Jonathan V. Sweedler, Ph.D. - US grants

This National Science Foundation Young Investigator project is in the general area of analytical and surface chemistry and in the subfields of bioanalysis and neurochemistry. During the tenure of this five-year award, Professor Sweedler and his students will develop the analytical instrumentation and methodology capable of identifying and quantifying neuronal releasates from a single nerve terminal and the contents of individual varicosities, and then use those techniques to better understand the mechanisms of cellular communication. Specific thrusts include the development of capillary zone electrophoresis/laser induced fluorescence instrumentation and methodology to assay and quantitate the contents of individual varicosities and vesicles; demonstration of the capabilities of that instrumentation using cultured neurons; study of the distribution of neuropeptides within individual neurons of the giant marine mollusk Aplysia; quantitative measurement of the peptide content of individual varicosities along a nerve process; measurement of the release of neuropeptides from a single nerve terminal; and determination of whether facilitation in Aplysia neurons is due to enhancement of neurotransmitter release. %%% The successful attainment of the goals of this project will afford protocols for the reliable quantitation of analytes in the zeptomole range, thereby extending the sensitivity of conventional cell sampling techniques by over a thousand fold. This research will also serve to further bridge the scientific domains of analytical chemistry and cellular neurobiology, lead to a description of the subcellular dynamics of neuronal signalling, and contribute to our basic understanding of the nervous system.

As our understanding of the nervous system increases, the questions posed by neuroscientists become more complex and require more sophisticated analytical schemes to answer them.. A major challenge of contemporary neurobiology is to understand the cellular mechanisms responsible for neurotransmitter targeting and release. Essential to an understanding of neurotransmitter release is quantitative knowledge of the amounts and locations of neuropeptides present in the neuron. The methods currently employed for the assay of small molecules are not sensitive enough to quantitate the neuropeptides found within small subsections of individual neurons nor to detect the release of neurotransmitters from a single electrical stimulation. The long term objective of this research program is develop and demonstrate new analytical instrumentation and methodology to allow the identification and quantitation of neuronal releasates from a single nerve terminal, and measurement of the contents of individual varicosities along a single nerve process. The approach used to accomplish these goals is capillary electrophoresis followed by radiochemical detection. Once the instrumentation and methodology is in place, individual cultured neurons of the giant marine mollusk Aplysia californica will be studied. These studies will answer the questions: do the neurons target different neuropeptides to specific release sites, and can the neuron release different neuropeptides at specific terminals? By using the latest advances in separation science and detector technology, significant gains can be made in our understanding of the differential packing, distribution, and release of neurotransmitters in Aplysia. In leading to a description of the subcellular dynamics of neuronal signalling, this work will contribute to the basic understanding of the nervous system. The symptoms of many mental disorders suggest that an imbalance of chemical messengers may be responsible for the disease state. By answering questions of neurotransmitter targeting and release, we will gain further insight into how complex systems of neurons interact in both healthy and diseased systems.

As our understanding of the nervous system increases, the questions posed by neuroscientists become more complex and require more sophisticated analytical schemes to answer them. A major challenge of contemporary neurobiology is to understand the cellular mechanisms responsible for neurotransmitter targeting and release. Essential to an understanding of neurotransmitter release is quantitative knowledge of the amounts and locations of neuropeptides present in the neuron. The methods currently employed for the assay of small molecules are not sensitive enough to quantitate the neuropeptides found within small subsections of individual neurons nor to detect the release of neurotransmitters from a single electrical stimulation. The long term objective of this research program is to develop and implement new analytical instrumentation and methodology to allow the identification and quantitation of the substances released from a single nerve terminal, and the measurement of the contents of individual varicosities along a single nerve process. The approach used to accomplish these goals is capillary electrophoresis followed by a unique multichannel laser-induced fluorescence detection system. Once the instrumentation and methodology is in place, neurotransmitter distribution and release will be studied using several different model systems including several identified neurons from the marine mollusks Aplysia californica and Pleurobranchaea californica and isolated nerve terminals from the cortex of the rat. These studies will answer the questions: do the neurons target different neurotransmitters to specific release sites, and can the neuron release different neurotransmitters at specific terminals? By using the advances in separation science and detector technology developed as part of this research, significant gains can be made in our understanding of the differential packing, distribution, and release of neurotransmitters. In leading to a description of the subcellular dynamics of neuronal signalling, this work will contribute to the basic understanding of the nervous system. The symptoms of many mental disorders suggest that an imbalance of chemical messengers may be responsible for the disease state. By answering questions of neurotransmitter targeting and release, we will gain further insight into how complex systems of neurons interact in both healthy and diseased systems.

DESCRIPTION: The combination of NMR and a separation method provide unmatched structural elucidation capabilities, based primarily on the wealth of chemical information that NMR provides. Unfortunately NMR is an inherently insensitive technique and thus requires large sample masses. The overall goal of this proposal is to improve the mass sensitivity of NMR by two orders of magnitude so that 5-500 nL volumes and picomole masses can be analyzed. The advance that enable such sensitivity improvements is development of miniaturized radiofrequency coils for signal detection in NMR. As the coil diameter is reduced in size it provides much higher signal-to-noise ratios for a given mass of sample. A major portion of this work is the optimization of coil fabrication and design geometry. The nanoliter volume NMR detector cells will be coupled to microseparations. Specifically, optimized flow cells, acquisition parameters, separation modes and preconcentration methods will be developed for capillary electrophoresis and capillary liquid chromatography. In addition to hardware development this proposal seeks to employ to nanoliter NMR method to study the mass limited, but high concentration environment of the interior of a series of peptide containing vesicles from the marine mollusk, Aplysia californica.

DESCRIPTION (provided by applicant): As our understanding of the nervous system increases, the questions posed by neuroscientists become more complex and require more sophisticated analytical schemes to answer them. A major challenge of contemporary neurobiology is to understand the mechanisms of cellular communication. Essential to an understanding of how neurons communicate is complete information concerning the neurotransmitters and neuropeptides present in and released from individual neurons. The methods currently employed for the assay of such molecules are not sensitive enough to quantitate the neuropeptides found within small subsections of individual neurons nor to detect the release of neuropeptides from a single neuron under most conditions. The goals of this proposal are to develop the analytical instrumentation and methodology capable of identifying and quantifying neuropeptides from single cells or cellular processes, and to use the model neuronal system Aplysia californica both to test these new methods, as well as to elucidate fundamental aspects of peptidergic transmission. Two approaches will be used - matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary electrophoresis. A significant portion of the research involves improving sampling techniques to allow mass spectrometry to study small neurons, sections of neurons and the release of material from specific cellular regions. In addition, the biological activity of a number of novel neuropeptides will be characterized and new neuropeptides identified. By using the advances in separation science and mass spectrometry developed as part of this research, significant gains can be made in our understanding of the processing, distribution, and release of intercellular signaling molecules. In leading to a description of the subcellular dynamics of neuronal signaling, this work will contribute to the basic understanding of the nervous system.

An important capability required by many biotechnology processes is the ability to separate specific chemicals from complex biological samples and identify, quantitate and manipulate them. As a trend in biotechnology over the last decade has been the ability to work with ever small and chemically more complex samples, separation techniques such as capillary electrophoresis (CE) have been developed that work with nanoliter to picoliter volumes. However, the ability to collect and manipulate the output of small-volume separations has not kept pace with the miniaturized separation itself. The overall goal of this proposal is to develop and interface molecular gates with CE. These molecular gates will have the ability to "capture" selected analytes either on-column or existing from the separation capillary for additional manipulation and analyses such as mass spectrometry and NMR. In essence, the molecular gate converts a nanoliter volume separation technique from an analytical method to a preparative method that allows manipulation of individual selected analytes. The proposed systems consists of a CE system with an imaging fluorescence detection to detect the analytes on-column, thus allowing the molecular gate to be turned on at the appropriate times to collect specific molecules. The performance of the system initially will be characterized using GABA, peptides, and DNA as model analytes. In addition to developing the CE/gate system, the capability to demount the gate and subject the analytes to assays such as mass spectrometry, nanoliter volume NMR, and PCR analysis will be developed. The ability to manipulate and identify ultra-small amounts of diagnostically useful compounds will be a significant advance for the biomedical community.

technology /technique development; tissue /cell culture

This project, carried out by Professor J. Sweedler of the University of Illinois, and supported by the Analytical and Surface Chemistry Program, will develop the analytical instrumentation and methodology capable of identifying and quantifying neuropeptides from single cells, and those neuropeptides involved in cellular processes. The model system Aplysia californica is used as a test bed to demonstrate these new and improved capabilities, and to elucidate the fundamental aspects of peptidergic transmission. Matrix-assisted laser desorption ionization mass spectrometry is used to image intact ganglion sections with molecular specificity and subcellular resolution. Capillary electrophoretic methods are used to analyze sub-nanoliter volumes of samples derived from these systems. Electrospray ionization mass spectrometry is used to monitor peptide releases from a single cell under precisely controlled stimulation conditions.

Professor Sweedler will use advanced analytical methods to identify and quantify the peptides released from individual nerve cells on stimulation, and to image the distribution of these compounds within the cell itself. Signal transmission between cells depends on the release and uptake of these small peptides. Careful study of the model system will provide, for the first time, a complete understanding of the fundamentals of nerve impulse transmission. This example can then be used as a starting point for the study of nerve impulse transmission in more complex organisms.

Nuclear magnetic resonance (NMR) spectroscopy is an analytical method that provides a wealth of chemical and structural information from complex biological samples. Unfortunately, NMR is an inherently insensitive technique which requires larger sample amounts than other chemical characterization methods. The overall goal of this proposal is to continue nanoliter-volume NMR probe development so that 5 - 1000 nanoliter volumes and picomole masses can be analyzed. The fundamental advance that enables such sensitivity' improvements is miniaturized radio- frequency coils for NMR signal detection. A major portion of this work involves the optimization of coil geometry and fabrication to maximize sensitivity and minimize spectral linewidth, to add a broad range of heteronuclear NMR capabilities, and to develop multiple microcoil probes. Probes with up to 16 microcoils will dramatically improve NMR throughput. The combination of NMR and separation methods provides unmatched structural elucidation capabilities. Specifically, optimized flow cells, acquisition parameters, separation modes and preconcentration methods will be developed for capillary electrophoresis and capillary liquid chromatography. A unique series of on-line NMR techniques will be developed to monitor the separation process including on-line temperature, flow rate and imaging techniques. The implementation of this technology greatly expands biological applications where mass limitations currently prevent NMR structural determinations. Specifically, diffusion-ordered NMR will be used to obtain quantitative information on the diffusion rates in specific populations of cellular organelles from a series of model cells from the marine mollusk Aplysia californica. Secondly, single cell NMR spectroscopy will be developed for identified neurons in Aplysia californica which will allow the major osmolytes and cytoplasmic and nuclear components to be measured in intact neurons, as well as the physico-chemical environment of these compartments. Thus, the nanoliter- volume NMR probes developed during this research will offer significantly improved mass sensitivity for a widening range of NMR analysis, enable NMR to be used with a variety of microseparation methods and allow a new range of biological applications.

[unreadable] DESCRIPTION (provided by applicant): An important goal of drug abuse research is to detect and characterize changes in the biochemistry of the nervous system on upon exposure to drugs of abuse, and to relate these changes to higher neuronal functions in normal and pathological conditions. Amongst numerous signaling molecules, intercellular signaling peptides (ISPs) are a dramatic measurement challenge; there are literally hundreds of peptides expressed in biological systems, many of which are only physiologically active when posttranslationally modified. The proposed research both develops and implements matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) based technologies for the discovery and characterization of ISPs, including determination of their chemical form and spatial localization in the mammalian nervous system. The overall strategy is to develop new technologies and sampling protocols to detect and identify new ISPs in very small biological samples, including individual freshly prepared or cultured single mammalian cells, small groups of cells, and those released from spatially restricted regions of the nervous system. These technologies promise to uncover hundreds of potential ISPs that will be filtered for functional relevance by studies designed to: (A) find peptides with specific post- translational modifications (e.g., amidation); (B) determine the peptides that undergo long- distance transport in the nervous system; (C) discover which peptides are released in an activity-dependent manner. This technology development ideally matches the cutting-edge basic research award solicitation. By developing powerful new proteomics-based technologies to answer questions about the most complex and elusive set of neuromodulators, the neuropeptides, basic insight into the effects of drugs of abuse on the CNS will be gained. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The development of innovative measurement tools can provide new information on brain function, often allowing a range of novel questions to be addressed. Proteomics is one such exciting new tool. NIDA called for the establishment of neuroproteomics centers with several major objectives, including: (1) to provide neurobiologists with the ability to benefit from proteomics experiments, (2) to build a cadre of proteomics experts who will develop expertise in analyzing neural tissues, and (3) to develop new or improve existing proteomics technologies as they relate to neurobiology or tissues of the nervous system. The UIUC Neuroproteomics Center on Cell-Cell Signaling addresses these three key areas. The Center specifically provides peptidomics, proteomics and bioinformatics technologies to the UIUC and national neuroscience communities while simultaneously advancing the performance of state-of-the-art proteomics technologies to new levels of performance. The Center is built around the overarching theme of cell-cell signaling. Extracellular signaling peptides and proteins-neuropeptides, trophic factors, cytokines, and hormones-represent a critical part of the cell proteome that has been implicated in almost all aspects of organism function;they influence behavior, learning and memory, and addiction phenomena. The Center is divided into three scientific cores: the Sampling and Separation Core, the Protein Identification Core, and the Bioinformatics Core, plus the Administrative and Users Cores. There are twelve major users representing 28 individual grants, with their Center research projects concentrating on three overarching scientific thrusts: (1) signaling peptide discovery, (2) relating peptide signaling to function, and (3) non-traditional aspects of cell-cell communication, including glia to neuron signaling and dendritic/axonal RNA transport. The high level of synergy between the neuroscientists and technologists ensures progress in this broad suite of projects, and offers tremendous promise for advancing our knowledge of how systems of neurons interact in both the healthy nervous system and on exposure to drugs of abuse. CENTER CHARACTERISTICS

DESCRIPTION (provided by applicant): In the post human genomic era, genetic information and functional assays of gene expression have become indispensable for a significant fraction of NIH-funded research. We are requesting funds to purchase two automated capillary sequencers to improve sequencing operations provided by the University of Illinois at Urbana-Champaign's W. M. Keck Center for Comparative and Functional Genomics. New sequencers have greatly improved throughput, reliability, performance, and are operated at greatly reduced costs for labor, consumables, and maintenance. These cost savings will be reflected in lower sequencing costs for users, stretching NIH-funded lab's budgets, and producing shorter turnaround times and higher quality data. Also, as the cost of operating and maintaining aging sequencers increases, upgrading to more efficient and economical sequencers becomes essential. With the funding of several NIH-funded functional genomics projects at the UIUC, the continuous increase in sequencing services performed since the establishment of the Keck Center, and the major expansion of campus biomedical researchers with the establishment of the UIUC Post-Genomic Institute, large increases in our sequencing needs are occurring. The Keck Center's High Throughput Sequencing and Genotyping lab is part of an integrated genomics facility providing comprehensive, state-of-the-art instrumentation, consultation, and training opportunities for researchers, and occupies a unique niche not provided by large sequencing centers.

DESCRIPTION (provided by applicant): Understanding brain function in healthy and diseased brains requires an understanding of the biochemistry occurring within the neurons and supporting cells making up the brain. It is well known that individual cells in the brain have distinct signaling molecule complements and protein complement, but the effects on the cellular metabolome are much less well understood. What is the cell to cell variation of the cellular metabolome in the mammalian CNS? For most metabolites, the answer is unknown. Using invertebrate models, large differences in the metabolome of adjacent neurons have been measured such as millimolar levels of nitrate in nitrergic neurons with no detectable nitrate in non-NO producing neurons. Similarly, large changes in the amino acid complement occur depending on the signaling molecules used by the neuron. Unfortunately, technology limitations do not currently allow the major metabolites to be measured within individual mammalian neurons. A suite of technology development and hyphenated approaches are proposed to create instruments and protocols to measure the metabolites in neuronal clusters, groups of neurons and even individual neurons. These development efforts include unique sampling protocols, microfluidically-based sample conditioning unit with integrated electrophoretic separations, followed by native fluorescence and mass spectrometric detection, and where necessary, capture of the appropriate metabolites into nanolitervolume capillaries for nanoliter volume NMR spectroscopic characterization. This unique set of technology promises to open up a new volume regime to profile the cell to cell variations in the metabolites. The technology will be tailored to and validated on the most heterogeneous samples known - the mammalian brain.

The diversity and complex regulation of intercellular signaling protein (ISP) networks in the biochemical makeup of heterogeneous nervous tissues presents a formidable challenge in the investigation Of the cell-to-cell signaling roles in sophisticated behavioral phenomena such as addiction. To address this issue, a core facility dedicated to customizing sampling and separation methods to profile the ISPs in single cells, specific brain regions, and extracellular fluids will be established within the proposed UIUC center. These technologies wilt be applied to ISP profiles within the nervous system in the study of fundamental neuroscience as well as pathological conditions such as drug-of-abuse-induced changes. To profile ISP in specific brain tissues and cells this Core will specialize in: 1.) collecting and fractionating biological samples including simultaneous preparation of large numbers of rapidly acquired, nanoliter volume biological samples; 2.) applying directed sampling methodologies to single cells, spatially distinct brain regions, and biological fluids to collect ISPs; and 3.) optimizing preparation of fractionated samples and tissues for mass spectrometry including imaging MS approaches. Established techniques such as 2-D electrophoretic separations as well as state-of-the-art sampling, separation, and quantitative approaches will be used to prepare a large range of ISP-containing fractions ready for MS analysis at the Protein Identification Core. This overarching strategy provides the flexibility to individually tailor isolation and detection methodology for ISP profiles inherently unique to the neurobiological phenomena investigated by the center's biological user base.

The goals of the Administrative Core are clearly distinct from those of the scientific cores. Its major tasks are to organize, support, and manage the UIUC Neuroproteomics Center on Cell-Cell Signaling. Primary activities include resource management, education and outreach, and datasharing oversight. The Administrative Core brings together the individual core Pis and the biological users with the Center's internal advisory committee and external advisory board. The first area described is the Center's organization and operational plans, with an emphasis on facilitating communication between Center personnel and the users. After all, the goal is to establish a logically interconnected neuroproteomics center and not simply to manage separate projects undertaken by individual investigators. Successful and continuing efforts to acquire and upgrade our measurement infrastructure are described. In addition, efforts in outreach and training, both for users of the Center and for the entire neuroscience and drug abuse research communities are highlighted. We also present an overview of the UIUC Center and the relationship between the cores. Several neuroproteomics centers were initiated four years ago by NIDA with the stated goals to provide neurobiologists with the ability to (1) benefit from proteomics experiments, (2) build a cadre of proteomics experts who will develop expertise in analyzing neural samples, and (3) develop and improve existing technologies as they relate to neurobiology. The Administrative Core is the cohesive force that binds all three of these efforts in the UIUC Neuroproteomics Center on Cell-Cell Signaling.

Professor Jonathan Sweedler of the U of Ill Urbana-Champaign is supported by the Analytical and Surface Chemistry Program to analyze a chiral post-translational modification in neuropeptides, namely, an L to D isomerization thought to activate the peptide biologically. The biological system under study is the single neuron of Aplysia californica. There are three proposed approaches: instrumental, enzymatic and immunological. In the first, chiral separation methods will be used to try to resolve diastereomeric peptides that differ by D/L isomerization of one of the amino acids. Secondly, N-terminal peptidases that cleave only L-amino acids will be used followed by analysis of fragments by matrix-assisted laser desorption ionization mass spectrometry (MALDI). A third approach is to attempt to grow antibodies to peptides containing D-amino acids. The goal is to discover new D-amino acid containing peptides that function neurologically. Students in this laboratory are trained in analytical chemistry and neurobiology.

Signaling in the brain and nervous system is accomplished to a large extent by a vast number of peptides, many as yet undiscovered. The well studied Aplysia snail system gives researchers one model system that a great deal is already known about. Success of this project will allow a better understanding of peptides in neurochemistry, as well as in neurological diseases such as Alzheimer's, and will also contribute to advances in analytical methods for other questions in biology and pharmacy.

DESCRIPTION (provided by applicant): Understanding brain function in healthy and diseased brains requires an understanding of the biochemistry occurring within the neurons and supporting cells making up the brain. It is well known that individual cells in the brain have distinct signaling molecule complements and protein complement, but the effects on the cellular metabolome are much less well understood. What is the cell to cell variation of the cellular metabolome in the mammalian CNS? For most metabolites, the answer is unknown. Using invertebrate models, large differences in the metabolome of adjacent neurons have been measured such as millimolar levels of nitrate in nitrergic neurons with no detectable nitrate in non-NO producing neurons. Similarly, large changes in the amino acid complement occur depending on the signaling molecules used by the neuron. Unfortunately, technology limitations do not currently allow the major metabolites to be measured within individual mammalian neurons. A suite of technology development and hyphenated approaches are proposed to create instruments and protocols to measure the metabolites in neuronal clusters, groups of neurons and even individual neurons. These development efforts include unique sampling protocols, microfluidically-based sample conditioning unit with integrated electrophoretic separations, followed by native fluorescence and mass spectrometric detection, and where necessary, capture of the appropriate metabolites into nanolitervolume capillaries for nanoliter volume NMR spectroscopic characterization. This unique set of technology promises to open up a new volume regime to profile the cell to cell variations in the metabolites. The technology will be tailored to and validated on the most heterogeneous samples known - the mammalian brain.

Irving R. Epstein (Brandeis University), Rustem F. Ismagilov (University of Chicago), Anna Lin (Duke University) and Jonathan Sweedler (University of Chicago) are jointly supported to study the chemical processes involved in glial-neuronal signaling. The collaborative project includes fabrication of topologically well-defined networks of microchannels to support neuronal and neuron-glial networks, time-resolved imaging of the calcium levels in both neurons and glial cells of rat hippocampus cultures, specific stimulation of spatially and temporally defined glial cells using microfluidic tools, advanced data analysis and modeling.

Recent research suggests that glial cells are actively involved in chemical communication with and between neurons and other glia. Goals of this project include a better understanding of the role of glial cells in individual synapses and in neural networks, elucidation of the interaction between the network topology and the dynamics of individual elements (cells), and the development and evaluation of methods that can be applied to the analysis of complex chemical and biological networks. This project is funded through the Collaborative Research in Chemistry Program (CRC) and provides outstanding opportunities for undergraduate, graduate and postdoctoral students to acquire knowledge and skills in neurochemistry and bioanalytical chemistry.

DESCRIPTION (provided by applicant): Understanding brain function in healthy and diseased brains requires an understanding of the biochemistry occurring within the neurons and supporting cells making up the brain. It is well known that individual cells in the brain have distinct signaling molecule complements and protein complement, but the effects on the cellular metabolome are much less well understood. What is the cell to cell variation of the cellular metabolome in the mammalian CNS? For most metabolites, the answer is unknown. Using invertebrate models, large differences in the metabolome of adjacent neurons have been measured such as millimolar levels of nitrate in nitrergic neurons with no detectable nitrate in non-NO producing neurons. Similarly, large changes in the amino acid complement occur depending on the signaling molecules used by the neuron. Unfortunately, technology limitations do not currently allow the major metabolites to be measured within individual mammalian neurons. A suite of technology development and hyphenated approaches are proposed to create instruments and protocols to measure the metabolites in neuronal clusters, groups of neurons and even individual neurons. These development efforts include unique sampling protocols, microfluidically-based sample conditioning unit with integrated electrophoretic separations, followed by native fluorescence and mass spectrometric detection, and where necessary, capture of the appropriate metabolites into nanolitervolume capillaries for nanoliter volume NMR spectroscopic characterization. This unique set of technology promises to open up a new volume regime to profile the cell to cell variations in the metabolites. The technology will be tailored to and validated on the most heterogeneous samples known - the mammalian brain.

[unreadable] DESCRIPTION (provided by applicant): An important goal of drug abuse research is to detect and characterize changes in the biochemistry of the nervous system on upon exposure to drugs of abuse, and to relate these changes to higher neuronal functions in normal and pathological conditions. Amongst numerous signaling molecules, intercellular signaling peptides (ISPs) are a dramatic measurement challenge; there are literally hundreds of peptides expressed in biological systems, many of which are only physiologically active when posttranslationally modified. The proposed research both develops and implements matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) based technologies for the discovery and characterization of ISPs, including determination of their chemical form and spatial localization in the mammalian nervous system. The overall strategy is to develop new technologies and sampling protocols to detect and identify new ISPs in very small biological samples, including individual freshly prepared or cultured single mammalian cells, small groups of cells, and those released from spatially restricted regions of the nervous system. These technologies promise to uncover hundreds of potential ISPs that will be filtered for functional relevance by studies designed to: (A) find peptides with specific post- translational modifications (e.g., amidation); (B) determine the peptides that undergo long- distance transport in the nervous system; (C) discover which peptides are released in an activity-dependent manner. This technology development ideally matches the cutting-edge basic research award solicitation. By developing powerful new proteomics-based technologies to answer questions about the most complex and elusive set of neuromodulators, the neuropeptides, basic insight into the effects of drugs of abuse on the CNS will be gained. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Understanding the healthy and the diseased brain requires knowledge of the biochemistry occurring within the neurons and supporting cells that make up our brain. It is well known that individual cells in the nervous system have distinct protein complements, but the consequences of this heterogeneity on the cellular metabolome are much less well understood. While neurotransmitters used in a network vary neuron-by-neuron, what are the cell-to-cell variations of the metabolome in the mammalian nervous system? For most metabolites, the answer is unknown. Using a suite of analytical technologies based on small-volume sampling, capillary electrophoresis with laser- induced fluorescence detection, single cell mass spectrometry, and capillary separations coupled to Fourier-transform mass spectrometry, the neurometabolome of a network of dorsal root ganglion (DRG) sensory neurons will be studied with unmatched chemical detail. DRG neurons are involved in the mechanisms of pain - one of most devastating side effects of many disorders. The neurotransmitters and metabolites in individual neurons and their functional neuronal compartments such as dendrites, axons, and cell bodies, will be characterized. The first stage of this work is to compile information on the metabolome of the DRG neurons, and examine this for unusual molecules. Next, using the DRG and its neurometabolome as the model, several specific questions are addressed: (1) Does the neurometabolome of functionally distinct neuronal compartments differ? (2) What subset of the small-molecule complement is released upon electrical or chemical stimulation, and thus may participate in cell-to-cell communication? (3) How does inflammation- induced pain change the neurometabolome of the DRG neurons? These efforts fit within the scope of the metabolomics roadmap initiative by developing innovative methodologies to investigate neurotransmitter and other small molecule compartmentalization on the function of a model sensory neuron network. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A major challenge of contemporary neurobiology is our incomplete understanding of the mechanisms and plasticity involved in cell-to-cell communication. Determining how neurons communicate with other neurons within a network requires complete information about the neurotransmitters and neuropeptides present in and released from individual neurons. This proposal uses the well-known animal model Aplysia californica, with its well-defined neuronal networks, to characterize the suite of molecules used in individual neurons, as well as their release, in an activity dependent manner. By taking advantage of new analytical tools that allow single neurons and neuronal subcompartments to be assayed for their chemical constituents, combined with the data becoming available as part of the Aplysia genome and transcriptome projects, a nearly complete list of signaling molecules will be characterized from specific identified neurons involved in important physiological functions. For the classical transmitters, capillary electrophoresis with several selective detection schemes (ranging from radionuclide detection to native fluorescence) will be used. In particular, significant efforts will determine the roles of the unusual amino acids, d-glutamate and d-aspartate, in neurotransmission. While prior work has demonstrated that in specific neurons, these molecules are synthesized from their L-amino acid counterparts, transported to release zones and likely released, here the details of their functional roles in cell-to-cell signaling will be explored. In addition, the complete set of peptides (the peptidome) used in these neuronal networks will be characterized;the methods to be used include single cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, a variety of small volume electrospray mass spectrometric approaches, and several bioinformatics approaches. The outcome of this work will be a well-defined neurochemistry to complement the well-known physiology and behavior in the neuronal networks of Aplysia. By using the advances in separation science and mass spectrometry, significant gains can be made in our understanding of the synthesis, posttranslational processing, distribution, release and function of known and novel cell-to-cell signaling compounds. In leading to a description of the subcellular dynamics of neuronal signaling, which plays a crucial role in coordinating neuronal network activities, this work will contribute to furthering our basic understanding of the nervous system.

The Bioinformatics Core will work closely with the major users by providing a customized laboratory sample tracking system, an efficient and quick data pipeline, a tailored project management system, and a web portal to facilitate sharing of experimental results. The Core will convert the data acquired from a range of mass spectrometric platforms into qualitative and quantitative information to address the questions posed by our users. Besides facilitating the analysis of the data for the users using a range of commercial software, several new bioinformatics tools will be created to benefit both local users and the broader neuroscience communities. We will continue the development of NeuroPred, a currently available web-based tool that predicts prohormone cleavages and the resulting signaling peptides. This predictor provides a valuable link between genetic information coding for the protein prohormones and the peptide products one observes. NeuroProSightPTM is another important bioinformatics tool that identifies post-translational modifications in intact proteins from "top-down" data analysis of absolute masses. The top-down approach will enable investigators to use absolute masses of intact proteins to identify neuropeptides, cytokines, hormones, and other neuron-specific proteins and peptides. Lastly, a unique method of shot-gun peptidomics that combines bioinformatics tools and high accuracy mass measurements will enhance peptide characterization and identification, and will be used across a range of important neuroscience models. Together, the well-planned experiments, existing tools, and new bioinformatics capabilities will create novel approaches and new data on the intercellular signaling molecules found in the brain.

The overall goal of the Protein Identification Core of the UIUC Neuroproteomics Center on Cell-Cell Signaling is to characterize the protein and peptide complements of a range of samples, with a special emphasis on those involved in intercellular signaling. The intracellular signaling molecules expressed in the cells and tissues under investigation present an extremely complex analytical challenge with a vast number of components, varying dramatically in size and concentration. To address these challenges, and to provide our users with a battery of state-of-the-art protein identification techniques, a range of mass spectrometric approaches are used, including differential gel electrophoresis, mass fingerprinting, tandem sequencing, accurate mass measurements, and top-down intact protein analysis. In addition, spatial localization of intercellular signaling molecules is performed using a variety of mass spectrometric imaging techniques. Our user's needs for intercellular signaling molecule identification and quantitation in the brain are quite different from what is required from proteomics measurements of unicellular organisms and homogenous tissues, particularly with regard to sample size and complexity. Thus, the advanced measurement strategies used in our Center reflect this complexity. With the combined expertise and facilities in the research groups associated with this core, a unique opportunity exists to identify and characterize intercellular signaling peptides and proteins across a surprising range of models and scales.

UNIVERSITY OF ILLINOIS The University of Illinois at Urbana-Champaign has one of the leading Chemistry Departments in the world and combines this with two interdisciplinary research institutes with expertise in bioengineering and neuroscience, with these multidisciplinary programs enabling many our Core's research projects. More specifically, Sweedler is a member of the faculty of the School of Chemical Sciences (SOS), the Institute for Genomic Biology (IGB), and the Beckman Institute for Advanced Science and Technology (BI) at the University of Illinois at Urbana-Champaign (UIUC). The facilities in these units offer strong support for the research described in the proposal.

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, and co-funding from the Instrument Development for Biological Research Program in the Division of Biological Infrastructure, Prof. Jonathan Sweedler and his group at the University of Illinois at Urbana-Champaign are developing a suite of measurement approaches to characterize d-amino acids within individual cells and other small-volume domains within the nervous systems of multiple animals. Using capillary electrophoresis for separation and fluorescence and mass spectrometry for detection, along with several unique high-throughput cell sampling devices, information on the presence of d-proline, d-glutamate and d-alanine is being determined. In addition to development of new enzymatic imaging approaches and single cell characterization measurements, application to several carefully selected animal models will provide information relevant to the function of the d-amino acids throughout Metazoan life.

This research project not only promises to train students to be analytical chemists/measurement scientists, it also provides them with significant education and research experience in the biological sciences. Undergraduate and graduate students with diverse backgrounds, including many women scientists, will be actively involved and mentored in these interdisciplinary efforts. The broad-based dissemination of the research through publications, conferences, outreach, and training that includes undergraduates and graduates from multiple disciplines assures that this research advances our country's needs in science education and research.

DESCRIPTION (provided by applicant): The three-dimensional structure of a cell-cell signaling peptide determines how it interacts with its cognate receptor and with various degradation enzymes. An enigmatic, poorly understood peptide modification-the enzymatic epimerization of a single amino acid residue-results in the formation of a D-amino acid-containing peptide (DAACP). Despite significant progress in DAACP characterization, discovery efforts are hampered by shortfalls in current technology. Why do we think there are unknown DAACPs? More than 30 have been reported in a surprising range of animals and organs including the brain; in mammals an undetermined enzyme activity converts several all-L-amino acid-containing peptides into DAACPs, and a unique aminopeptidase in nervous tissue is capable of degrading DAACPs. Accordingly, we hypothesize that DAACPs are widely present in nervous and endocrine tissues throughout the Metazoa. The overarching goal is to characterize the DAACP peptidome and determine the function of the newly uncovered DAACPs. We will create a comprehensive three-stage DAACP discovery funnel (Aim 1): (1) putative DAACP candidates are identified without the need for peptide standards; (2) the presence of the D-amino acid in the putative DAACP is confirmed; (3) using appropriate peptide standards and semi-purified peptides, the DAACPs are sequenced for absolute confirmation. Specifically, the funnel consists of linked analytical approaches: separation and characterization of isobaric peptides, analyses of peptides resistant to enzymatic digestion, separation/digestion of the peptides into their component amino acids for characterization via multiple reaction monitoring and chiral amino acid capillary electrophoresis, and finally, chiral tandem mass spectrometric peptide sequencing hyphenated to in silico structure determination. After funnel optimization, several model organisms will be used in the discovery phase: the exceptional neurophysiological model Aplysia californica with its known and putative DAACPs, and the regenerative planarian model with its ease of genetic manipulations (Aim 2); followed by experiments in mice and rat endocrine and selected nervous tissues (Aim 3). Most signaling peptide systems are ancient and well conserved; it appears that DAACPs are also. These comparative studies allow common biochemical pathways and physiologies to guide subsequent peptide bioactivity tests. Characterizing unknown DAACPs, mapping them to specific locations, correlating their levels to an animal's physiological state, and even electrophysiological testing combines to provide unparalleled information on their function (Aim 4). These research efforts are timely given the wealth of genomic and peptidome information available for our selected animal models and the recent discovery of enzymatic and biochemical data suggesting the presence of DAACPs. The discovery of new signaling DAACPs will improve our understanding of the functioning of the nervous and endocrine systems, and the discovery funnel will have application to a range of fundamental and applied investigations.

DESCRIPTION (provided by applicant): Glia comprises the majority of the cells that make up the central nervous system. These cells are integral to maintaining normal cellular activity and have been implicated in the development of many pathological conditions. The functional, positional, and biochemical heterogeneities of neurons are crucial aspects in regulating a myriad of physiological activities within the larger neuronal context; glia likely have similar localized and individual cell diversity. Here we propose to develop a unique mass spectrometry (MS)-based toolset combined with imaging to facilitate the discovery of the metabolome of thousands of individual glia. A key objective is the multifaceted investigation of multiple individual glia isolated from three specific brain regions in mouse, the hippocampus, cerebellum and brainstem, followed by transcriptional biomarker-guided glia sub-type identification. The technology allows the same cell to be characterized by its morphology, metabolic profile, and distinctive transcriptomic expression profile. Integrative analyses of the resulting data enable a unique determination of the molecular basis for glia heterogeneity. We expect to uncover characteristic metabolite biomarkers and biomarker patterns for glia subtypes. Our armamentarium includes a variety of MS technologies, including secondary ion MS and matrix-assisted laser desorption/ionization MS, as well as in-situ hybridization guided cell identificatio. Besides tool development, glia from 4- and 56-day old C57BL/6J mice will be characterized, and the results correlated to data assembled in the Allen mouse brain atlas and referenced in the Neuroscience Information Framework. This study will also reveal age- related differences in glia heterogeneity. When unknown metabolites are detected, we will characterize them via off-line approaches such as capillary electrophoresis hyphenated to MS. This innovative integration of single cell MS characterization approaches provides the capability to target individual glial cells and characterize them. These efforts are well matched to the goals of RFA-HD-12-211 Tools to Enhance Studies of Glial Cell Development, Aging, Disease and Repair. The approaches are general and adaptable to a range of glial cell types. The data obtained and the technology suite created will be used by laboratories and research groups that engage in clinical diagnostic measurements, fundamental scientific investigations, and pharmaceutical discovery.

The development of innovative measurement and analysis tools enables new information on brain function, often allowing a range of novel questions to be addressed. Rapidly evolving metabolomics, peptidomics and proteomics tools facilitate new findings in both discovery and targeted modes. The Neuroproteomics and Neurometabolomics Center on Cell-Cell Signaling provides high-end 'omics-scale characterization of the small molecules, peptides and proteins for samples obtained from brain sub-regions like defined nuclei and even specific single cells. Our sampling methods allow molecular localization via discrete cell isolation, mass spectrometry imaging, measurement of activity dependent release, and quantitation of level changes as a function of exposure to drugs. We then characterize the most important molecular targets in these samples using metabolomics, peptidomics and proteomics via a broad array of mass spectrometry-based technologies. Finally, we provide the critical expertise for capturing the value of data via expert bioinformatics support that integrates disparate data types, develops advanced analytical approaches for complex metabolomics and proteomic experiments, and provides community support through several web platforms. At the beginning of the next granting period, we will be supporting an initial group of 17 major users representing 23 separately funded research projects across the fields of neuroscience, including projects targeting neuropeptides, transmitters and proteins that are involved in multiple aspects of drug escalation, exposure and addiction. We also will address fundamental questions of neuron/glia communication, dendritic protein expression and neuronal plasticity. The Neuroproteomics and Neurometabolomics Center on Cell- Cell Signaling is divided into three scientific cores: Sampling and Separation, Molecular Profiling and Characterization, and Bioinformatics and Systems Biology (plus an Administrative Core). The high level of synergy between the neuroscientists and technologists affiliated with the Center ensures progress in our broad suite of supported research projects, and promises continued advancements in the knowledge of how systems of neurons interact in both the healthy nervous system and upon exposure to drugs of abuse Lastly, a series of outreach initiatives assures that our protocols and approaches are broadly available to the appropriate scientific communities.

FACILITIES, EQUIPMENT AND OTHER RESOURCES BIOINFORMATICS AND SYSTEMS BIOLOGY CORE UNIVERSITY OF ILLINOIS Rodriguez Zas is a faculty member in the Department of Animal Sciences and Department of Statistics, as well as an affiliate of the Institute for Genomic Biology (IGB), the Center for Advanced Study, and the UIUC Neuroscience Program. The facilities in these units and the UIUC library (the nation's largest state university library) will be available to the interdisciplinary research described in the proposal. For more information on these facilities please visit http://wvwv.ansci.illinois.edu, http://www.igb.illinois.edu, and http://www.stat.illinois.edu. The University of Illinois is home to the National Center for Supercomputing Applications (http://www.ncsa.illinois.edu/) and Blue Waters supercomputer infrastructure (http://www.ncsa.illinois.edu/BlueWaters/), which are made available to local researchers. Laboratory and Office: Rodriguez Zas' bioinformatics laboratories are housed in three rooms totaling 600 sq. ft in the Animal Sciences Laboratory at the University of Illinois at Urbana-Champaign. These labs house multiple dry lab resources and computer equipment, and benefit from the extensive infrastructure of the University of Illinois at Urbana- Champaign. A state-of-the-art computer network is maintained across campus and provides distributed data storage, automated backup, library access, and shared software. Desk-space at Dr. Rodriguez Zas' lab is available for the personnel in this project. Rodriguez Zas has an office adjacent to the labs

This award is being made jointly by the Neural Systems Cluster in the Division of Integrative and Organismal Systems and the Instrument Development for Biological Research program (IDBR) in the Division of Biological Infrastructure.

Understanding how the brain enables us to think, act, learn, and remember is challenging. Progress has been impeded by lack of a dynamic picture of interactions and properties that emerge when tiers of interconnected brain cells (neurons) are activated in response to experiences. These interactions cause changes in our behaviors and can affect subsequent activities of these neurons, a process called plasticity. This proposal will develop and use newly created, complementary technologies that will non-invasively control, measure, and analyze brain network dynamics and change in real time. Neuroscientists, engineers, and chemists from the University of Illinois at Urbana-Champaign will work together, each bringing cutting-edge methods to bear on this problem. Approaches include: 1) analyzing slices of brain tissue that maintain dynamic properties in a dish; 2) real-time, label-free imaging of neuron activity by novel optical methods; 3) activating and measuring neuronal activity with flexible, clear electrodes that interface directly with cells; and, 4) measuring and identifying patterns of brain chemicals released by experiences. These approaches will be applied together to better understand the dynamic geography of brain information processing and plasticity. Such comprehensive studies of brain dynamics in space and time have never been done. In the future, these technologies can be applied to many brain regions to advance understanding, broadening their impact. Students will be trained beyond usual disciplines, so that neuroscience, imaging technology, engineering of new materials for electrodes, and high-resolution analysis of neuron-to-neuron signals will be taught and used together. Outcomes will contribute to a workforce trained in new ways to tackle problems beyond current boundaries.

What dynamic interactions and emergent properties of neuronal cells and circuits encode experience and generate changes in complex behaviors? Understanding the temporal and spatial dynamics of signal flow and evolution in multi-tiered neuronal circuits has been elusive. The proposed study will address this gap through transformational research that bridges excellence in fundamental neuroscience with innovative technologies in non-invasive imaging, materials development, and neurochemical analysis. Focus will be on processing of a surrogate sensory signal in the suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, that generates long-term behavioral change. This initiative will enable a pioneering program to develop and integrate novel non-invasive imaging of action potentials assessed by quantitative phase imaging of optical signals, stimulation/sensing by original, transparent, biocompatible electrodes, and chemical analyses of complex peptide-release signatures to understand the spatiotemporal dynamics of information flow in rat SCN circuits. These approaches will be applied together to better understand the dynamic geography of brain information processing and plasticity. Such comprehensive studies of brain dynamics in space and time have not been done previously. In the future, these technologies can be applied to many brain regions to advance understanding, broadening their impact. Students will be trained beyond usual disciplines, so that neuroscience, imaging technology, engineering of new materials for electrodes, and high-resolution analysis of neuron-to-neuron signals will be taught and used together. Outcomes will contribute to a workforce trained in new ways to work beyond current boundaries.

FACILITIES, EQUIPMENT AND OTHER RESOURCES MOLECULAR PROFILING AND CHARACTERIZATION CORE NORTHWESTERN UNIVERSITY The research enterprise at Northwestern University is distinguished by its ability to define and lead interdisciplinary research. From the University culture of cooperation and collaboration, as laid out in the University's strategic plan, interdisciplinary research is identified as one of Northwestern's hallmarks and a foundation upon which Northwestern will build its future. Laboratory: The Kelleher research team develops next-generation proteomics using whole proteins (i.e., the Top-Down approach) and efficiently translates this frontier approach to Northwestern. The team uses a diversity of commercial and custom sample handling approaches, mass spectrometers and software to apply high performance and cutting-edge proteomics to a variety of cell lines, tissues and mammalian tissues/fluids. The research group has two wet labs equipped with chemical fume hoods, biosafety cabinets, vacuum lines, gas lines, ice machines, four-80C freezers, five -20C freezers, two refrigerators and a walk-in cold room. Further, the group has all of the instruments necessary for producing nano-chromatographic columns as well as equipment and tools for both basic machining and electronic test and measure.

? DESCRIPTION (provided by applicant): The BRAIN Initiative seeks to understand the spatial, temporal and chemical nature of the brain. RFA-MH-15- 225 calls for the development of new tools and technologies with a number of goals, including methods to obtain cell type and chemical information from individual cells and their connections. While many imaging approaches exist that use specific probes to image subsets of cells and their interconnections, this project will create a chemical information-rich approach that advances the emerging technique of stimulated Raman scattering microscopy (SRSM). SRSM provides vibrational spectral data from every location of a living brain slice so that dynamic chemical changes can be followed. The Raman spectra contain tremendous chemical information but the data is coded in complex overlapping molecular vibrational bands. With appropriate training sets-derived from the Raman data and comparing it to the chemical contents of individual cells-a series of mathematical models will be developed that create unlimited Computational Histology maps. In order to (a) inform the mathematical model in the development phase and (b) greatly augment the chemical information obtained from these studies, dissociated cells will be subjected to another measurement-high throughput single cell mass spectrometry (MS)-on tens of thousands of cells. Single cell MS provides detail on hundreds of components in each cell, effectively mapping each cells' peptidome and metabolome. The MS data includes unique information on the metabolic state of these cells and allows us to define known and unknown cell types. Computational models will be used to correlate the SRSM data to the MS-derived chemical content as well as deliver strategies to examine the dynamic changes and heterogeneity in brain tissue. These technologies will be validated using the dentate gyrus. The focus of the work will be on the hippocampal neurons and glia of the dentate gyrus and their involvement in memory formation, and issues related to astrocyte morphology changes. By performing patch clamp physiological measurements and detailed MS-based metabolomic profiling on the patched cells of the dentate gyrus, the SRSM and single cell MS technology platform will be validated by investigating this complex area of the brain containing many cell types, heterogeneous morphologies, and chemical characteristics. These technologies will provide unmatched detail on the chemical content and dynamics within this defined brain region, answer long intractable questions related to cellular heterogeneity, and relate this information to organization and functional processes such as long term potentiation.

? DESCRIPTION (provided by applicant): The BRAIN Initiative seeks to understand the spatial, temporal and chemical nature of the brain. RFA-MH-15- 225 calls for the development of new tools and technologies with a number of goals, including methods to obtain cell type and chemical information from individual cells and their connections. While many imaging approaches exist that use specific probes to image subsets of cells and their interconnections, this project will create a chemical information-rich approach that advances the emerging technique of stimulated Raman scattering microscopy (SRSM). SRSM provides vibrational spectral data from every location of a living brain slice so that dynamic chemical changes can be followed. The Raman spectra contain tremendous chemical information but the data is coded in complex overlapping molecular vibrational bands. With appropriate training sets-derived from the Raman data and comparing it to the chemical contents of individual cells-a series of mathematical models will be developed that create unlimited Computational Histology maps. In order to (a) inform the mathematical model in the development phase and (b) greatly augment the chemical information obtained from these studies, dissociated cells will be subjected to another measurement-high throughput single cell mass spectrometry (MS)-on tens of thousands of cells. Single cell MS provides detail on hundreds of components in each cell, effectively mapping each cells' peptidome and metabolome. The MS data includes unique information on the metabolic state of these cells and allows us to define known and unknown cell types. Computational models will be used to correlate the SRSM data to the MS-derived chemical content as well as deliver strategies to examine the dynamic changes and heterogeneity in brain tissue. These technologies will be validated using the dentate gyrus. The focus of the work will be on the hippocampal neurons and glia of the dentate gyrus and their involvement in memory formation, and issues related to astrocyte morphology changes. By performing patch clamp physiological measurements and detailed MS-based metabolomic profiling on the patched cells of the dentate gyrus, the SRSM and single cell MS technology platform will be validated by investigating this complex area of the brain containing many cell types, heterogeneous morphologies, and chemical characteristics. These technologies will provide unmatched detail on the chemical content and dynamics within this defined brain region, answer long intractable questions related to cellular heterogeneity, and relate this information to organization and functional processes such as long term potentiation.

Understanding the structure and chemical makeup of the human brain has remained an enduring intellectual challenge for more than a century. More recently, the nation has devoted considerable resources, tools, and efforts to advancing knowledge of the brain, as exemplified by the White House BRAIN Initiative (Brain Research through Advancing Innovative Neurotechnologies). While a number of projects have been undertaken to explore specific directions, from fundamental science to a greater understanding of human health, the role of chemistry in meeting this grand challenge is underexplored. This workshop brings together leading stakeholders in the chemical and measurement sciences with those in the neurosciences. The goal is to examine and articulate a path forward for creating new chemistry tools, representing varied experiences and disciplines. Given the central role of the brain as a key topic in modern society, science, and research, the subject of this workshop presents a rich opportunity for achieving broad impact by bringing together a multidisciplinary group of scientists. Together they are providing unique perspectives and creating a research blueprint for the role of chemistry in advancing neuroscience over the coming decade. The results of the workshop are disseminated widely through web publication of both technical and lay reports. A workshop summary and other outcomes are disseminated via symposia organized at analytical chemistry and neuroscience meetings so as to make both communities aware of the workshop results.

Advancing the understanding of the brain has become a significant challenge using a considerable fraction of the nation's research efforts, as exemplified by the multi-institutional efforts of the BRAIN Initiative. From fundamental science to a greater understanding of human health, the charge has been led by the biological sciences. With unprecedented knowledge and measurement tools, now is the time to chart a strategic path forward in terms of the roles that the chemistry community can play in helping to meet this grand challenge. This workshop generates a plan to enable new understanding of brain organization, activity, and function across the metazoan. The output is a targeted set of goals, outlining opportunities and novel directions for neuroscience within the chemical sciences. Furthermore, it identifies areas of focus and the investments required to address the chemical and measurement challenges over the coming decade. As an example, the full parts list of the brain is still unknown, much less how, when, and where these parts work together. Obtaining this information represents a major analytical frontier. The workshop is identifying such challenges and proposing strategies that are at the heart of advances in chemical measurements.

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Jonathan V. Sweedler and his research group at the University of Illinois at Urbana-Champaign are developing new measurement technologies (based on mass spectrometry imaging and chemical separations) to probe the chemical composition of individual cells in a high throughput manner. In a test application, the new methods are used to analyze and classify all the cells within the central nervous system of the marine sea slug, Aplysia californica, an important neuroscience/physiological animal model. This research promises to provide the most complete description of cellular chemistry of the brain for any animal model, providing fundamental details linking neurochemistry to brain function. This proof-of-concept study is ultimately expected to lead to measurement of more complex brains. The interdisciplinary research bridges the worlds of analytical chemistry and cellular neurobiology. The broad-based dissemination of the results through scientific and lay publications, conferences, high school and open house outreach programs, and training across multiple disciplines assures that this research advances NSF's mission in science education and research.

The analytical platform being developed integrates mass spectrometry-based chemical imaging, capillary electrophoresis separations hyphenated to mass spectrometry, and optical microscopy to enable high-throughput characterization of tens of thousands of cells at the single-cell level, with an aim of global characterization of the chemical constituents of complex tissues such as the brain. The first goal is to create novel cell isolation approaches and then high-throughput, multiplex single-cell chemical analyses based on laser desorption/ionization and secondary ionization mass spectrometry imaging to examine individual cells within larger cell populations. An enhanced method of off-line coupling of selected cells of interest to a capillary electrophoresis mass spectrometry system is used to perform follow-up assays. In a model test system, the experiments provide detailed information on the cell types and chemical heterogeneity within the Aplysia central nervous system. Undergraduate and graduate students with diverse backgrounds are actively involved in these interdisciplinary efforts, and are involved in multiple outreach programs in both public and educational settings.

This National Science Foundation Research Traineeship award to the University of Illinois at Urbana-Champaign will address the next frontier in biotechnology: to engineer, and then decipher and harness, the living three-dimensional brain. The program will provide doctoral students with the skills and knowledge base to develop and utilize miniature brain machinery in an effort to understand and regulate brain activities. To achieve the goals of developing cross-disciplinary researchers, trainees will learn diverse fundamentals in biology, mathematics, engineering, and cognitive science, relevant to miniature brain machinery. The training grant anticipates providing a unique and comprehensive training opportunity for sixty (60) PhD students, including thirty four (34) funded trainees. Trainees will be recruited from neuroscience, cell and developmental biology, molecular and integrative physiology, chemistry, chemical and biomolecular engineering, bioengineering, electrical and computer engineering, and psychology. The training program will foster a culture of innovation and translational research, and will produce a new generation of scientists and engineers prepared to tackle major problems in brain studies that can improve the quality of human life.

The research and training program will bridge two dominant, non-overlapping brain research paradigms: i) cognitive and behavioral studies, focused principally on understanding of adaptation, decision-making, psychology, and learning of an individual using bioimaging and computational tools vs. ii) cell and tissue studies, focused on activities of multiple neuronal cells by altering their internal and external microenvironments comprised of biomolecules, extracellular matrix, and external stimuli. The goal of this NRT training program is to unite these two dominant paradigms in brain science studies and bridge the expertise of cell and molecular biologists, physiologists, chemists, nano/micro technologists, and cognitive neuroscientists. This training program prepares students for studies that enable control over the networks producing behavior and thus to study causal relations. The overarching goal of the program is to provide students with an interdisciplinary curriculum grounded in problem-based learning and an immersive research experience that blends techniques from multiple disciplines. A second goal is to increase the participation of women, underrepresented minorities, and students with disabilities in neuroscience, life sciences, chemical sciences, and engineering fields. A third goal is to train students in communication skills with the public. Evaluative studies conducted throughout this research traineeship project will explore the dynamics and efficacy of interdisciplinary collaboration by students in this program. Project outcomes will be a demonstrated, evaluated model for transformative graduate training that is effective in developing broadly trained professionals.

The NSF Research Traineeship (NRT) Program is designed to encourage the development and implementation of bold, new potentially transformative models for STEM graduate education training. The Traineeship Track is dedicated to effective training of STEM graduate students in high priority interdisciplinary research areas, through comprehensive traineeship models that are innovative, evidence-based, and aligned with changing workforce and research needs.

Project Summary In the effort to understand brain function in both healthy and disease states, it is important to identify the active structures of cell-to-cell signaling neuropeptides and elucidate their cellular signaling pathways. This information enables the design of therapeutic compounds to modulate these pathways for treating a variety of human health conditions. Neuropeptides can undergo a subtle post-translational modification (PTM) that isomerizes one amino acid residue from the L-stereoisomer to the D-stereoisomer. L- to D-residue isomerization alters the three-dimensional structure of the resulting D-amino acid-containing peptide (DAACP), often leading to significantly higher biological potency and stability relative to the all-L-residue analogue. A related PTM is the formation of isoaspartate from aspartate residues to form isoaspartate-containing peptides (IsoAspPs), a modification that has been implicated in a number of neurological disorders. However, both L- to D-residue isomerization and isoaspartate formation are difficult to detect because these modifications do not change a compound?s mass or chemical composition, rendering these PTMs ?invisible? to most peptide characterization approaches. The central hypothesis is that DAACPs and IsoAspPs are present as cell-to-cell signaling peptides in many animals, including mammals, but have been mischaracterized due to technological deficiencies in detecting peptide residue isomerization. There is currently an unmet requirement for methods to detect and predict the occurrence of these functionally important PTMs. This need is addressed with a DAACP and IsoAspP discovery funnel, a new technology designed to study the synthesis and signaling of peptides that undergo isomerization, with the long-term objective being to use this information to establish the neurobiological role these PTMs play in healthy organisms and in neurological disorders. Aim 1 will develop a non-targeted method to screen for DAACPs and IsoAspPs in a variety of animals and biological tissues. The method will be used to fully characterize the suite of DAACPs and IsoAspPs present in the central nervous system of the model organism Aplysia californica, as well as in central nervous, endocrine, and heart tissues of mouse. Simultaneously, Aim 2 will create a method to identify DAACPs and IsoAspPs at the level of the single cell using on-slide enzymatic digestion coupled to sensitive single cell mass spectrometry techniques. Finally, Aim 3 will fully characterize the biosynthesis and signaling of known DAACPs in Aplysia, including identifying the first L/D-isomerase enzyme that acts on cell-to-cell signaling peptides, which will allow for the identification of homologous enzymes in other animals, including mammals. Together, these efforts will define the importance of cell-to-cell signaling DAACPs and IsoAspPs and characterize their synthesis and function throughout the nervous system. The tools developed will have wide applicability to many future investigations of cell-to-cell signaling molecules.

As part of the UIUC Neurometabolomics and Neuroproteomics Center on Cell-Cell Signaling, the Sampling and Separation Core develops and applies methods to enhance all aspects of sample preparation and fractionation for the comprehensive chemical analysis of brain neurochemistry at the tissue, cell, and subcellular levels. Precise, accurate, and careful sampling is crucial in successful proteome, peptidome, and metabolome studies. In contrast to DNA/RNA-based molecular techniques, direct measurement of proteins and metabolites in volume- and mass-limited samples lacks a significant signal amplification step. This Core offers unmatched capabilities in the sampling of tissues, cells, and organelles, enabling the chemical characterization of various classes of molecules from an ever-increasing range of samples and animal models. Infused throughout this Core is the philosophy that directed sampling is fundamental to reducing the intrinsic chemical and morphological complexity of heterogeneous neuronal systems to achieve effective measurement of dynamic brain systems. The methodological advances we have developed already provide better results for experiments that, in some cases, would have been considered impossible to perform without these approaches. A unifying strategy for studies employing separation methods is that they seek to fractionate complex tissue samples into components that make them more accessible for in-depth chemical characterization via mass spectrometry analysis in the Molecular Profiling and Characterization Core. Since our inception, we have been expanding our analytical toolset to include a number of both well-established and innovative approaches for analyte sampling and separation, thus providing robust processing of nervous system samples for metabolite, peptide, and protein characterization. Obviously, this Core is closely linked to the needs of our users, and exists within the framework of the entire Center. Only when integrated with the Pilot Research Projects Core, the Molecular Profiling and Characterization Core, and the Bioinformatics, Data Analytics, and Predictive Modeling Core, are we able to generate the biological information required by our users. Simply stated, the Sampling and Separation Core links the biological user base to the rest of the Center and lays the foundation for successful Center to user project outcomes. The close interactions between the neuroscience users and the Sampling Core chemists and biologists enables a robust synergy. The neuroscience users have become increasingly aware of the advantages provided by the analytical tools and expertise available to them through our Core. So too have the facility chemists gained greater familiarity with the diversity of sample types, from a honey bee brain to a rat brain, with sample sizes ranging from whole brain extracts to single cells, to subcellular compartments. We tightly couple the methodologies to the neuroscience to ensure that we will continue to advance analytical methods of significant value to the broader NIDA research community.

The UIUC Neuroproteomics and Neurometabolomics Center on Cell-Cell Signaling provides high-end chemical characterization services to a broad user community, as well as develops innovative measurement and analysis tools, and provides these tools to the NIDA research community. More specifically, the Center provides high-end 'omics-scale characterization of the small molecules, peptides, and proteins in samples obtained from brain sub-regions like defined nuclei and even specific single cells. The Center consists of three research cores, a Pilot Research Project Core and this Administrative Core. This Core is tasked with ensuring the seamless operation of the Center and the flow of information between the research cores, providing administrative support, and facilitating Center interactions with the internal and external advisory boards. The Core also evaluates the user research projects, including Pilot Projects, in consultation with our advisory teams. These evaluations are used to select new users and to terminate support for non-productive projects. The Administrative Core ensures the Center runs smoothly and efficiently, particularly with regard to scheduling and resource allocation, coordinates Center activities to achieve optimum productivity in supporting the users in our research project base and in developing new technologies, helps the other cores disseminate our output widely to appropriate scientific communities (such as the technology development and drug abuse research communities), facilitates the training and education of our staff and user base as needed, and schedules presentations and sessions related to Center activities at local and national meetings. The Administrative Core has successfully implemented these responsibilities over the past 14 years and will continue to provide the same level of support to Center in the coming period. The high level of synergy between the neuroscientists and technologists affiliated with the Center ensures we will enable exciting scientific advances in understanding how systems of neurons interact in both the healthy nervous system and upon exposure to drugs of abuse. Lastly, a series of outreach initiatives enabled through this Core confirms that our protocols and approaches are widely available to the appropriate scientific communities.