the ejection current polarity. In contrast, neutral molecules are ejected by electroosmotic flow (EOF) at a rate proportional to the barrel solution concentration. Furthermore, the presence of EOF was observed from barrels containing high ionic strength solutions. In practice, use of a retaining current draws extracellular ions into the barrel and will alter the barrel solution composition. Even in the absence of a retaining current, diffusional exchange at the barrel tip will occur. Thus behavior of successive ejections may slightly differ. To account for this, electrochemical or fluorescence markers can be incorporated into the barrel solution in order to compare ejection quantities. These may also be used to provide an estimate of the ejected amount and distribution provided accurate use of calibration procedures.

Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits.

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinson's disease.

Voltammetric detection of 5-hydroxytryptamine release in the rat brain.

5-Hydroxytryptamine (5-HT) is an important molecule in the brain that is implicated in mood and emotional processes. In vivo, its dynamic release and uptake kinetics are poorly understood due to a lack of analytical techniques for its rapid measurement. Whereas fast-scan cyclic voltammetry with carbon fiber microelectrodes is used frequently to monitor subsecond dopamine release in freely moving and anesthetized rats, the electrooxidation of 5-HT forms products that quickly polymerize and irreversibly coat the carbon electrode surface. Previously described modifications of the electrochemical waveform allow stable and sensitive 5-HT measurements in mammalian tissue slice preparations and in the brain of fruit fly larvae. For in vivo applications in mammals, however, the problem of electrode deterioration persists. We identify the root of this problem to be fouling by extracellular metabolites such as 5-hydoxyindole acetic acid (5-HIAA), which is present in 200-1000 times the concentration of 5-HT and displays similar electrochemical properties, including filming of the electrode surface. To impede access of the 5-HIAA to the electrode surface, a thin layer of Nafion, a cation exchange polymer, has been electrodeposited onto cylindrical carbon-fiber microelectrodes. The presence of the Nafion film was confirmed with environmental scanning electron microscopy and was demonstrated by the diminution of the voltammetric signals for 5-HIAA as well as other common anionic species. The modified microelectrodes also display increased sensitivity to 5-HT, yielding a characteristic cyclic voltammogram that is easily distinguishable from other common electroactive brain species. The thickness of the Nafion coating and a diffusion coefficient (D) in the film for 5-HT were evaluated by measuring permeation through Nafion. In vivo, we used physiological, anatomical, and pharmacological evidence to validate the signal as 5-HT. Using Nafion-modified microelectrodes, we present the first endogenous recording of 5-HT in the mammalian brain.

Voltammetry with microscopic electrodes in new domains.

Voltammetric electrodes of microscopic dimension, termed ultramicroelectrodes, can be used to make measurements that are difficult or impossible with conventional electrochemical techniques. Measurements of chemical concentration can be made with these electrodes on a microsecond time scale and with micrometer spatial resolution. In addition, measurements can be made in highly resistive solutions.

Observation of individual chemical reactions in solution.

Discrete chemical reaction events occurring in solution have been observed by single photon detection of a bimolecular, chemiluminescent reaction. The reactants were generated from 9,10-diphenylanthracene in acetonitrile with potential pulses applied to an ultramicroelectrode. Electrogenerated radical ions of opposite sign react to yield the excited singlet state of the parent compound. The chemical reactions were restricted to a 20-femtoliter volume adjacent to the electrode by the use of rapid potential pulses. Individual chemical reaction events were stochastic and followed the Poisson distribution, and the interarrival time between successive reaction events was exponentially distributed.

Direct in vivo monitoring of dopamine released from two striatal compartments in the rat.

Microvoltammetric electrodes were used to monitor dopamine released in the caudate nucleus of the rat after electrical stimulation of the medial forebrain bundle. The time resolution of the technique is sufficient to determine in vivo concentration changes on a time scale of seconds. Direct evidence identifying the substance released as dopamine was obtained both voltammetrically and pharmacologically. Administration of alpha-methyl-p-tyrosine terminates the release of dopamine, although tissue stores of dopamine are still present. Thus there appears to be a compartment for dopamine storage that is not available for immediate release. This compartment appears to be mobilized by amfonelic acid, since administration of this agent after alpha-methyl-p-tyrosine returns the concentration of dopamine released by electrical stimulation to 75 percent of the original amount.

Liquid chromatographic analysis of endogenous catecholamine release from brain slices.

A simple new liquid chromatographic technique has been applied to transmitter release studies in brain slice preparations. This method, which gives direct readings of picomoles of endogenous transmitter released, has been shown to yield reliable results with a variety of brain slice manipulations.

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.

Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine.

Vesicular monoamine transporters are known to transport monoamines from the cytoplasm into secretory vesicles. We have used homologous recombination to generate mutant mice lacking the vesicular monoamine transporter 2 (VMAT2), the predominant form expressed in the brain. Newborn homozygotes die within a few days after birth, manifesting severely impaired monoamine storage and vesicular release. In heterozygous adult mice, extracellular striatal dopamine levels, as well as K+- and amphetamine-evoked dopamine release, are diminished. The observed changes in presynaptic homeostasis are accompanied by a pronounced supersensitivity of the mice to the locomotor effects of the dopamine agonist apomorphine, the psychostimulants cocaine and amphetamine, and ethanol. Importantly, VMAT2 heterozygous mice do not develop further sensitization to repeated cocaine administration. These observations stress the importance of VMAT2 in the maintenance of presynaptic function and suggest that these mice may provide an animal model for delineating the mechanisms of vesicular release, monoamine function, and postsynaptic sensitization associated with drug abuse.

Loss of autoreceptor functions in mice lacking the dopamine transporter.

Autoreceptors provide an important inhibitory feedback mechanism for dopamine neurons by altering neuronal functions in response to changes in extracellular levels of dopamine. Elevated dopamine may be a component of several neuropsychiatric disorders. However, evidence concerning the state of autoreceptors in such conditions has remained elusive. The function of dopamine autoreceptors was assessed in mice lacking the dopamine transporter (DAT). Genetic deletion of the DAT gene in mice results in a persistent elevation in levels of extracellular dopamine. Direct assessment of impulse-, synthesis- and release-regulating autoreceptors in these mice reveals a nearly complete loss of function. These findings may provide insight into the neurochemical consequences of hyperdopaminergia.

Paracrine neurotransmission in the CNS: involvement of 5-HT.

While GABA and glutamate have an established synaptic function in the CNS, recent evidence suggests 5-HT neurotransmission is predominantly paracrine. As the amino-acid neurotransmitters interact with receptors that produce effects rapidly, electrophysiological approaches can be used to assess the time delay between transmitter release and the postsynaptic response directly. However, this approach cannot be used for studies of 5-HT-mediated neurotransmission, because the majority of its receptors react more slowly, so anatomical and voltammetrical approaches have been used to provide insight into 5-HT-mediated events. These studies have revealed that extrasynaptic receptors and transporters for 5-HT exist, and that 5-HT escapes readily from the synaptic cleft. Attenuation of 5-HT binding by 5-HT-receptor antagonists and 5-HT-uptake inhibitors does not affect the synaptic efflux elicited by transient stimuli, although the effects of such drugs are apparent at later time points. Once it is extrasynaptic, 5-HT has a concentration that is similar to those estimated to be optimal for receptor and transporter activation, and it can diffuse a few micrometers until removed by its transporter. These properties of 5-HT raise the possibility that it can act on receptors that are distant from its release site and function as a paracrine transmitter.

Facile oxidative decarboxylation of 3,4-dihydroxyphenylacetic acid catalyzed by copper and manganese ions.

Under physiological conditions, we observed the rapid, pH- and temperature-dependent, oxidative decarboxylation and hydration of 3,4-dihydroxyphenylacetic acid (DOPAC) to form 3,4-dihydroxybenzyl alcohol (DBAlc). This product was oxidized and underwent tautomerization to form 3,4-dihydroxybenzaldehyde (DBAld). This reaction did not occur in the presence of EDTA, was catalyzed by copper (CuI, CuII) and manganese (MnII) and was oxygen dependent. A variety of mono- and dihydroxyphenyl carboxylic acids were tested and the reaction producing DBAlc as an intermediate was observed to be unique to DOPAC. 3.4-Dihydroxymandelic acid (DOMA) was rapidly oxidatively decarboxylated to form DBAld directly. The substrate and catalyst selectivity of this reaction suggest that this may have physiological relevance in the neurotoxic consequences of manganese and copper to the dopaminergic system in man.

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.

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.

Intracyclization rates of 6-hydroxydopamine and 6-aminodopamine analogs under physiological conditions.

It is agreed that the neurotoxic action of 6-hydroxydopamine and 6-aminodopamine is related to their ease of oxidation. The initial oxidation products, the p-quinone and p-quinone imine, readily undergo 1,2-intracyclization. These reactions could represent an important loss of active neurotoxic agent available uptake. A variety of substituted 6-aminodopamine analogs was prepared and their formal potentials and cyclization rates were measured accurately. The effect of the balance of ease of oxidation vs. rate of cyclization on their neurotoxicity was examined. The results are in general accord with in vivo lifetimes for 6-hydroxydopamine and 6-aminodopamine in rat caudate nucleus.

Comparison of cytosolic Ca2+ and exocytosis responses from single rat and bovine chromaffin cells.

The relationship between cytosolic Ca2+ and catecholamine secretion during stimulus-secretion coupling has been examined at individual chromaffin cells isolated from the cow and rat. Vesicular catecholamine exocytosis was determined via amperometric measurements with carbon fibre microelectrodes and fura-2 was used for simultaneous fluorescent monitoring of cytosolic Ca2+ at the same cell. Individual secretory vesicles in cells from the two species were found to release similar amounts of catecholamine. In addition, the time courses for secretion from individual vesicles was similar with rat and bovine chromaffin cells. The total quantity of catecholamine released and the change in cytosolic Ca2+ were also similar in response to exposure to K+ (60 mM), 1,1-dimethyl-4-phenylpiperazinium (50 microM), and histamine (50 microM), although both responses were more prolonged following 1,1-dimethyl-4-phenylpiperazinium and histamine at bovine cells. Agents that mobilize intracellular Ca(2+)-stores such as methacholine, caffeine and bradykinin resulted in different cytosolic Ca2+ and exocytosis responses at the rat and bovine chromaffin cells. Results indicate a heightened Ca(2+)-store activity or a more filled state in chromaffin cells from the rat. The results of this study clearly show that single-cell techniques can be used to characterize stimulus-secretion coupling. The requirement for lower numbers of cells with these techniques means that chromaffin cells from rodents can be routinely employed. This can be advantageous to minimize biological variability which occurs with organs obtained from slaughter houses and enables the investigation of genetically-altered animals.

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.

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.

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.