Floyd E. Bloom, M.D. - US grants

To understand how albumin production by liver parenchymal cells is inhibited directly by ethanol, we are characterizing this specialized function during the growth cycle of proliferation competent adult rat hepatocytes in primary "monolayer" culture. Ethanol's actions will be examined at varying doses after acute and chronic exposure. The dependence of albumin secretion, synthesis and "turnover" on (a) cell density, (b) growth state, (c) growth rate, and (d) nutritional conditions will be explored. Albumin will be measured by specific radioimmunoassay, immunoprecipitation and slab gel electrophoretic procedures. Cell growth will be measured quantitatively and qualitatively as described in Leffert et al. (PNAS 71:1834-1838, 1978) and Koch and Leffert (Cell 18:153-163, 1979). The total numbers of albumin mRNA-like sequences during the growth cycle also will be measured by nucleic acid hybridization of cellular RNA to specific albumin 32p-labelled cDNA probes. Since the cultures show a growth-state dependent change in pyrazole-sensitive alcohol dehydrogenase activity, we will see if ethanol metabolism is required for its function-inhibiting effects.

To understand how albumin production by liver parenchymal cells is inhibited directly by ethanol, we are characterizing this specialized function during the growth cycle of proliferation competent adult rat hepatocytes in primary "monolayer" culture. Ethanol's actions will be examined at varying doses after acute and chronic exposure. The dependence of albumin secretion, synthesis and "turnover" on (a) cell density, (b) growth state, (c) growth rate, and (d) nutritional conditions will be explored. Albumin will be measured by specific radioimmunoassay, immunoprecipitation and slab gel electrophoretic procedures. Cell growth will be measured quantitatively and qualitatively as described in Leffert et al. (PNAS 71:1834-1838, 1978) and Koch and Leffert (Cell 18:153-163, 1979). The total numbers of albumin mRNA-like sequences during the growth cycle also will be measured by nucleic acid hybridization of cellular RNA to specific albumin 32p-labelled cDNA probes. Since the cultures show a growth-state dependent change in pyrazole-sensitive alcohol dehydrogenase activity, we will see if ethanol metabolism is required for its function-inhibiting effects.

brain mapping;

This award will support collaborative research in neurobiology between Dr. George Koob, Scripps Clinical and Research Foundation, La Jolla, California, and Professor Michel LeMoal, University of Bordeaux II, France. The objective of the proposed research is to study the role of the peptide neurotransmitter corticotronin releasing factor (CRF) as a primary mediator of the behavioral state of stress and the behavioral responses to stress in the central nervous system. Recent studies have shown that CRF liberated directly into the central nervous system may have neurotropic actions important for mobilizing behavioral responses to stress. The investigators have designed a set of experiments to identify the specific neural substrates for these behavioral actions by direct local interacerebral injection of CRF and CRF antagonists. In addition experiments are proposed to explore the possible mediation of these CRF effects via the central and peripheral noradrenergic systems. An attempt will be made to modify behavioral responses to CRF using specific pharmacological and neurotoxin induced inactivation of the central and peripheral noradrenergic systems. These studies have important implications for the role of pituitary peptides in brain function, and may provide important clues to the pathophysiology associated with behavioral disorders as diverse as depression, anxiety, and obesity. Over the past ten years Dr. Le Moal and his research group have developed and explored systematically behavioral and endocrine correlates of stress, coping and behavior. Dr. Koob's expertise is in classic behavioral pharmacology and basic neurobiology. The expected benefits to U.S. science from this project include not only the exchange of special behavioral techniques, but also the gain of valuable expertise regarding the measurement of brain function via behavioral techniques.

In response to NIMH announcements, we propose to create a multidisciplinary comprehensive Research Center for AIDS Dementia Complex Research. This Center will be based primarily within the Department of Neuropharmacology of the Research Institute of Scripps Clinic (RISC), with additional collaborating investigators based within the RISC Departments of Immunology and Molecular Biology, and the Department of Psychiatry, University California-San Diego. This Center will investigate the molecular and cellular mechanisms underlying the AIDS Dementia Complex by a highly focussed comparative evaluation of brain pathophysiology in selected AIDS patients with that found in 3 animal model systems suitable for invasive experimental investigation: Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV) and persistent selected neuronal infection with a murine neurotropic arenavirus, lymphocytic choriomeningitis virus (LCMV) capable of eluding immunodetection and clearance from host cells. With these animal models, the Center will correlate virus-dependent changes in immune system and nervous system function with the clinical profile of AIDS. In collaboration with the UCSD-based, NIMH-sponsored HIV Neurobehavioral Research Center and the US Naval Hospital, we will perform electrophysiological studies on HIV patients sampled throughout their clinical courses. These profiles will serve as a comparison for neuropsychological, macro-and microelectrode electrophysiological, and in vivo and in vitro evaluation of immune system and cytokine function in SIV and FIV. Murine studies will determine the functional alterations of persistent virus infections as expressed by neurobehavioral, electrophysiological, and molecular biological alterations within specific cell types of the cns (e.g., neuronal, glial, microglial, lymphocytic and vascular elements), and will evaluate the use of unique, genetically engineered anti-virus treatments as well as azidothymidine to reverse discrete cellular and behavioral sequelae of virus effects. These multidisciplinary studies will define the biological actions of persistent virus infections of the brain, the profiles of cell-cell signals activated by these infections, and the nature of the effects of these signals on neuronal function. In addition to their relevance tot he nature of AIDS Dementia Complex, these studies may illuminate other unexplained neuropsychiatric disorders for which infectious agent-caused immune dysfunction may be suspected.

biomedical equipment purchase;

biomedical equipment purchase;

This P20 application under Phase I of the Human Brain Project will support a group collaboration to develop computer-assisted quantitative microscopy data acquisition and analysis software, to construct a complex hierarchical, relational, object oriented database based on brain atlas templates, and establish means to make these data accessible through high speed electronic networks. Each of the scientific teams will use the data acquisition and analysis systems on microscope system and computers of their preference on inter-related molecular and cellular mapping studies designed to reveal neurons vulnerable to degeneration in human diseases. Six scientists will participate in the collaboration by extensions of already funded research projects: 1) Dr. Floyd Bloom, The Scripps Research Institute (TSRI), La Jolla will be Principal Investigator, and Dr. Warren Young, TSRI, La Jolla will be co-PI with overall responsibility for software development. The Bloom/Young Project will develop 3 critical software tools: a) digital atlases of the primate, feline, and rodent nervous systems, (using as an experimental base, animal models of human immunodeficiency virus syndrome); b) a comprehensive neuronal circuitry database for the major vertebrate central nervous systems; c) a cross-platform neuroscience workstation environment that operates on both local and wide area networks, interconnecting the 5 research projects, and the required programs for data acquisition, storage, analysis, and communication. 2) Dr. John Morrison, Mt. Sinai Medical School, New York, NY, will develop the fourth essential tool with Young: a computerized microscopy data acquisition system which integrates neuroanatomical paradigms of experimental observation at all levels of resolution from macroscopic to ultrastructure. The Morrison Project will generate quantitative data on primate and human neocortex glutamate receptor distribution and other molecules related to neurodegenerative vulnerability (e.g., neurofilament protein) or resistance (e.g., calcium binding proteins). 3) Dr. Harvey Karten, UCSD Medical School, La Jolla will develop a database of the molecular, cellular and physiological characteristics of vertebrate retinas, and link this database using our group microscope data collection methods. This retina database will include both normative adult and developmental data and pathological changes, and compare species. 4) Dr. Steve Foote, UCSD Medical School, La Jolla will evaluate quantitative long term changes in dopamine receptor expression (mRNA and protein) in the primate neocortex consequent to repeated psychostimulant administration using our group microscope data collection methods. These structural parameters of dopamine re-ceptor alteration will be integrated int he object oriented database with electrophysiological, behavioral, and in situ microdialysis dopamine release data. 5) Dr. Edward Jones, UC-Irvine California College of Medicine, will employ the computer- assisted microscopy methods to quantify specific neuronal populations in the major nuclei of the human reticular nucleus and dorsal thalamus and their immunocytochemically defined monoamine and neuropeptide afferent fiber systems in brains from adult patients with schizophrenia, Parkinson's disease and Alzheimer's disease and matched controls and in mid-trimester fetal material. This normative and disease-related data collection will quantitatively test the hypothesis that activity- dependent regulation of gene expression and cellular morphology can reveal functional disconnection of these particular thalamic nuclei. In the Morrison, Foote, Karten, and Jones Projects, data will be collected in common formats modified from existing software that are easily accessed and specifically prepared for incorporation into the large scale atlases and databases to be prepared by the Bloom/Young Component. We will utilize the quantitative data atlases and databases to establish norms of cellular, circuitry, and neurochemical information which will then serve to detect and define experimental and pathological variations, and to generate explanatory hypotheses.

Under this ADAMHA grant, the Scripps-Salk-UCSD-San Diego State University Alcohol Research Center (SSU-ARC) proposes to continue its long term, cooperative, interdisciplinary research focussed on the theme of "CNS Effects of Alcohol". For this renewal application, the SSU-ARC will consist of 9 Components: The Administrative Core resource (supervising 3 supporting resource units), 8 independent collaborating research Components as well as a separate Pilot Project Component, supervised by the Administrative Core designed to stimulate new directions and recruit new investigators to ethanol centered research. The SSU-ARC research program remains strongly interdisciplinary, employing molecular, cellular, systematic and behavioral research methods on human and experimental animal subjects. Three major subthemes emerging from our past 14 yrs of work within this Center will be continued: 1) the molecular and cellular mechanisms of short term ethanol intoxication, and its endocrine, metabolic and behavioral concomitants 2) the molecular, cellular and behavioral changes underlying alcohol neurotoxicity on chronic exposure to ethanol (including aversive behavioral changes) in the adult and developing rodent and human nervous system, such as the Fetal Alcohol Syndrome; 3) the molecular, cellular and neuropsychological basis for susceptibility to alcoholism in humans, including the re- inforcing actions of ethanol. Individual research components range from studies of molecular genetic neuronal development through radiological and electrophysiological analysis of human FAS neuropathology, cellular and intracellular indices of intoxication, neuronal and endocrine indices of tolerance, dependence and toxicity. The Center also supports a participating unit in the NIAAA sponsored Consortium on the Genetics of Alcoholism, as well as other Center-funded, and RO-1 funded research on human CNS indices of short term and long term ethanol sensitivity. Research funded within this Center provides knowledge which will reduce the burden of illness associated with the consequences of alcohol abuse, and help define factors than can reduce the susceptibility to, and thereby prevent, alcohol abuse and alcoholism.

In response to NIMH announcements, we propose to create a multidisciplinary comprehensive Research Center for AIDS Dementia Complex Research. This Center will be based primarily within the Department of Neuropharmacology of the Research Institute of Scripps Clinic (RISC), with additional collaborating investigators based within the RISC Departments of Immunology and Molecular Biology, and the Department of Psychiatry, University California-San Diego. This Center will investigate the molecular and cellular mechanisms underlying the AIDS Dementia Complex by a highly focussed comparative evaluation of brain pathophysiology in selected AIDS patients with that found in 3 animal model systems suitable for invasive experimental investigation: Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV) and persistent selected neuronal infection with a murine neurotropic arenavirus, lymphocytic choriomeningitis virus (LCMV) capable of eluding immunodetection and clearance from host cells. With these animal models, the Center will correlate virus-dependent changes in immune system and nervous system function with the clinical profile of AIDS. In collaboration with the UCSD-based, NIMH-sponsored HIV Neurobehavioral Research Center and the US Naval Hospital, we will perform electrophysiological studies on HIV patients sampled throughout their clinical courses. These profiles will serve as a comparison for neuropsychological, macro-and microelectrode electrophysiological, and in vivo and in vitro evaluation of immune system and cytokine function in SIV and FIV. Murine studies will determine the functional alterations of persistent virus infections as expressed by neurobehavioral, electrophysiological, and molecular biological alterations within specific cell types of the cns (e.g., neuronal, glial, microglial, lymphocytic and vascular elements), and will evaluate the use of unique, genetically engineered anti-virus treatments as well as azidothymidine to reverse discrete cellular and behavioral sequelae of virus effects. These multidisciplinary studies will define the biological actions of persistent virus infections of the brain, the profiles of cell-cell signals activated by these infections, and the nature of the effects of these signals on neuronal function. In addition to their relevance tot he nature of AIDS Dementia Complex, these studies may illuminate other unexplained neuropsychiatric disorders for which infectious agent-caused immune dysfunction may be suspected.

tissue /cell culture; glia; biomedical facility; microglia; vascular endothelium; astrocytes; animal genetic material tag; flow cytometry; tissue /cell preparation; polymerase chain reaction; animal tissue;

biomedical facility; animal colony; animal care; animal tissue; genetically modified animals; laboratory mouse; cats; Macaca mulatta; laboratory rat; tissue /cell culture; postmortem;

neuropsychology; fetal alcohol syndrome; developmental neurobiology; pharmacogenetics; developmental genetics; ethanol; brain electrical activity; genetic regulation; regulatory gene; gene expression; adolescence (12-20); teratogens; RNase protection assay; magnetic resonance imaging; immunocytochemistry; laboratory rat; electroencephalography; in situ hybridization; northern blottings; neuropsychological tests;

The need for methods to extract accurate, reliable quantitative data on brain structure rapidly has increased substantially, especially with the proliferation of genetically manipulated mice as models_of C.N.S. disorders. Software tools developed under our Human Brain Project P20 consortium MH/DA 52154 define the structural bases for normal and pathologic brain function in animal models of Neuro-AIDS and others neurodegenerative models. In this Phase I STTR application, we will: 1) extend our existing NeuroZoom computer-assisted microscopy application with additional software stereological probes; 2) develop cross-platform access to a quantitative, neuroanatomic database; 3) initiate the development of a stereologically-derived database of key mouse brain structures in the normal brain; and 4) perform a prototypic analysis of a transgenic mouse model of spinal neurodegeneration to document the feasibility of such stereological approaches to pathology in genetically- manipulated mice. We envision three classes of potential commercial customers for these products: 1) laboratories possessing sufficient expertise to carry out the analyses in-house with our software, but require ongoing access to our normative database against which to compare our own mutant mice; 2) biotechnology firms or laboratories developing lines of transgenic mice and outsourcing for their comprehensive structural characterization; and 3) pharmaceutical companies that need assistance in developing an on-site program for their analyses, and require access to the normative database. PROPOSED COMMERCIAL APPLICATION: There is no available software that incorporates mapping, stereology, databases, Internet access, 3D visualization, and neurocircuitry data in one package. There is also no stereology database for either select regions of the mouse brain, or for a transgenic mouse. There are 3 classes of potential customers: l) laboratories possessing sufficient expertise to carry out the analyses in-house with our software; 2) biotechnology laboratories developing transgenic mice and outsourcing for their comprehensive structural characterization; and 3) pharmaceutical companies that need assistance in developing an on-site program for their analyses.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The need to extract quantitative brain structural data has increased substantially with the proliferation of genetically manipulated mice as models of CNS disorders. Software developed under their Human Brain Project consortium define the structural bases for normal and pathologic brain function in animal models. Under the Phase I grant, they have established the need for a commercial version of NeuroZoom with networking and integrated atlases. They have developed and validated new stereology probes by performing stereological analyses of transgenic mice with a point mutation in the mouse superoxide dismutase (SOD-1) gene. In this Phase 2 application, they will: 1) develop NeuroZoom into a commercially-supported, cross-platform application and extend it to the Web, 2) develop software to integrate an electronic mouse atlas on C57BU6 and SV1 29, and 3) map out a related set of proteins in transgenic and normative mouse models. They believe that the potential customers for these products are: 1) laboratories possessing sufficient expertise to carry out the analyses in-house with their software; 2) biotechnology firms developing lines of transgenic mice and outsourcing for their comprehensive structural characterization; and 3) pharmaceutical companies that need assistance in developing an on-site program for their analyses. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE