Robert P. Elde - US grants

This application outlines the request for advanced image processing equipment by an interdisciplinary group of investigators at the University of Minnesota. The equipment requested will form the basis for a Biomedical Image Processing Facility at the University of Minnesota which will be used to expedite and enhance image-intensive investigations supported primarily by NIH. In particular, the image processing equipment proposed herein will be interfaced as directly as possible with image producing technology that is used especially in structurally-oriented research questions. The tasks to be accomplished by acquisition and use of this equipment fall into four general categories: 1) Two-dimensional stereology; 2) Two-dimensional feature extraction; 3) Real time, two-dimensional video-enhanced imaging; 4) Pseudo three-dimensional image reconstruction. These tasks will be accomplished with a general-purpose image processing computer that will be provided with high-resolution video and photodiode array camera input. Imaging tasks that are computationally intensive will be aided by the link to a minicomputer and a high speed telecommunications link to the University of Minnesota Cray 1-S supercomputer facility.

The neuropeptides are a class of substances recently isolated from neural tissue and have been found to influence the activity of the nervous system at the level of the membrane, the neuron and the behaving organism. Immunohistochemistry of these substances has revealed discrete and rather extensive neuropeptide immunoreactivity in neuronal perikarya, axons and terminals. This proposal focuses on determining the details of the differential distribution of two of these peptides --methionine- and leucine-enkephalin. In addition to the extensiveness of enkephalinergic systems revealed in our preliminary studies, the enkephalins are of interist in light of their morphinomimetic properties at opiate receptors as well as on an organismic level. This proposal seeks to determine the differential distribution of enkephalins by purifying antisera to mono-specificity for each enkephalin by removing cross-reacting antibodies on a heterologous immunoabsorbent. The purified, monospecific antisera to each enkephalin will then be used in immunofluorescence histochemical procedures on sections of the rat brain to reveal the terminals, axons and perikarya containing either methionine- or leucine-enkephalin immunoreactivity. The results of these studies will be translated into detailed, schematic coronal maps of the rat brain with approximate stereotaxic coordinates. The connectivity of selected perikarya and terminals will be studied with immunofluorescence histochemistry on rat brains after stereotaxic lesions of enkephalinergic perikarya. Disappearance of normally-fluorescent terminals will indicate the perikarya of origin for these terminals. Selected regions will also be studied with antisera to other neuropeptides where convergence of enkephalinergic and other peptidergic systems has been reported. Finally, a small portion of this proposal (10%) aims to investigate possible changes in the differential distribution of the enkephalins in selected nuclei in the brains of mice made tolerant to and dependent upon opiates.

This award provides funds to expand the computing and instrumentation capabilities of the Biomedical Image Processing Laboratory at the University of Minnesota Medical School to meet research needs in the area of spectrofluorometry. The facility is dedicated to the use of digital image processing and three-dimensional graphics in basic biological research. Tasks for which the new equipment will be used include two-dimensional stereology and image classification, two- and three-dimensional digitally enhanced imaging, high speed video motion analysis, and optical sectioning microscopy. The types of image analysis tools funded with this award are key to research at the forefront of modern cell biology. The current revolution in light microscopy and the vastly expanded ability of the electron microscope to provide serial sections are among the most striking spinoffs of the development of digital image reconstruction by astronomers and space scientists.

Discoveries made over the last two decades have established that the nervous system produces opioid peptides, as well as several classes of opioid receptors coupled to intracellular effectors. It is likely that either endogenous or exogenous opioids interact with these receptors, although the physiological circumstances which lead to occupancy of receptors by endogenous opioids is far from clear. It is generally assumed that endogenous opioids are delivered to opioid receptors by cellular - arrangements and mechanisms similar to those established for classical neurotransmitters, that is, at a synapse. However, several observations suggest that the classical form of synaptic transmission may not hold for neuropeptides in general, and opioid peptides in particular. The overall goal of studies in this laboratory is to determine at the cellular level the spatial organization of the molecules functionally involved in opioid neurotransmission. This goal will be approached through experiments designed to investigate the following specific aims: 1) To determine the spatial relationship of nerve terminals which contain opioid peptides to specific regions of the dendritic tree and cell body of target neurons; 2) To determine the spatial distribution of opioid receptors within the membrane of the dendritic tree and cell body of "opioceptive" neurons. For certain neurons, the distribution of these receptors observed under physiological conditions will be compared with the distribution of receptors under the conditions usually employed in "binding" studies; 3) To determine the spatial distribution of G-proteins predicted to mediate opioid action within the membrane of the dendritic tree and cell body of opioceptive neurons; 4) To determine the spatial relations among the three features outlined above. In particular, the proximity of opioid nerve terminals to opioid receptors will be determined, as will the relationship between opioid receptors and G-proteins. The latter relationship will also be examined under conditions thought to increase or decrease G-protein/receptor coupling. In order to accomplish some of these aims, we propose the synthesis of new, highly fluorescent ligands for opioid receptors. These ligands will be designed not only for their ability to bind selectively to classes of opioid receptors, but also to exploit the imaging power of scanning laser confocal microscopy. Knowledge gained from these studies will determine if opioidergic neurotransmission obeys the organizational 'rules' proposed to exist for synaptic transmission. If opioidergic nerve terminals and opioid receptors are not compartmentalized as in a classical synapse, these studies will determine the cellular arrangements through which opioids act upon their targets.

9419233 Carlis Many of the observations made by brain scientists, from molecular physiology to behavior, can only be understood when they are placed within the context of the brain's anatomical structure. With this award, a group of computer scientists and neurobiologists, headed by Dr. John Carlis, will design a prototype architecture for storing high resolution images of brain structure. These images will be organized (1) so that they can be viewed globally, but also so that the operator can zoom in on very fine details, and (2) they will be organized as network accessible files so that they can be shared over the internet. This work will advance the emerging field of bioinformatics and will improve the ways in which scientists store, share, and analyze experimental data about the brain.

Distributed Collaborative Database for Brain Viewing This project will develop a distributed database system and the collaborative viewing tools necessary to overcome the difficulties inherent in imageintensive neurobiological research that is accomplished by collaborations between geographically separated researchers. Images containing morphological information either are, or have recently become, a primary form of data in many fields of biological research. For an individual laboratory, the iceberg metaphor is true. Published images are the tip, whereas the ever- expanding collection of raw image files stored on hard disks and removable media is the hidden body of the iceberg. Neuroscience is particularly image intensive. From the perspective of a single laboratory, managing the magnitude of the data collected in recent years is daunting. Neuroscientists often wish to compare images of brains from experimental groups with those from control animals. It is oftentimes a challenge to identify and understand the spatial relations of a brain region identified by a staining or labeling procedure. Furthermore, collaborations among researchers in the same or different institutions are impeded by lack of image viewing tools. This goals of this project present challenges for computer scientists, including the design and development of: (i) additional file header information that represents biological variables and is generated at the microscope during the capturing of the image using speech-to-text technology; (ii) viewing tools needed for collections of images that give perspective, while retaining the pristine data captured from the digital camera; (iii) multipleresolution image-handling techniques integrated with a DBMS to meet data intensive image requirements; (iv) collaboration support for on-going research discussion centered around images, across time and space boundaries; (v) techniques for integrating audio and text within the same discussion. This research wi ll yield two kinds of results: a) artifacts, software and databases, that will be demonstrated and made available to others, and b) reports in the literature on studies of a variety of research questions on the impact of these on neuroscientists' activity.

The Image Core facility will provide three categories of services to the Drug Abuse Research Center: 1) imaging of labelled cells and whole brains at high resolution and detail. 2) Live cell imaging and measurement capabilities. 3) Image processing and enhancement capabilities for better separation of regions of interest from background. The main purpose of the core is to provide investigators with training regions of interest from background. The main purpose of the core is to provide investigators with training and with appropriate imaging devices to see where markers have localized, look for differences in morphologies and colocalization, and record movement of calcium. The core will also train investigators ib software to measure areas and volumes, and to enhance images. This core also posts training materials on its web site for other researchers, updating as new information arises.

The cloning of each of the pharmacologically identified types of opioid receptors (i.e., delta, mu and kappa) has dramatically altered the nature of the questions that can be addressed in the problem of the mismatch between opioid receptors and their endogenous ligands. Two questions that arise are: "Do the cloned receptors account for all of the opioid receptors in the nervous system?" and "Do the identified endogenous ligands represent the complete set of ligands available for action at opioid receptors?" With the successful production of a mu opioid knockout mouse and the isolation and characterization of a new family of neuropeptides (the endomorphins) that are active in mu receptors, these questions can be addressed in novel ways. It is with this in mind that we propose experiments that will contribute to the realization of the following specific aims: 1) Determine if the cloned mu receptor, MOR1, is the mu receptor responsible for presynaptic inhibition of norepinephrine release from axons of locus coeruleus neurons. 2) Determine the biosynthetic precursor responsible for synthesis of endomorphin, and the regional distribution of cells that express the transcript for endomorphin and its biologically active products. 3) Determine the spatial relationship between the endomorphin peptides and MOR1. 4) Determine if the expression of endomorphin is altered in mice that lack the apparent cognate receptor (MOR1). These aims will be accomplished by several different types of experiments. Synaptosomal preparations of the forebrain of wild type and mu knockout mice will help to elucidate the extend to which MOR1 is responsible for the presynaptic inhibition of norepinephrine release. Also, proposed are expression cloning experiments to isolate the biosynthetic precursor of the endomorphins, followed by in situ hybridization studies. Finally, we propose the development of specific antisera to the endomorphins and subsequent two-color immunofluorescent experiments in wild type and knockout mice to determine the extent of match between the cloned mu receptor and this new family of endogenous ligands.

Description (provided by applicant): The interaction of keratinocytes and epidermal nerve fibers has emerged as an important interface in sensory transduction and pain and may be a factor in the mechanisms underlying painful peripheral neuropathies. Keratinocytes express receptors and channels that enable them to "sense" mechanical and thermal stimuli and transmit signals to epidermal nerve fibers through the release of messenger molecules. Growth factors and cytokines secreted from keratinocytes are also likely to impact long-term changes within the sensory neurons that innervate epidermis. Conversely, neuropeptides contained within sensory neurons have the ability to modulate keratinocytes, including their production and secretion of cytokines and growth factors. Partial peripheral nerve injury that leads to neuropathic pain is associated with degeneration of injured nerve fibers and altered function of adjacent uninjured fibers. The impact of altered epidermal innervation on the dynamic neuro-keratinocyte relationship is unknown. The objective of this R21 proposal is to determine the effects of epidermal innervation on keratinocyte protein expression and function. Specific Aim 1 will address the hypothesis that nerve injury-induced changes in innervation will alter keratinocyte protein expression and function. Epidermal innervation and nerve injury- induced hypersensitivity will be correlated with keratinocyte expression of receptors and channels implicated in sensory transduction as well as cytokines known to sensitize sensory neurons. Functional changes in epidermis will be evaluated based on measurement of keratinocyte ATP release. Specific Aim 2 will determine the effects of sensory neurons on protein expression and function of keratinocytes in organotypic culture where neuronal input will be introduced by co-culture with sensory neurons. These experiments will address the hypothesis that selected subsets of sensory neurons will have differential effects on keratinocytes. Biochemical approaches will be used to identify candidate mediators of neuro-keratinocyte signaling. Changes in the dynamic neuro-keratinocyte relationship following nerve injury may contribute to neuropathic pain mechanisms through sensitization of surviving epidermal nerve fibers and changes in keratinocyte transduction of sensory stimuli. Therefore, neuro-keratinocyte interactions and their modulation by nerve injury represent a previously unexplored component of the pathobiological consequences of nerve injury, and their understanding may lead to identification of novel peripheral therapeutic targets for neuropathic pain. PUBLIC HEALTH RELEVANCE: The proposed project will analyze the interaction between two important structures in the transmission of sensory information: sensory nerves and the epidermis of the skin. These studies will identify a pattern of keratinocyte changes associated with nerve injury that will provide a foundation for in-depth analysis of the contribution of neuro-keratinocyte interactions to pain. Manipulation of these interactions may be able to provide a peripherally restricted therapeutic strategy.

This Partnership for Innovation project from the University of Minnesota-Twin Cities will take a successful bioremediation technology previously used for pesticide removal from water and re-engineer it for use in microbiologically-enhanced coal-bed natural gas recovery from depleted gas wells and in bioremediation of the percentage of water returned via the well from waters generated during the hydraulic fracturing (fracking) method of drilling for shale oil, oil sands, and gas. The technology is based on encapsulating microorganisms in an inexpensive, safe, extended life-time, silica-based hybrid gel. The microbes are stabilized within the gel and continue to biodegrade pesticides for more than four months. In re-engineering the platform technology for enhanced recovery, communities of microorganisms that transform coal to natural gas will be stabilized by silica encapsulation for ease of transport, enhanced stability, and improvement of function. In re-engineering of the platform technology for bioremediation, microbes will be stabilized and rendered highly active to biodegrade organic contaminants within the percentage of water returned via the well from waters generated during the hydraulic fracturing (fracking) method of drilling.

The broader impacts of this research are on possible future applications to an efficient and safe oil and gas industry. There is a strong link between a robust domestic energy sector and the creation of new jobs. The U.S. has recently become a net exporter of energy, credited largely to advances in shale oil and gas extraction that use hydraulic fracturing. This proposal develops technologies to enhance gas and oil recovery and a platform that can minimize the environmental impacts from a percentage of the water that is returned via the well and then kept in settling ponds. Two of the knowledge enhancement partners (KEPs) will take away technology to develop and scale-up production of silica-encapsulated, biodegrading bacteria that can be used for treating fracking water in settling ponds. Biostabilization imparted by the silica materials is also important for preserving microbial communities that transform coal to natural gas. One of the KEPs will take away this technology to enhance the ability to deliver microorganisms that increase natural gas production throughout the U.S. and potentially in foreign markets. This application is important because the U.S. has enormous coal reserves and enhancing coal to methane conversion will contribute to a clean energy future for our country.

Partners at the inception of the project: the lead instituion, University of Minnesota-Twin Cities (Department of Biochemistry; Bioresource Center, Biotechnology Institute; Sponsored Projects Administration; Office of Technology Commercialization; Venture Center; and Office of the General Counsel; KEP core small businesses: 1) Luca Technologies (Boulder, CO) 3F LLC (St. Paul, MN) and 3) Tundra Companies (White Bear Lake, MN).