Selected recent in vivo studies on chemical measurements in invertebrates.

In vivo measurements of neurotransmitters and related compounds have provided a better understanding of the chemical interactions that are a major part in functioning of brains. In addition, a great deal of technology has been developed to measure chemical species in other areas of living organisms. A key part of this work has been sampling technologies as well as direct measurements in vivo. This is extremely important when sampling from the smallest animal systems. Yet, very small invertebrate systems are excellent models and often have better defined and more easily manipulated genetics. This review focuses on in vivo measurements, electrochemical methods, fluorescence techniques, and sampling and is further narrowed to work over approximately the last three years. Rapid developments of in vivo studies in these model systems should aid in finding solutions to biological and bioanalytical challenges related to human physiological functions and neurodegenerative diseases.

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.

Investigating lipid-lipid and lipid-protein interactions in model membranes by ToF-SIMS.

With the chemical imaging capability of ToF-SIMS, biological molecules are identified and localized in membranes without any chemical labels. We have developed a model membrane system made with supported Langmuir-Blodgett (LB) monolayers. This simplified model can be used with different combinations of molecules to form a membrane, and thus represents a bottom-up approach to study individual lipid-lipid or lipid-protein interactions. We have used ternary mixtures of sphingomyelin (SM), phosphatidylcholine (PC), and cholesterol (CH) in the model membrane to study the mechanism of domain formation and interactions between phospholipids and cholesterol. Domain structures are observed only when the acyl chain saturation is different for SM and PC in the mixture. The saturated lipid, whether it is SM or PC, is found to be localized with cholesterol, while the unsaturated one is excluded from the domain area. More complicated model membranes which involve a functional membrane protein glycophorin are also investigated and different membrane properties are observed compared to the systems without glycophorin.

Which is more important in bioimaging SIMS experiments-The sample preparation or the nature of the projectile?

Sample preparation is central to acquiring meaningful molecule-specific images with SIMS, especially when submicron lateral resolution is involved. The issue is to maintain the distribution of target molecules while attempting to introduce biological cells or tissue into the high vacuum environment of the mass spectrometer. Here we compare freeze-drying, freeze-etching, freeze-fracture and trehalose vitrification as possible strategies for these experiments. The results show that the prospects for successful imaging experiments are greatly improved with all of these methods when using cluster ion bombardment, particularly C(60) (+) ions, not only due to increased sensitivity of this projectiles, but also since it removes contamination overlayers without insult to the underlying chemistry. The emergence of 3-dimensional imaging capabilities also suggests that sample preparation should not perturb the 3-dimensional morphology of the cell, a situation not generally possible during freeze-drying. Hence, sample preparation and projectile type are strongly coupled parameters for bioimaging with mass spectrometry.

Relative Quantification of Cellular Sections with Molecular Depth Profiling ToF-SIMS Imaging.

We report the use of SIMS imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C(60) (+) ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.

The effects of vesicular volume on secretion through the fusion pore in exocytotic release from PC12 cells.

Many spikes in amperometric records of exocytosis events initially exhibit a prespike feature, or foot, which represents a steady-state flux of neurotransmitter through a stable fusion pore spanning both the vesicle and plasma membranes and connecting the vesicle lumen to the extracellular fluid. Here, we present the first evidence indicating that vesicular volume before secretion is strongly correlated with the characteristics of amperometric foot events. L-3,4-dihydroxyphenylalanine and reserpine have been used to increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles. Amperometry and transmission electron microscopy have been used to determine that as vesicle size is decreased the frequency with which foot events are observed increases, the amount and duration of neurotransmitter released in the foot portion of the event decreases, and vesicles release a greater percentage of their total contents in the foot portion of the event. This previously unidentified correlation provides new insight into how vesicle volume can modulate the activity of the exocytotic fusion pore.

Quantitative measurements of released amines from individual exocytosis events.

Chemical analysis of single cells is an area of great interest in the biological sciences. Single-cell systems are being utilized as a model to understand in vivo processes better. One method that is moving to the forefront in cellular analysis is electrochemistry. Owing to their rapid response time and small dimensions, voltammetric microelectrode techniques, such as amperometry and fast-scan voltammetry, have made it possible to monitor minute amounts of biological compounds and transiently occurring chemical events in cellular systems. The application of these methods to the quantitation of individual vesicular release events from single cells is overviewed here. The application of electrochemical monitoring to several types of cultured cells, including bovine adrenal chromaffin cells, rat pheochromocytoma (PC12) cells, beige mouse mast cells, superior cervical ganglion neurons, and human pancreatic beta-cells, as well as to the invertebrate systems, the leech Hirudo medicinalis, and pond snail Planorbis corneus has provided a wealth of new information concerning exocytosis. Results obtained from the studies highlight the potential of electrochemical techniques in cellular analysis to contribute to our understanding of molecular and pharmacological effects on exocytosis. This article overviews work done on all the above cell types with an emphasis on PC12 cells.

Chemical analysis of single cells and exocytosis.

Recent advances in the development of microscale analytical techniques have allowed minute amounts of compounds of biological interest to be monitored in microenvironments. Microcolumn separation techniques, such as microcolumn liquid chromatography and capillary electrophoresis, provide a means of obtaining high-efficiency separations of analytes in picoliter to femtoliter volume samples. Voltammetric microelectrode techniques, such as amperometry and fast-scan cyclic voltammetry, are ideally suited for monitoring transiently occurring chemical events in cellular and subcellular processes owing to their rapid response times and small structural dimensions. The principles and applications of these techniques in single-cell analysis are discussed throughout this review. Multicomponent separations and quantitations of large invertebrate neurons of the land snail Helix neurons and the pond snail Planorbis corneus dopamine neurons, and of small mammalian cells, bovine adrenal cells, rat PC12 cells, and human lymphocytes, by use of microcolumn liquid chromatography and capillary electrophoresis are presented. Electrochemical monitoring of neurotransmitter exocytosis from single adrenal cells, from PC12 cells, and from the cell body of the Planorbis dopamine neuron is highlighted. Results obtained from both separation and voltammetric techniques in single-cell analyses will provide a better understanding of cellular and/or neuronal chemistry and biology in complicated living environments.

Microcolumn separations of single nerve cell components.

Miniaturization of separation techniques is providing new methods to examine neurochemistry and neurophysiology at the single cell level. Open tubular liquid chromatography and capillary electrophoresis have been scaled down to levels where picoliter and femtoliter volume samples can be separated. This provides the means to profile the chemistry of single whole nerve cells and to sample subcellular regions.

Dopamine concentration in the cytoplasmic compartment of single neurons determined by capillary electrophoresis.

It has long been a goal of the biochemist to study the chemistry of single cells. This is particularly important in neuroscience, since each cell can maintain a specific function and identity. Investigations into neurotransmitter compartmentalization within the cell body of neurons should lead to an understanding of the role of the cell body in metabolism, uptake and storage of neurotransmitters. Using capillary electrophoresis, it has been possible to separate and detect attomole levels of neurotransmitter in picoliter volumes of cytoplasm withdrawn from single neurons of the pond snail, Planorbis corneus. This work demonstrates, for the first time, the direct determination of the cytoplasmic concentration of dopamine in single, intact neurons.

Capillary electrophoresis of single cells: observation of two compartments of neurotransmitter vesicles.

Capillary electrophoresis has been used to directly identify and measure the neurotransmitter, dopamine, in two vesicular compartments in a single nerve cell of Planorbis corneus. Dopamine in the cytoplasm and in easily released transmitter vesicles was separated from dopamine in what are apparently non-functional storage vesicles. In this method, the two peaks in the electropherogram attributed to dopamine were differentiated based on cell lyse time in a non-physiological buffer. The measurements presented here suggest that of the total dopamine present in the cell: 24% is in the cytoplasmic and easily released compartments and 76% is more centrally located, perhaps in a reserve compartment, in the cell. This methodology provides the means to determine molecular species in subcellular compartments and should allow kinetic parameters associated with membrane lysing to be evaluated at single nerve cells.

Amperometric analysis of exocytosis at chromaffin cells from genetically distinct mice.

Amperometry is a very powerful technique for investigating the role(s) specific proteins play in exocytosis at the single-cell level. In this study, amperometry has been used to investigate possible changes in exocytosis at chromaffin cells isolated from coloboma and tottering mutant mice. Coloboma mice possess a deletion mutation that encompasses the gene for the presynaptic protein SNAP-25 and tottering mice carry a mutation of the alpha(1A) subunit gene, which encodes the pore-forming region of P/Q-type calcium channels. Although amperometric data measured from tottering and coloboma cells are not significantly different from that measured at wild-type control cells, significant differences are found when groups of wild-type chromaffin cells are analyzed at room temperature and at 37 degrees C. Due to the large variability inherent to amperometric data, it is possible that changes in release resulting from some genetic differences cannot be detected. To fully exploit the technical advantages of using mouse chromaffin cells, experimental guidelines are described which should maximize changes in release resulting from genetic differences and increase the likelihood of detecting a change in amperometric data.

Demonstration of two distributions of vesicle radius in the dopamine neuron of Planorbis corneus from electrochemical data.

An electrochemical model to calculate the relative size and neurotransmitter concentration of individual nerve cell vesicles is presented to examine potentially different types of vesicles in Planorbis corneus. Amperometric current transients resulting from individual exocytosis events detected from single cells contain the information necessary to quantify vesicular neurotransmitter amount and to estimate other important cellular properties such as vesicular neurotransmitter concentration and vesicle radius. Use of a simplifying assumption that the cross-sectional area of the contents of each release event is the apparent electroactive area of the electrode and that the shape of the decreasing phase of each current transient follows Cottrell-like behavior, the Cottrell equation and Faraday's law can be combined to yield expressions for relative vesicle radius and neurotransmitter concentration. This analysis has been applied to data obtained from the cell body of the giant dopamine neuron of the pond snail P. corneus. The histogram of vesicular dopamine concentration reveals a single wide distribution and the histogram of vesicle radius reveals a bimodal radius distribution. These data strongly suggest two distinct classes of vesicle radius in the P. corneus neuron lead to the bimodal distribution of amount released reported earlier.

Calculation of transmitter concentration in individual PC12 cell vesicles with electrochemical data and a distribution of vesicle size obtained by electron microscopy.

A mathematical model is described to accurately calculate vesicle size and neurotransmitter concentration distributions from electrochemical data. This model uses parameters from electrochemical exocytosis data obtained from PC12 cells in culture to calculate a size distribution that is then correlated to the size of vesicles obtained by electron microscopy. The relative standard deviation of the size distribution calculated from electrochemical data is 25% which matches the relative standard deviation of the vesicle size distribution measured by electron microscopy. The distribution calculated from electrochemical data is normalized to the vesicle size distribution of PC12 cell vesicles obtained from electron microscopy. Calculation of a vesicular catecholamine concentration histogram from the normalized size data and electrochemical parameters is then possible for individual exocytosis events. The average vesicular catecholamine concentration for PC12 cells as calculated by this method is 148+/-7 mM. More importantly, there is a distribution of concentration rather than a constant value. Additionally, the model permits evaluation of the concentration of transmitter in each individual vesicle and vesicle size for each vesicle from electrochemical data when the overall vesicle size distribution is known.

In vitro comparison of the selectivity of electrodes for in vivo electrochemistry.

The properties of 4 types of carbon electrodes designed for use as in vivo sensors of easily oxidized species in the mammalian brain have been evaluated in aqueous solutions at physiological pH. The electrodes are formed from a graphite-epoxy mixture, carbon paste, or carbon fibers, and have the geometries of a disk or a cylinder. The voltammetric properties of several catecholamines, some of their metabolites and precursors, uric acid, and ascorbic acid are reported at unmodified carbon surfaces. The problem of overlap of the voltammetric waves of ascorbate and catechols is addressed, and two different methods which minimize this problem are examined. These are the use of disk-shaped electrodes fabricated from carbon fibers, which facilitate the use of subtracted voltammograms to determine small changes in the concentration of catecholamines in the presence of ascorbic acid, and the use of electrochemically modified, cylindrically-shaped electrodes also fabricated from carbon fibers. Voltammetry at the modified electrodes gives evidence the catechols can be resolved from ascorbate, and that catechols, but not ascorbic acid, adsorb to the electrode surface.

Multiple classes of catecholamine vesicles observed during exocytosis from the Planorbis cell body.

The heterogeneous nature of vesicles has been studied by evaluating exocytotic events released from the cell body of a dopamine-containing neuron of Planorbis corneus. Vesicular exocytosis has been elicited by stimulation with in situ application of elevated potassium and has been monitored electrochemically with a carbon fiber microelectrode placed on the cell body. These electrodes allow individual release events to be monitored and quantitated to reveal vesicular dopamine content. Statistical analysis of individual release events demonstrates that two classes of vesicles with specific bimodal distributions in dopamine content and vesicle size are observed after cell stimulation. The effect of a psychostimulant on individual vesicular dopamine level has been studied by treating the cells with D-amphetamine. After a 20-min application of 10 microM amphetamine, changes in both vesicle content and size distributions are obtained with an overall decrease in vesicular dopamine level of 40%. The altered distribution show trimodal shapes indicating a third class of vesicles is created by amphetamine treatment. Our data appears to indicate that multiple classes of vesicles are released from the cell body of the dopamine-containing neuron and vesicular dopamine level can be manipulated by the application of the lipophilic weak base amphetamine.

Electrochemical monitoring of individual exocytotic events from the varicosities of differentiated PC12 cells.

Rat pheochromocytoma (PC12) cells have been used as a model of developing neurons to study exocytosis during differentiation. Upon treatment with nerve growth factor, PC12 cells become more neuronal-like. Using amperometric detection at carbon fiber microelectrodes, time-resolved exocytosis of electroactive catecholamines can be observed. The site of exocytosis has been compared for differentiated and undifferentiated cells. Upon differentiation, cells release catecholamines primarily from varicosities along their neurites with no release from the cell body. In addition, the mean vesicular content is not significantly altered upon differentiation although it appears that the distribution of vesicle content becomes more narrow. The number of release events observed also decreases as the cells become more neuronal in character. It is possible that the smaller range of vesicle dopamine content and the decreased number of release events observed after differentiation are a result of the relocation of the site of exocytosis.

Electrochemical monitoring of bursting exocytotic events from the giant dopamine neuron of Planorbis corneus.

We have discovered a neuronal system that fires bursting exocytotic events. In the giant dopamine neuron of the fresh water snail Planorbis corneus, bursting exocytotic events are evoked following in situ stimulation with elevated potassium. Amperometric detection using carbon fiber microelectrodes, which provides high temporal resolution, has been used to record exocytotic events released from the neuron. Evaluation of the time interval between consecutive exocytotic events (inter-spike interval) recorded from about 80% of the neurons reveals the occurrence of distinct bursting patterns defined by transients having an equal interval among them. Statistical analysis of these bursting exocytotic events shows three distinct distributions of inter-spike intervals with mid points occurring at 5, 22 and 45 ms. This bursting release behavior is not observed from cultured pheochromocytoma cells although they show calcium-dependent exocytosis following in situ stimulation with elevated potassium. Our data appear to indicate that the Planorbis dopamine neuron in vivo is actively involved in specific modes of neural communication and may represent an important phenomenon in understanding single cell activities.

Dopamine levels of two classes of vesicles are differentially depleted by amphetamine.

Differential depletion of neurotransmitter by amphetamine in two classes of vesicles, termed large vesicles and small vesicles, has been studied with amperometry. Carbon fiber microelectrodes have been used to monitor and quantify exocytotic events. Current transients, corresponding to individual exocytotic events, have been obtained from the cell body of the dopamine-containing neuron of Planorbis corneus. The dopamine released from individual vesicles of these cells has been compared for cells treated with D-amphetamine vs. control cells. Our results show that amphetamine has differential effects on the release of dopamine from the two classes of vesicles. Thus, it is concluded that at low concentrations, amphetamine preferentially depletes the large vesicles with a minimal effect on the small vesicles. At high concentrations, amphetamine depletes small vesicles more strongly than large vesicles although amphetamine continues to deplete the large vesicles in a dose-dependent manner. Our data appear to indicate that the two classes of vesicles observed in the Planorbis dopamine neuron might have different mechanisms associated with transmitter depletion.