Andrew G. Ewing - US grants

Renewed support for Ewing's research in sensing of neurotransmitters and related analytical chemistry comes from the Analytical and Surface Chemistry Program in the Chemistry Division and the Synaptic Mechanisms Program in Behavioral and Neural Sciences. Research will include assembly of an electrospray time-of-flight mass spectrometer, which will be used to identify and quantify lipids and phospholipids from intact cell membranes. In addition, neuropeptides from single transmitter vesicles will be identified. Mass spectral measurements will complement microelectrochemical detection of neurotransmitters for characterization of neural transmission and transduction. %%% While a variety of small molecules are known to transmit signals from one nerve cell to the next, a variety of larger molecules are also important in understanding neurotransmission and nerve cell function. Among these are fatty substances (lipids and phospholipids) in cell membranes, whose composition varies along the cell membrane and with the state of the cell. The use of mass spectrometry will allow identification and structure elucidation of small numbers of these molecules from single cells. In addition, peptides (small chains or oligomers of amino acids) which transmit or modify transmission of neuronal signals will be identified and sequenced.

This project is in the general area of analytical and surface chemistry and in the subfield of bioelectrochemistry. This award will provide funds for the purchase of a video enhanced microscope with which Professor Ewing can perform some truly innovative experiments in bioelectrochemistry. Specifically, this young investigator will employ the video enhanced microscope to optically visualize synaptic junctions between cultured neurons to perform the first in vivo measurement of neurotransmitter release from a single synaptic bouton. Success in these experiments would demonstrate the electrochemist's ability to make measurements at the same cellular level that the bioscientist thinks in terms of. This achievement would represent an important milestone in bioelectrochemistry and its possibility has been facilitated by this investigator's advances in microelectrode fabrication and neuronal cell culture experiments. The ability to perform analytical voltammetry wholly within synaptic gaps will enable the identification of catecholaminergic neurotransmitters by voltammetry and, therefore, by cell type. Additionally, this new experimental capability will permit correlation of neuronal stimulation type and strength with neurotransmitter release from a single neuron. This research could lead to procedures for identifying and characterizing cultured neurons as well as new fundamental insights into neuronal communication.

This project is in the general area of analytical and surface chemistry and in the subfields of electrochemistry and separation science. Three areas of technological and fundamental importance are addressed by this research. In the first area, extremely small microvoltammetric probes will be disigned and characterized and their use for the dynamic in vivo quantification of intracellular neurotransmitters will be evaluated. In a second area, capillary zone electrophoretic and microcolumn open-tubular liquid chromatographic protocols will be developed for the analysis of ultra-small-volume samples. Coupled with newly developed electrochemical detectors, these methods should permit the detection of sub-femtomolar concentrations of redox-active analytes and provide a static quantitative method for neurotransmitter analysis to complement the dynamic approach already described. Thirdly, the unique electrochemical behavior of metallocene-derivatized phosphazenes and phosphazene polymers will be investigated and their potential utility as charge storage media evaluated. This five year continuing grant to The Pennsylvania State University for the support of Professor Ewing as a Presidential Young Investigator is designed to enable his rapid and effective initiation of this broad and innovative research program in chemistry.

This proposal concerns the development and characterization of bioanalytical methods based primarily upon microcolumn separation techniques. The systems we are interested in are neurochemical in nature. This is a methods development oriented proposal. We plan to investigate the use of a carbon film electrode as an on-column electrochemical detector for microcolumn liquid separation schemes. This detector is extremely sensitive, should be applicable to any size column and application to capillary zone electrophoresis is feasible. The use of capillary zone electrophoresis for schemes involving neurochemically interesting compounds is planned with both laser fluorescence and electrochemical detection. Lastly, we propose to use electroosmotic flow to acquire extremely small volume samples and to "inject" these samples onto the capillary electrophoresis column. A similar scheme will be used to carry out in vivo perfusion experiments on rats. These experiments can be carried out with side-by-side capillaries drawn out to only a few microns in diamter.

Professor Andrew G. Ewing and other members of the Chemistry Department of Pennsylvania State University are being supported to continue a Research Experiences for Undergraduates (REU) site in Chemistry. For the period 1990-92, ten (10) undergraduate students will spend ten (10) weeks each summer actively engaged in a variety of research projects. Projects range from laser spectroscopy and thermochemistry to template synthesis of oligomers and computer modeling of semiconductors. Students will attend two weekly seminar series on research and on instrumentation and will prepare oral and written final reports.

We have pioneered the development of amperometric detection in narrow-bore capillary electrophoresis and the use of this technique to detect neurotransmitters in the cytoplasm of single nerve cells. The goals of this proposal involve the miniaturization of capillary electrophoresis and development of detection schemes for separation and detection of neurochemicals removed from the cytoplasm of single nerve cells. A major portion of this proposal deals with methods development for the neurochemical experiments. 5-micro(m) i.d. capillaries will be used with amperometric detection and a novel injection scheme to sample the cytoplasm of single invertebrate neurons. The use of 2-micro(m) and even 1-micro(m) i.d. capillaries will be investigated for sampling the cytoplasm of single mammalian cells. Detection schemes based on pulsed amperometry and copper/copper oxide electrodes will be investigated for detecting removed intracellular second messengers. In addition to systems involving electrochemical detection, schemes based on laser fluorescence and electrogenerated chemiluminescence will be examined for detection of neuropeptides. Finally, low-volume capillary electrophoresis will be coupled to flow SIMS TOF MS for acquisition, separation and detection of the major components of cell membranes. This will lead to the development of technology to sample a small segment (2-5 micro(m) diameter) of membrane and provide analysis of localized membrane regions of single cells. The neurochemical goals of this proposal emphasize the study of neurotransmitter and the intracellular second messenger levels in single cell cytoplasm. These experiments are meant to complement and expand our experiments involving dynamic monitoring of neurotransmitters and intracellular messengers in cytoplasm with voltammetry.

We propose to investigate the feasibility of a novel approach to DNA sequencing involving the combination of electrophoresis in thin slab gels with a method to rapidly and sequentially sample fom an array of microvials containing Sanger sequencing reaction products. The use of thin enclosed slab gels will provide a means to carry out multiple separations in parallel using a staggered sample introduction method developed in this laboratory. Under optimum conditions, up to 100 separations can be carried out per hour. Eventually, separations providing up to 600 to 1000 bases of sequence information should result in a sequencing rate on the order of 10(5) bases per hour. A key part of this system will be coupling the slab gel to a capillary for sample introduction. The capillary will be used to electrophoretically sample sets of nested fragments from nanoliter or picoliter microvials and deposit these along the entrance of the enclosed thin slab gel. Continuously moving the capillary along the entrance to the slab gel will provide a means to carry out continuous separations over hours to days. This format represents an alternative to the multi capillary approach wherein multiple gel-based separations will be carried out across the thin slab gel. The sampling method proposed will provide a means to transfer picoliter solutions of nested fragments to the thin slab gel without the use of relatively large sample wells. This will enhance the spatial resolution across the gel permitting additional separation lanes and will permit the use of thinner gels without sample overload. Gels with thickness of 22 micromoles are planned. Improved heat dissipation in the thinner gels leads us to speculate that single-base-resolved separations will be attained for 600 up to 1000 bases. Finally, the enclosed slab-gel format will permit pressurization facilitating the use of replaceable linear gels. A library of different oligonucleotide primers of known sequence will be placed in arrays of microvials for use in sequencing reactions. Initially, primers will be chosen from segments of the M13mp18 DNA template which will be used as a model system to demonstrate the method. The 600 to 1000 bases of sequence information obtained from each reaction vial will be used to reconstruct the total sequence. As the ease of overall sequence reconstruction increases dramatically with the length of individual sequences obtained from each separation, achieving separations with single base resolution of DNA larger than 500 bases is critically important. Large fragments of DNA (10 to 50 kb) can potentially be sequenced with this methodology without digestion into smaller fragments that must be reconstructed later. It should be possible to obtain a 106 base sequence in a matter of days at a cost easily under $.50 per base.

This proposal concerns the use of capillary electrophoresis as an ultrasmall volume analysis method to investigate specific issues in neurobiology requiring quantitative single cell analysis. We have pioneered the use of capillary electrophoresis for single cell and subcellular analysis and propose to apply several new strategies to investigate these issues. First, the two compartment model of neurotransmitter storage and the effect of psychostimulants on autoreceptors and catecholamine release will be studied by capillary electrophoresis with electrochemical detection. Pharmacological and temperature manipulation will be used to examine the kinetics of neurotransmitter transfer between compartments and the generality of this model to other cell systems. Experiments are proposed to identify and quantitate substances released from cells immobilized in micro-vials having volumes in the 8 to 95 picoliter range. Second, capillary electrophoresis with fluorescence detection will be used to determine the levels of transferrin in single myelinating and nonmyelinating oligodendrocytes in culture. Combined with assays of proteins and peptides in myelin-producing vs myelin-inhibiting culture systems, these measurements should provide important information concerning oligodendrocyte growth and proliferation. Third, noncompetitive, heterogeneous, enzyme-amplified immunoassays are proposed in picoliter microvials, or conversely, to carry out detection by capillary electrochemical immunoassay. The goal is to determine the peptides, beta- endorphin and corticotropin releasing hormone, at zeptomole levels in individual cerebrospinal fluid lymphocytes involved in the etiology of the experimental disease autoimmune encephalomyelitis - a model for multiple sclerosis. Finally, it is proposed to determine these target peptides in lymphocytes of schizophrenic and normal patients.

This Small Grant for Exploratory Research (SGER) is supported in the Analytical and Surface Chemistry Program. During the tenure of this twelve-month standard grant, Professors Ewing and Day and their students at Pennsylvania State University will attach single oligonucleotide molecules of poly dT to small monodispersed beads arranged in arrays on a flat substrate. The arrays will be hybridized with micro samples of messenger RNA and then reverse transcription will produce an array of cDNAs for differential screening and quantification for gene expression. Using this approach single gene products can be screened and identified by sequencing in a single automated process. Many genomic applications involve either small amounts of sample or expression of low abundance genes. Amplification by the polymerase chain reaction is then required. The work of Professors Ewing and Day and their students at Pennsylvania State University will permit single molecule detection on an array of cDNAs permitting differential screening and quantification for gene expression in a single automated process. This work will have immediate impact on problems in genomic fields.

This renewal project, supported in the Analytical and Surface Chemistry Program, focusses on the development of new continuous electrophoretic separations in narrow channels coupled to capillary sample introduction. Professor Ewing and his students at Penn State University will pursue work to improve detection schemes for these separations using electrochemical arrays and post-separation derivatization for fluorescence detection. The goal of this work is to develop a fast continuous or sequential analysis protocol for very small samples taken from neuronal environments. Another focus of this work will be in using these methods to monitor enzymatic reactions in order to do drug screening. This project has both fundamental and applied goals that will advance the sensitivity and speed of small sample analyses. New developments in continuous electrophoretic separations of very small samples in narrow channels will be pursued in this project. Professor Ewing and his students at Penn State University will focus on substantial improvements in the detection limits for these separations using both electrochemical and fluorescence methods of analysis. An important aspect of this work will be enhancing the speed of these analyses using very small samples so that screening methods can be developed in fields such as neurochemistry and drug development. This project has both fundamental scientific components and important applied goals that will be of societal value.

DESCRIPTION: (adapted from applicant's abstract) This proposal concerns the development and application of electrochemical methods for measurements of single exocytosis events. The overall goal of the proposed work is to further understanding of neurotransmitter exocytosis. This is the basic process by which neurotransmitters and hormones are released from cells to initiate chemical communication. Yet, a great deal is still unknown about the function of the molecular mechanism of exocytosis. The applicants propose to develop new strategies for manufacturing smaller electrodes that can be placed in the synapse and should theoretically be useful to make enzyme electrodes with millisecond response time. The experiments proposed involve 1) investigation of events that involve cellular control of the timing of exocytosis; 2) investigation of sites of exocytosis outside the synapse and the target receptors for this release; 3) use of a recently developed electrochemical model of exocytosis to examine the effects of pharmacological agents on neurotransmitter concentration and vesicle size; 4) development of methodology to examine the role of vesicle-docking proteins on exocytotic release; 5) development of strategies to electrochemically measure exocytosis events in single synapses and 6) preparation of enzyme electrodes with submicron tip diameter. The successful completion of the work proposed will further develop the technology available for measurements of exocytotic events and will provide a significant step toward understanding the molecular basis of exocytosis.

DESCRIPTION: Development of high throughput DNA sampling, separation, and detection strategies are described for use in sequencing and genotyping. New technology is proposed for massively parallel, low-volume DNA separations. This will be accomplished by combining capillary sample introduction parallel separations in microfabricated chips, and electrochemical array detection to provide a single integrated system for separation and detection. Ultimately, this will be connected via a sampling capillary to an array of nanovials for introduction of large numbers of samples. The proposal is divided into four major parts which include 1) optimization of separations of DNA fragments in ultrathin channels with sieving buffers, 2) development of an improved interface to allow efficient sample transfer across the capillary-channel interface, 3) development of electrochemical array detection using Ru(bpy)32+ as an electrophore for dsDNA and ssDNA, and 4) application of the capillary transfer method and electrochemical array detection to DNA separations on chips. This will provide the means to rapidly place multiple samples in a very large number of channels (1000 parallel channels is feasible) on a single chip. The proposed technology promises to increase sample throughput by 10- to 50-fold and reduce sample size by 2 to 3 orders of magnitude relative to currently available systems.

With support from the Chemistry Research Instrumentation and Facilities (CRIF) Program, the Department of Chemistry at Pennsylvania State University will acquire a X-ray diffractometer with CCD detector and stereo microscope for small molecule diffractometry. This equipment will enhance research in a number of areas including the following: single crystal and fiber diffraction of phosphazene-based compounds and molecular inclusion clathrates; design and characterization of multi-dimensional covalent networks; novel methods of molecular recognition and the preparation of fluorescent chemical sensors; structural relationships between bioinorganic analogues and metalloprotein active sites; molecular recognition and artificial photosynthesis in lamellar and zeolitic solids; relating structural details to the mechanistic aspects of organometallic chemistry and homogeneous catalysis; structural characterization of complex natural products and their synthetic precursors; and structure-function relationships in enantioselective catalysts.

The X-ray diffractometer allows accurate and precise measurements of the full three dimensional structure of a molecule, including bond distances and angles, and it provides accurate information about the spatial arrangement of the molecule relative to the neighboring molecules. These studies will have an impact in a number of areas, including the preparation of more efficient catalysts, chemical sensors, and biochemistry.

Professor Andrew Ewing of Pennsylvania State University is supported by the Analytical and Surface Chemistry Program to fabricate unilamellar liposomes to use as models for biological cells. The idea is to study fusion of the liposomes using advanced electrochemical methods in order to learn about fusion itself, and as a model for cell exocytosis. Microelectrode work as well as scanning electrochemical microscopy (SECM) using nanotube tips will be performed. Parameters to be examined include vesicle size, mode of fusion, membrane composition, pH and ionic strength and development of an artificial dense core with hydrogels.

Exocytosis is a fundamental process underlying communication between cells, for example between neurons. A better understanding of exocytosis could lead to improved models of brain function and treatment. Fusion of vesicles is an important process in drug delivery. There are applications of this work in microfluidics and possibly for biosensors as well.

DESCRIPTION (Provided by applicant): The overall objective of this research is to develop new microanalytical approaches to study the neurochemistry of Drosophila melanogaster, the fruit fly. Drosophila is arguably one of the most studied organisms in biology offering many advantages as a model system. However, there is a difficulty with a need for low-volume and sensitive analytical methods to examine the chemical changes associated with genetic and behavioral aspects of the fly. The fly nervous system contains approximately 200,000 cells and occupies only about 8 nL. We propose to further develop and apply small sample handling and capillary electrophoresis methods we have recently reported for studies with the fly. These methods have produced some of the richest electropherograms or chromatograms for electrical detection of a biological sample reported to date and there are many compounds eluted that we have not yet identified. In addition, we propose to use one Pi's background in in vivo voltammetry to develop protocols for in vivo voltammetry in the fly. This will present the smallest in vivo approach in an intact organism and is an exciting analytical challenge. Current and newly developed methods will be brought to bear on the chemical mechanisms that underlie chronic alcohol tolerance and the analysis of single fly outliers. The aims of the proposal are to 1) enhance sampling methodologies for analytical separations of fly homogenates, single heads, and sections of nervous tissue, 2) develop in vivo electrochemical methods for the chemical analysis of dynamics in distinct fly brain structures, and 3) apply the newly developed techniques to identify neurochemicals associated with chronic tolerance to alcohol.

DESCRIPTION (provided by applicant): The long term goal of this research is to develop a microfluidic platform to investigate the intercommunication of nerve cells in networks and the influence of the interconnections between cells on the neuronal response to physical trauma and exposure to chemical toxins. A key aspect of this work will be to develop and utilize three-dimensional microfluidic systems to precisely control delivery of reagents to individual cells in a network and to use this system to further our understanding of cell-to-cell communication. Cell networks in vitro will be used to model complex communication in the larger network of the brain. Special emphasis will be placed on understanding pre- vs. postsynaptic receptors, plasticity in patterns and screening for pharmacological efficacy with multiple cells at once. It is normally difficult to carry out detailed studies of drugs, toxins or damage to specific cells in neural networks owing to an inability to simultaneously stimulate (or expose to drug, toxin) and record from several specific cells in an array or network. The system proposed will provide a solution to this problem. We expect the work will expand existing knowledge concerning molecule-based cell-to-cell communication pathways and provide new avenues for screening drugs and toxins involved in the brain. There are four specific aims for this proposal. First, an integrated system will be designed and fabricated to allow linear cell arrays to be non-invasively stimulated and continuously monitored in real-time. Second, a three-dimensional microfluidic device combining small apertures with cross flow will be used to address more complicated two-dimensional cell networks. Third, the microfluidic system combined with fluorescence monitoring of cell activity will be tested on PC12 and P19 cell networks as models. Fourth, neuronal cell cultures with functional synapses will be monitored. The long-term goal is to use this system to examine the effects of physical and chemical damage to specific cells in a neuronal network. We propose to test the hypotheses that 1) neuronal connectivity changes to compensate for neuronal loss following physical damage, 2) activity in neuronal networks affects the rate of degradation following exposure to toxins, 3) glutamate neurons play an active role in dopaminergic neuronal cell loss following exposure toxins, and 4) L-DOPA plays a role in the progression of cell loss in Parkinson's Disease and might be involved in the mechanism of action of neuroprotective agents on dopamine cells. The proposed research has the overarching goal of establishing microfluidics as a valuable tool for biology.

The goal of the proposed research is to develop and apply analytical methods in extremely small environments to probe specific mechanisms that regulate and modulate exocytosis. A key aspect of this work will be to use amperometric measurements simultaneously with fluorescence imaging to test the hypothesis that control of calcium homeostasis and exocytosis are part of the mechanism by which estrogen is neuroprotective, to use small-volume separations to determine the extent to which the vesicle contents are released during an exocytosis event, and to use amperometric experiments and confocal fluorescence imaging to examine a new and potentially controversial hypothesis that lipid nanotubes play a role in regulating vesicle state and release. This work will be done with pheochromocytoma (PC12) cells in culture. Thus, the experiments proposed here are targeted at understanding exocytosis at the molecular level and understanding how cells are "wired" in a way that might be more general in cell biology. The specific aims of the proposal are:1) to use amperometry and calcium imaging to examine the neuroprotective effects of estrogen by measuring catecholamine release and calcium entry;2) to develop electrophoresis with electrochemical detection in small capillaries to determine the level of neuromessenger in vesicles and by comparison to amperometric measurements at cells to determine the fraction released during exocytosis;3) to identify lipid nanotube structures in cells by fluorescence and to investigate the mechanism of membrane trafficking to and from vesicles;and 4) to use electrochemistry and fluorescence to examine and develop models of transmitter release via the fusion pore prior to full exocytosis and an alternative hypothesis for "kiss and run"release. This proposal captures the work that we envision as necessary to truly understand the function of vesicles and exocytosis in neurotransmission and synaptic plasticity. The highly preliminary data we have obtained suggest that lipid nanotubes exist connecting vesicles to other structures, perhaps each other. If this is correct, it represents a new idea in cell biology and could be incredibly important. Overall, the use of amperometry, fluorescence and separations is proposed to investigate cellular chemistry and new ideas in cell biology related to neuronal communication.