Andrew G. Ewing

Chemical Analysis of Single Cells

In this Review, we provide an overview of methods developed for chemical analysis of single cells over the last two years. Many biological systems contain an ensemble of cells with heterogeneous chemistry; therefore, it is important to analyze them on an individual basis in order to elucidate the role each cell plays in the function of these systems. In clinical diagnostics, the development of extremely sensitive measurements, down to single cells, may provide the best ability for diagnoses. Single cell analysis has, in fact, been present for quite some time. Investigators in life sciences consider the cell as the unit of life and so the pursuit to quantify, image, and modulate the cell has been ongoing for decades.

The PC12 cell as model for neurosecretion

This review attempts to touch on the history and application of amperometry at PC12 cells for fundamental investigation into the exocytosis process. PC12 cells have been widely used as a model for neural differentiation and as such they have been used to examine the effects of differentiation on exocytotic release and specifically release at varicosities. In addition, dexamethasone‐differentiated cells have been shown to have an increased number of releasable vesicles with increased quantal size, thereby allowing for an even broader range of applications including neuropharmacological and neurotoxicological studies. PC12 cells exhibiting large numbers of events have two distinct pools of vesicles, one about twice the quantal size of the other and each about half the total releasable vesicles. As will be outlined in this review, these cells have served as an extremely useful model of exocytosis in the study of the latency of stimulation‐release coupling, the role of exocytotic proteins in regulation of release, effect of drugs on quantal size, autoreceptors, fusion pore biophysics, environmental factors, health and disease. As PC12 cells have some advantages over other models for neurosecretion, including chromaffin cells, it is more than likely that in the following decade PC12 cells will continue to serve as a model to study exocytosis.

In vivo voltammetry with electrodes that discriminate between dopamine and ascorbate.

Untreated carbon-fiber voltammetric electrodes have been employed as chemical sensors of easily oxidized compounds in the brains of rats anesthetized with chloral hydrate. These electrodes can be used to distinguish dopamine from ascorbate and dihydroxyphenylacetate by the shape of the voltammograms. The electrodes are shown to provide a reproducible response to different neuronal stimuli. The rapid release of dopamine in the caudate nucleus can be measured following a local application of potassium chloride. Intraperitoneal injections of amphetamine also induce an increase of easily oxidized compounds; however, the voltammetry suggests that ascorbic acid, rather than dopamine, is the primary substance detected. Measurements in the cortex or in the caudate nucleus of animals lesioned by prior injection of 6-hydroxydopamine show that a substance with voltammetric properties identical to those of ascorbic acid also increases in concentration in these areas as a result of amphetamine administration.

Monitoring the stimulated release of dopamine with in vivo voltammetry. I: Characterization of the response observed in the caudate nucleus of the rat.

Abstract: Microvoltammetric electrodes were employed in the brain of an anesthetized rat to monitor chemical substances in extracellular fluid following electrical stimulation of the medial forebrain bundle. An increase in concentration of an easily oxidized substance is observed in the caudate nucleus and in the nucleus accumbens. A large amount of evidence suggests that the substance that is observed following stimulation is dopamine. (1) The location of the stimulating electrode must be in known dopaminergic tracts to induce release. (2) Release is most easily observed in brain regions that contain significant numbers of dopamine‐containing neurons. (3) Two voltammetric electrodes with very different electrochemical responses provide voltammograms of the released species that are unique for catechols in one case and catecholamines in another case. (4) The amount of 3,4‐dihydroxyphenylacetic acid found in striatal tissue by postmortem analysis correlates with the calculated amount of dopamine released. (5) Inhibition of tyrosine hydroxylase, and thus dopamine synthesis, decreases the observed release while inhibition of monoamine oxidase, and thus formation of dopamine metabolites, does not (6) The dependence of release on stimulation parameters agrees with results obtained with perfusion techniques. Thus, a new method has been developed to characterize endogenous dopamine release in the rat brain and can be used on a time scale of seconds.

Retention of ionic and non-ionic catechols in capillary zone electrophoresis with micellar solutions.

The use of micellar solutions in capillary zone electrophoresis has been primarily relegated to separations of non-ionic solutes, while its applicability to cationic species has been unexplored. We have found that the use of sodium dodecyl sulfate micelles in phosphate buffer allows for tremendous gains in selectivity for several cationic and non-ionic catechols over what can be obtained with normal capillary zone electrophoresis. Complexation of catechols with boric acid alters the net charge on the solutes and changes the partitioning behavior to produce adequate selectivity with improved analysis times. Although the mechanisms of solute interaction with the micellar phase for the cationic species are not decisively known, evidence is presented supporting the existence of ion-pairing equilibria simultaneously accompanied by micellar solubilization.

Catalysis of slow charge transfer reactions at polypyrrole-coated glassy carbon electrodes

Abstract Oxidation of ascorbic acid, dihydroxyphenylacetic acid and dopamine are compared at polypyrrole-coated glassy carbon and naked glassy carbon electrodes. These currents are mass-transport limited and not limited by permeation into or through the polypyrrole film. Ascorbic acid oxidation occurs at potentials 300 mV more negative at polypyrrole-coated electrodes and the rising slope of rotated disk voltammograms changes by over 100 mV. A similar enhancement in electrochemical reversibility is observed for dihydroxyphenylacetic acid, whereas dopamine is oxidized at slightly more positive potentials at polypyrrole-coated electrodes. Comparing the electrochemistry of dopamine and dihydroxyphenyl-acetic acid, it appears that the electrochemical reversibility differences for these substances are to some degree result of electrostatic interactions between the anionic solutes, or anionic reaction intermediates, and anionic functional groups on carbon or cationic fixed sites in oxidized polypyrrole.

Estimation of Free Dopamine in the Cytoplasm of the Giant Dopamine Cell of Planorbis corneus by Voltammetry and Capillary Electrophoresis

Voltammetric electrodes having a tip diameter of 2–12 μ and microscale capillary zone electrophoresis have been used for dynamic and static monitoring of dopamine in the somal cytoplasm of the giant dopamine neuron of Planorbis corneus. Intracellular dopamine levels can be altered by extracellular application of dopamine or ethanol. The latter experiment provides a means to estimate total stores of endogenous dopamine, and the evidence presented suggests that at least 98% of these stores are bound and not directly accessible to the cytoplasm.

Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic β-cells

Abstract: Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic β‐cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for <6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine‐containing vesicles at later days in culture is responsible for this alteration.

Spatially and Temporally Resolved Single-Cell Exocytosis Utilizing Individually Addressable Carbon Microelectrode Arrays

We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-mum-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 microm) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of high-density individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight spaces, as well as for developing multifunctional electrochemical microsensors.

Catecholaminergic suppression of immunocompetent cells

Abstract The synthesis of catecholamines by immunocompetent cells has now been demonstrated. In addition, catecholamines have been discovered inside the nuclear membrane and may therefore interact in the transcription process. Here, Jonas Bergquist and colleagues describe the powerful impact that catecholamines exert on the immune system by downregulation of proliferation and differentiation, and induction of apoptosis.