Gregg Roman, Ph.D. - US grants

DESCRIPTION (provided by applicant): The objective of this proposal is to develop a genetic framework for the processes controlling alcohol sedation. The framework will be produced through epistasis analysis on several defined signaling mutants in Drosophila. This framework will center on the Drosophila non-visual arrestin kurtz, and will define genetic interactions that modulate behavioral sensitivity to alcohol. Mutants in kurtz are hypersensitive to the sedative effects alcohol. This sensitivity is rescued by the targeted expression of a kurtz cDNA within the nervous system, demonstrating a neural requirement for arrestin activity in the normal resistance to alcohol's intoxicating properties. The proposed experiments will examine whether this requirement is a developmental function of kurtz, or whether the absence of arrestin activity leaves the nervous system physiologically sensitized to the sedative effects of alcohol. The experiments will also map the neural foci for the function of kurtz in regulating alcohol sedation through gain-of-function rescue experiments. With this deeper understanding of where and when kurtz functions to modulate alcohol sensitivity, we will define additional molecules that interact with kurtz in the development of alcohol intoxication. One such interaction, in which the krz1 mutation can repress the alcohol sensitivity phenotype of the rutabaga typeI adenylyl cylase, has already been demonstrated. The data gained from these expriements will provide a functional dissection of the molecular processes that modify an animal's sensitivity to alcohol.

DESCRIPTION (provided by applicant): Anxiety Disorders are the most common mental illness in the US. Currently it is believed that many of these disorders are be triggered by environmental factors, but a predisposition to the disorder is strongly controlled by genetics factors. The ability to identify novel anxiety susceptibility genes in humans has been limited in part by the absence of endophenotypes for anxiety, and by the complexity of the constellation of diseases that comprise anxiety disorders. Since anxiety and fear represent ancient and evolutionarily conserved emotional states, animal models should be extremely valuable in dissecting the underlying neurobiology of anxiety and for identifying candidate genes. Simple animal models will allow for the identification of "core" anxiety responses that have since expanded and multiplied through evolution. A core anxiety response would also illuminate possible endophenotypes for anxiety disorders. The proposed experiments will identify novel behavioral responses to anxiogenic stimuli in Drosophila melanogaster. In these experiments the animals will be subjected to innately and conditionally aversive stimuli, and their responses in three behavioral measures will be determined. Drosophila is an extremely powerful genetic model system for the dissection of the behavioral and neurogenetic underpinnings of anxiety-like responses. Ultimately, the knowledge gained on these responses in this simple and agile genetic model system will permit the development of new and more detailed hypothesis on how different forms of anxiety may have evolved, which genes control the magnitude of responses, possible endophenotypes, and the potential functions and causes of pathological anxiety states in humans. PUBLIC HEALTH RELEVANCE: The proposed project investigates how anxiety-producing stimuli lead to changes in the behavior of Drosophila melanogaster in a novel open field arena. The studies will produce a foundation for further dissecting the genetic and neural basis of anxiety in both invertebrates and vertebrates.

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism affect 4.5% of the United States population causing an economic burden of approximately 184 billion dollars/year. The ability to develop new pharmacotherapies to help fight the descent into alcohol dependence and recidivism requires an understanding of mechanisms of alcohol actions on the nervous system. It is particularly important to define the targets of ethanol binding as these may bring about the most complete therapeutic effect. Although alcohol is known to have distinct and profound effects on presynaptic function, the mechanisms underlying this large impact are essentially unknown. The long-term goal of this proposal is to define the molecular mechanism by which alcohol exerts its action on presynaptic function. The objective of this exploratory application is to examine the physiological and behavioral interactions between Munc13.1 protein and ethanol. Munc13.1 is a presynaptic active zone protein essential for neurotransmitter release in brain. In Caenorhabditis elegans, the homologous Unc13 protein is responsible for the sensitivity to volatile anesthetics. The C1 or diacylglycerol binding domain of the Munc13 family of proteins is structurally similar to that of protein kinase C (PKC) which regulates behavioral effects of alcohol and has alcohol binding site(s). Preliminary data demonstrate that ethanol will also bind to this C1 domain in Munc13.1. The central hypothesis to be tested is that a significant effect of ethanol on nervous system function is due to the binding of ethanol to the Munc13.1 C1 domain. The first aim of this proposal will examine the hypothesis that ethanol binding to the C1 domain of the Munc13.1 modifies the activity of this presynaptic protein. This will be accomplished by photolabeling and mass spectrometry to identify alcohol binding residues, and by elucidating the effects of this binding on Munc13.1 activity in membrane translocation assays. The second aim is to determine how a reduction in Dunc13 activity changes the behavioral and physiological responses to ethanol in Drosophila melanogaster. This invertebrate model system was chosen for its ability to rapidly and economically alter Dunc13 levels and to monitor the functional consequences. These consequences of the ethanol-Dunc13 interaction will be revealed by measuring ethanol preference, stimulation, and sedation in wild type and Dunc13 reduction of function animals. Moreover, the effect of the interaction will be examined using the synapto-pHluorin sensor to image vesicle release from a specific subset of GABAergic neurons in intoxicated and sober flies. These GABAergic neurons are critical modulators of the stimulatory and sedative effects of ethanol in Drosophila. The approach is innovative as it applies the strengths of ultrasensitive biochemistry and powerful neurogenetic techniques for the first time to dissect the function of an ethanol-receptor interaction. This proposal is significant as it is expected to uncover a novel primary mechanism for an effect of ethanol on presynaptic function. Ultimately, this understanding may lead to new drugs designed to disrupt the ethanol-unc13.1 interaction providing a valuable weapon in the fight for sobriety.

This Major Research Instrumentation grant will support the acquisition of a fixed-stage upright confocal microscope and supporting computational infrastructure for Life Science research at the University of Houston. The acquired Leica confocal microscope will permit leading edge research into diverse areas of the life sciences including neurobiology and behavior, cell signaling, biochemistry and even evolution. Many established and junior investigators will use the advanced capabilities of the confocal microscope to address important questions, including: understanding the cellular mechanisms of memory formation, the dynamics and regulation of cytoskeleton function, and the biophysics of protein synthesis. This microscope is necessary both to maintain confocal capabilities of the University of Houston, and to expand the ability for investigations into new areas in biology and biochemistry. The state-of-the-art confocal microscope will permit advanced techniques such as uncaging neurotransmitters, fluorescence resonant energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). The microscope will also be used to establish collaborations with Computer Scientists to develop new solutions for computer assisted image analysis research. The collaboration between Life and Computer Scientists will facilitate new solutions for increasing the quality and quantity of data extracted from imaging experiments.

The acquired confocal microscope and biological image analysis computer facility will also be used to train postdocs, graduate and undergraduate students, including many underrepresented minorities. The University of Houston is the US?s second most ethnically diverse research university, and the 23rd largest in the United States. The Department of Biology and Biochemistry is the primary life science department at the University of Houston and has almost 3,000 undergraduate majors. Approximately 1/3rd of these majors are underrepresented minorities (11% African Americans, 21.5% Hispanic, 2% Native American). Many more of the Biology and Biochemistry undergraduates are also first generation college students. The confocal microscope will be used in undergraduate courses to train more than 400 undergraduate students per year and two graduate levels courses in cellular neuroscience, providing yearly training to approximately 20 graduate students in advanced microscopic techniques. The confocal microscope will be used further in new summer workshops on biological imaging for ~40 underrepresented minority undergraduate students. These trained undergraduates will be recruited into faculty labs for research projects that will use the acquired confocal microscope. The microscope will provide an attractive focus for engaging students to actively participate in life science and computer image analysis research programs. The confocal microscope and computer analysis facility will transform the scientific education of the Biology & Biochemistry and the Computer Science Students at the University of Houston.

There is an unequivocal need to develop and employ advanced techniques in microscopy toward understanding the cellular functions of glycans. Glycans participate in many cellular properties, including cell structure, cell-cell recognition, protein trafficking, and protein-protein interaction. Hence, changes in glycan abundance or composition can have considerable effects on cellular function, and as a consequence human health. Advanced imaging techniques are becoming a vital part of an integrative approach to understanding the roles of glycans in health and disease. These techniques provide insights into molecular composition, while preserving the spatial context of the chemical, structural, and physiological responses to changes in glycans. Recent advances in identify lectin specificities and the availability of a broad range of fluorescently labeled and biotinylated lectins has rapidly advanced our ability to identify changes in the location and abundance of specific glycans in cells and tissues. Moreover, several powerful approaches in confocal microscopy, including FRET, FRAP, and FLIP, permit highly sensitive measures of molecular dynamics within living cells. These measures will help researchers understand the fundamental effect glycans have on cellular function. The central goal of this proposal is to bring the ability to employ advanced imaging techniques to the GlyCORE investigators in their diverse research programs and to help develop new approaches in the cell biology of glycans. These goals will be accomplished in three aims: 1) establish an imaging core that contains three different imaging platforms, including a powerful new confocal microscope; 2) support the GlyCORE investigators in their individual projects by working to develop new imaging applications; and 3) develop an extensive training program to develop expertise and awareness of new methods in imaging and image analysis. This proposal will transform research in the glycosciences at the University of Mississippi by providing access to advanced instrumentation and expertise and training in the use of these imaging systems.