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.

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.

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.

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.

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.

Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core vesicles.

Large dense core vesicles in rat pheochromocytoma cells are morphologically distinct from dense core vesicles in mast and chromaffin cells in that the dense core occupies a much smaller fraction of the vesicular volume, allowing for a much larger vesicular clear space, or halo. In this work, we present evidence indicating that upon treatment with L-DOPA the majority of the dopamine loaded into these vesicles is preferentially compartmentalized into the halo portion of the vesicle. Amperometry was used to monitor release of loaded neurotransmitter from cells in both isotonic and hypertonic extracellular conditions, with the latter condition causing inhibition of dense core dissociation. In combination with this we have used transmission electron microscopy to determine the morphological characteristics of dense core vesicles before and after treatment with L-DOPA in solutions of varied osmolarity. The results provide a more complete understanding of the complex interaction of molecules within dense core vesicles, suggesting that newly loaded dopamine is located in the halo of the vesicle. This finding has fundamental significance for studies of neurotransmitter release from dense core vesicles, as the core appears to have a function involving more than simple storage of neurotransmitter and associated molecules, and the often overlooked vesicular halo appears to be an important storage compartment for neurotransmitter.

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.

Electrophoresis in nanometer inner diameter capillaries with electrochemical detection.

Separations have been achieved in 770- and 430-nm-inner diameter capillaries. The extremely low sample volumes involved in the study of biological microenvironments such as single cells has led to the desire to develop separation techniques in these ultrasmall capillaries. Total sample volumes as low as 12 fL have been injected using these nanometer inner diameter capillaries. Separations of several catecholamines have been accomplished in these submicrometer capillaries using both capillary zone electrophoresis and micellar electrokinetic chromatography with end-column amperometric detection.

DNA separations in microfabricated devices with automated capillary sample introduction.

A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention.

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.

Measurement of the dynamics of exocytosis and vesicle retrieval at cell populations using a quartz crystal microbalance.

The quartz crystal microbalance-dissipation technique (QCM-D) is used in two different measurement strategies to monitor the mass change and rigidity of populations of excitable cells during exocytosis and subsequent retrieval of dense-core vesicles. Two cell lines, NG 108-15 and PC 12, were grown to confluence on piezoelectric quartz crystals and were examined separately to demonstrate differences in release and retrieval with cells of different morphology, size, and number of dense-core vesicles. Stimulating the cells to exocytosis with media containing an elevated potassium concentration resulted in an increase in the frequency response corresponding to loss of mass from the cells owing to release of vesicles. In Ca2+-free media, the response was completely abolished. The amplitude and peak area in the frequency response corresponding to mass change with stimulated release was larger for PC 12 cells than for NG 108-15 cells, whereas the initial rate constants for the frequency responses were similar. The data suggest (1) that a greater number and larger size of vesicles in PC 12 cells results in a greater amount of release from these cells vs NG 108-15 cells, (2) the recycling of vesicles utilizes similar fusion/retrieval mechanisms in both cell types, (3) that the control of excess retrieval might be related to the number and size of released vesicles, and (4) that measured retrieval has a rapid onset, masking exocytosis and implying a rapid retrieval mechanism in the early stages of release. These results demonstrate that measurements of complex dynamic processes relating to dense-core vesicle release and retrieval can be simultaneously accomplished using the QCM-D technique.

Quantitative chemical analysis of single cells.

A fundamental perspective can be achieved by targeting single cells for analysis with the goal of deconvoluting complex biological functions. However, single-cell studies have their own difficulties, such as minute volumes and sample amounts. Quantitative chemical analysis of single cells has emerged as a powerful new area in recent years due to several technological advancements. The development of microelectrodes has allowed the measurement of redox-active species as a function of cellular dynamics. This miniaturization trend is also evident in the separation sciences with the application of small column separations to single cells. Desorption ionization methods with mass spectrometric detection have shown single-cell capability owing to numerous technological developments. Finally, fluorescence imaging has also progressed to the point where single-cell dynamics can be probed by native fluorescence utilizing either single or multiple photon excitation. The results of these studies are reviewed with an emphasis on the quantitation of single-cell dynamics.

VMAT-Mediated changes in quantal size and vesicular volume.

It has been well established that the volume of secretory vesicles can be modulated. However, we present the first data demonstrating that the amount of transmitter in a vesicle can regulate its volume. Amperometry and transmission electron microscopy have been used to determine that l-3,4-dihydroxyphenylalanine and reserpine increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles as well as their catecholamine content. Because changes in vesicular catecholamine content are tracked by changes in vesicle volume, our results indicate that when quantal size is altered via the vesicular monoamine transporter the concentration of catecholamines within the vesicles remains relatively constant. This previously unidentified cellular response provides new insight into how catecholamines can be packaged in and released from secretory vesicles.

Quantitative and statistical analysis of the shape of amperometric spikes recorded from two populations of cells.

Previously used methods of comparing amperometric spike characteristics from two separate groups of cells have entailed pooling all the values for a spike characteristic from each group of cells and then statistically comparing the two samples. Although this approach has indicated that there are significant differences between the spike characteristics from coloboma and control mouse chromaffin cells, the results are not consistent between experiments. We have reexamined the assumptions of the statistical tests used as well as the variability inherent in amperometric data measured from two groups of cells. Our findings indicate that when comparing amperometric spike characteristics between groups of cells, it is more appropriate to compare samples of mean spike values. This method consistently indicates that there is no difference between coloboma and control amperometric spikes. These results have been validated by using samples of mean spike characteristics to detect changes in the shape of amperometric spikes from both mouse chromaffin cells at 37 degrees C and PC12 cells previously exposed to 50 microM L-3,4-dihydroxyphenylalanine and by the use of an additional analysis method, the nested ANOVA. Together, these results indicate that pooled samples of amperometric spike characteristics can give results that may confound the interpretation of amperometric data.

Spatially resolved detection of attomole quantities of organic molecules localized in picoliter vials using time-of-flight secondary ion mass spectrometry.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been utilized to detect femtomole and attomole quantities of organic species from within silicon nanovials. By using high-density arrays (10,000 nanovials/cm2) it is possible to chemically characterize diverse sample sets within a single chemical image. Molecular sensitivities, for the compounds investigated, very between 85 attomoles and 25 femtomoles, and typical acquisition times are approximately 100 ms per nanovial. These vials are fabricated using photolithography and KOH etching of Si[001] wafers to create wells, with a pyramidal cross section, ranging in size from 25 to 5625 micron 2. The volume ranges from 30 femtoliters to 100 picoliters, respectively. A drawn glass microinjector and solenoid-driven dispenser are utilized to array picoliter volumes of organic compounds into individual silicon nanovials. Solution concentrations typically range from 1 x 10(-2) to 1 x 10(-4) M allowing femtomole and even attomole quantities of material to be dispensed into each vial.

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.

Chemical profiles and monitoring dynamics at an individual nerve cell in Planorbis corneus with electrochemical detection.

The identified dopamine cell of Planorbis corneus is described as a model system to study neurotransmitter storage and dynamics. Techniques developed with this model system include capillary electrophoresis with electrochemical detection and microelectrochemistry at single cells. These techniques provide a powerful combination to examine single cell neurochemistry. Whole cell and cytoplasmic dopamine concentrations have been quantified with capillary electrophoresis. Additionally, this technique has been used to profile amino acids and to quantify two compartments of neurotransmitter in a single cell. Individual exocytosis events have been monitored at the cell body of the dopamine cell of P. corneus with microelectrodes. In this case, two different types of vesicles have been identified based on the amount of transmitter released. The psychostimulant, amphetamine, has been shown to selectively affect the amount of dopamine in these vesicles with lower to higher doses affecting the larger to smaller vesicle types, respectively. Microelectrochemistry at single nerve cells has also been used to demonstrate reverse transport of dopamine across the cell membrane and to suggest a role of this process in the molecular mechanism of amphetamine.

Static time-of-flight secondary ion mass spectrometry imaging of freeze-fractured, frozen-hydrated biological membranes.

The study of cell membrane lipid and steroid composition and distribution is important for the understanding of membrane dynamics and function. Here we present efforts to chemically image phospholipid distributions on a submicron scale on freeze-fractured and frozen-hydrated liposomes and red blood cells using time-of-flight secondary ion mass spectrometry. Sample preparation by freeze fracturing of membranes is described. Fragments representative of phospholipid headgroups are found to be localized on both liposomes and red blood cells. In addition, the cholesterol molecular ion [M + H] is localized on liposome surfaces.