News and Publications
DEPARTMENT OF NEUROPHARMACOLOGY
|Floyd E. Bloom, M.D. ||Member and Chairman |
|David Amaral, Ph.D. ||Adjunct Associate Member |
|Roger Beachy, Ph.D.* ||Member |
|Michael Buchmeier, Ph.D. ||Associate Member |
|Iain L. Campbell, Ph.D. ||Associate Member |
|Francis Chisari, Ph.D.** ||Member |
|Neil Cooper, Ph.D.*** ||Member |
|George Dailey III, M.D. ||Adjunct Associate Member |
|Donald Dalessio, M.D. ||Adjunct Member |
|Denis Darko, M.D. ||Associate Member |
|Arthur D. Dawson, M.D. ||Adjunct Member |
|Juan Carlos de la Torre, Ph.D. ||Associate Member |
|Cindy L. Ehlers, Ph.D. ||Associate Member |
|John Elder, Ph.D.**** ||Member |
|Milton K. Erman, Ph.D. ||Adjunct Member |
|Howard S. Fox, M.D., Ph.D. ||Associate Member |
|Klaus Früh, Ph.D. ||Adjunct Assistant Member |
|Jeannie Giacchino, M.D, Ph.D. ||Assistant Member |
|Lisa Gold, Ph.D. ||Associate Member |
|Donna L. Gruol, Ph.D. ||Associate Member |
|Rosa Hayduk, M.D. ||Adjunct Assistant Member |
|P. Kahler Hench, Ph.D. ||Adjunct Assistant Member |
|Steven J. Henriksen, Ph.D. ||Associate Member |
|Harvey Karten, Ph.D. ||Adjunct Member |
|George F. Koob, Ph.D. ||Member |
|Cary Lai, Ph.D. ||Assistant Member |
|Michel Le Moal, M.D. ||Adjunct Member |
|Gail Lewandowski, Ph.D. ||Assistant Member |
|W. Ian Lipkin, M.D. ||Adjunct Member |
|Pierre J. Magistretti, M.D., Ph.D. ||Adjunct Member |
|Athina Markou, Ph.D. ||Assistant Member |
|James C. Miller, Ph.D. ||Adjunct Associate Member |
|Merrill M. Mitler, Ph.D. ||Member |
|Glen Nemerow, Ph.D.*** ||Adjunct Associate Member |
|Michael B.A. Oldstone, M.D. ||Member |
|Shirley Otis, M.D. ||Adjunct Member |
|Tom R. Phillips, D.V.M., Ph.D. ||Assistant Member |
|J. Stephen Poceta, M.D. ||Adjunct Assistant Member |
|John Polich, Ph.D. ||Associate Member |
|Anne Prieto, Ph.D. ||Assistant Member |
|Luigi Pulvirenti, Ph.D. ||Adjunct Assistant Member |
|David Redfield, M.D. ||Adjunct Assistant Member |
|Catherine Rivier, Ph.D. ||Adjunct Associate Member |
|Michael G. Rosenfeld, M.D. ||Adjunct Member |
|Pietro P. Sanna, M.D. ||Assistant Member |
|Paul Schweitzer, Ph.D. ||Assistant Member |
|George R. Siggins, Ph.D. ||Member |
|Scott Steffensen, Ph.D. ||Assistant Member |
|Karen Thatcher-Britton, M.D. ||Adjunct Associate Member |
|Matthias G. von Herrath, M.D. ||Assistant Member |
|Tammy Wall, Ph.D. ||Adjunct Assistant Member |
|Matthew Weinger, M.D. ||Adjunct Associate Member |
|Friedbert Weiss, Ph.D. ||Associate Member |
|J. Lindsay Whitton, M.B., Ch.B., Ph.D. ||Associate Member |
|Michael Wilson, Ph.D. ||Adjunct Associate Member |
|Warren Young, Ph.D. ||Director, Neuropharmacology Computing |
SENIOR RESEARCH ASSOCIATES
|Claire Evans, Ph.D. |
|Thomas E. Lane, Ph.D. |
|Marianne Manchester, Ph.D. |
|Jeffrey Netzeband, Ph.D. |
|Loren Parsons, Ph.D. |
|Zhihua Qiu, M.D. |
|Serge Ahmed, Ph.D. |
|Yvette Akwa, Ph.D. |
|Ling-Ling An, Ph.D. |
|Valerie Asensio, Ph.D. |
|Margaret C. Barr, D.V.M., Ph.D. |
|Ana Maria Basso, Ph.D. |
|Olivier Billet, Ph.D. |
|Dietmar Berger, M.D. |
|Jean-Noel Billaud, Ph.D. |
|Manuel Buttini, Ph.D. |
|Wei Cao, Ph.D. |
|Rocio Carrera, Ph.D. |
|Zhong Chen, MD |
|Jose R. Criado, Ph.D. |
|Francoise Dellu, Ph.D. |
|Mark Epping-Jordan, Ph.D. |
|Roberto Gianani, MD |
|Daniel Gonzalez-Dunia, Ph.D. |
|Wei Guo, Ph.D. |
|Mark Hansen, Ph.D. |
|Daniel Hassett, Ph.D. |
|Charles Heyser, Ph.D. |
|Andreas Holz, Ph.D. |
|Dirk Homann, M.D. |
|Thomas Horn, Ph.D. |
|Salvador Huitron-Resendiz, Ph.D. |
|Aaron Ilan, Ph.D. |
|Simon Katner, Ph.D. |
|Thomas Krucker, Ph.D. |
|Rong-Sheng Lee, Ph.D. |
|Daniel Lin, Ph.D. |
|Carmen Maldonado-Irizarry, Ph.D. |
|Gilles Martin, Ph.D. |
|Ignacio Mena, Ph.D. |
|Denise Naniche, Ph.D. |
|Thomas Nelson, Ph.D. |
|Zhiguo Nie, M.D. |
|Isabel Novella, Ph.D. |
|Ross O'Shea, Ph.D. |
|Axel Pagenstecher , Ph.D. |
|John Patterson, Ph.D. |
|Amanda Roberts, Ph.D. |
|Fernando Rodriguez, Ph.D. |
|Christian Sauder , Ph.D. |
|Amanda Smith, Ph.D. |
|Mariarosa Spina, Ph.D. |
|Anna Stalder, Ph.D. |
|Melanie Tallent, Ph.D |
|John Walker, Ph.D. |
|James B. Waters, Ph.D. |
|Michael Weed, Ph.D. |
|Naichen Yu, M.D. |
|Thomas Dyrberg, M.D. ||Novo Nordisk, Bagsvaerd, Denmark |
|Robert S. Fujinami, Ph.D. ||University of Utah School of Medicine, Salt Lake City, UT |
|Jean E. Gairin, Ph.D. ||CNRS, Toulouse, France |
|Leonard Glass, M.D. ||San Diego, CA |
|Jun-ichi Katayama, Ph.D. ||Hokkaido University, Sapporo, Japan |
|Rolf Kiessling, Ph.D. ||Karolinska Institute, Stockholm, Sweden |
|Lennart Mucke, M.D. ||Gladstone Institute, University of California, San Francisco, CA |
|Robert Purdy, Ph.D. ||University of Texas Health Science Center, San Antonio, TX |
|Barry T. Rouse, D.V.M., Ph.D. ||University of Tennessee, Knoxville, TN |
|Alexandra Valsamakis, M.D., Ph.D. ||Johns Hopkins Hospital, Baltimore, MD |
| * Joint appointment in Department of Cell Bology |
| ** Joint appointment in Department of Molecular and Experimental Medicine |
| *** Joint appointment in Department of Immunology |
|**** Joint appointment in Department of Molecular Biology |
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Floyd E. Bloom, M.D.
Their achievements in the past year continue to illustrate the significant productivity of our faculty members, once again making my task of summarizing their efforts and long-term goals a difficult one. Our essential departmental research strategy remains unchanged: We continue to focus our main efforts on the mechanisms by which infectious, environmental, and inheritable causes lead to psychiatric, neurologic, or endocrine disorders of the brain. We start here to acquire the information needed to devise treatments or preventions for these diseases. Given the finite amounts of space, human resources, and financial opportunities for serious engagement of the issues, we have elected to focus on a selected subset of human brain problems.
Although the disease-related implications of our research are more clear in some cases than in others, we also engage in fundamental biomedical research into the mechanisms by which infectious microbes can gain access to the brain or can initiate inflammatory and immune responses in the brain (see the reports by Drs. Oldstone, Buchmeier, de la Torre, and Whitton). This situation also applies to the basic mechanisms by which neurons (see the studies by Drs. Lai and Prieto) or glial cells (see the reports by Drs. Gruol, Lane, and Phillips) in the nervous system in developing or adult organisms express basic genetic information.
In keeping with the themes discussed in previous annual overviews, it is again with pleasure that I note the continued progress in the refinement of our whole-animal models of the CNS dysfunctions (so-called neuro-AIDS) produced in subjects infected with HIV. Those studies, emphasized in previous annual reports, have remained highly productive and insightful thanks to the efforts of Drs. Fox, Gold, Gruol, Henriksen, Phillips, and Siggins (see their specific project reports) and strengthen the already-powerful interdisciplinary collaborations within the department. In the past year, as newer medications have been released to suppress the effects of HIV infection on the peripheral immune system, the scientific community has come to recognize the increasing importance of the residual reservoir of virus within the endothelial cells of the brain's blood vessels (described first by members of this department) and of the brain's microglial cells and macrophages. Our investigators continue to focus on the ability to recognize the earliest phases of chronic CNS infections caused by feline and simian lentiviruses and to evaluate treatments that can reduce the consequences of, if not directly eradicate, the infections within the brain.
We are also moving ahead with drugs that can be evaluated in humans as well as in our experimental animal models. We have numerous examples of pathophysiologic changes that cross species, such as the neuropsychologic, sleep, and plasma cytokine disruptions in HIV-infected patients (see the reports by Drs. Darko, Henriksen, and Mitler). These changes are replicated on much shorter timescales in the feline and simian models of AIDS (see the reports by Drs. Fox, Gold, Henriksen, and Phillips). We are also continuing research with transgenic mice that overexpress inflammatory cytokines or selected glycoproteins of the lentivirus envelope (see the report by Dr. Campbell) and collaborations with former departmental member Lennart Mucke (see the report from Dr. Siggins). The similarity of the neurophysiologic alterations in infection caused by feline immunodeficiency virus and those caused by HIV and the reproducible progression of CNS signs and quantitative changes in both the simian and feline models of AIDS provide strong evidence of meaningful animal models of the neurologic aspects of AIDS.
This year, I also wish to call attention to another series of studies, undertaken as an important series of collaborations, that focus on our efforts to understand the mechanisms by which drugs of abuse can produce in specific persons long-lasting dependence or long-lasting craving for the use of these legal (alcohol and tobacco) or illegal (heroin, cocaine, amphetamines, and tetrahydrocannabinol) substances. Our studies again rely heavily on animal models (see the reports from Drs. Koob, Weiss, Gold, and Markou) in which drug self-administration is readily acquired and can be used to probe the molecular and cellular mechanisms of drug actions and the adaptations that the brain undergoes with continued drug exposure or after drug withdrawal (also see the reports by Drs. Siggins, Henriksen, Steffensen, and Schweitzer).
To investigate the basis by which some persons are genetically endowed with either susceptibility or resistance to the actions of alcohol and dependence, our faculty have shown the importance of parallel human studies on the relatives of male alcoholics (see the report from Dr. Polich) or in Native Americans (see the report by Dr. Ehlers). Our focus for the immediate future is to define the cellular changes that the brain undergoes after initial exposure to these drugs that may enhance the chances of self-administration when drug is again made available.
In addition, we have initiated several collaborations with scientists at Novartis to begin to examine animal models of Alzheimer's disease. We will examine lesions of specific chemical systems in the brain and will use transgenic mice that overexpress different genetic constructs of the ß-amyloid protein.
Our studies on these important interfaces between fundamental neuropharmacologic research and the need for medications to alleviate human brain diseases exemplify the hope that research brings to those who now have these illnesses.
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NeuroZoom: Neuroanatomic Atlases and Databases for Acquisition and Display of Stereologic Data
F.E. Bloom, W.G. Young, M. Morales, P. Sanna, E.L.F. Battenberg, J.G. Sutcliffe,* J.H. Morrison,** P. Hof,** E. Nimchinsky**
* Department of Molecular Biology, TSRI
** Mt. Sinai School of Medicine, New York, NY
Our efforts focus on using computer-assisted microscopy to obtain quantitative information on the expression of specific gene products in specific brain locations and to determine how these patterns of expression differ in discrete experimental models. In the past year, we have devoted our attention to the light and electron microscopic patterns of expression of the type 3 serotonin receptor (5-HT3R) and to the further refinement of the computer software NeuroZoom for data acquisition.
THE TYPE 3 SEROTONIN RECEPTOR
The 5-HT3R is a ligand-gated ion channel whose presence in the CNS has been established by radioligand binding, in situ hybridization, and immunohistochemical analysis. To analyze further the role of 5-HT3R, we used immunocytochemical studies with a polyclonal antibody to a synthetic peptide from the deduced amino acid sequence of cloned 5-HT3R to determine the overall patterns of expression of the receptor in the brain in rats.
Neurons expressing 5-HT3R were found in the forebrain, brain stem, and spinal cord, but within each region, the intensity of the immunoreactivity differed considerably. Within the forebrain, intensely immunoreactive cells were found in layers II--III of the neocortex, anterior olfactory nucleus, hippocampal formation, and amygdala. A few strongly immunoreactive neurons were consistently observed in the caudate putamen, and moderately or weakly labeled neurons were occasionally found in the nucleus accumbens. Within the brain stem, intensely labeled neurons were found in the trigeminal motor (V) and facial (VII) nuclei. Immunostained neurons were detected in the dorsal and the ventral horn of the spinal cord. These results show that the 5-HT3R--immunoreactive neurons are broadly distributed throughout the brain and spinal cord in rats and suggest that this receptor participates significantly in CNS neurotransmission.
We have since used in situ hybridization and immunocytochemical studies to show that neurons expressing 5-HT3R in rats are mainly neurons that contain -aminobutyric acid (GABA) in the telencephalon. We determined that neurons containing 5-HT3R and GABA do not express somatostatin but often contain cholecystokinin. Cells that expressed both 5-HT3R and cholecystokinin were observed in the neocortex, olfactory cortex, hippocampus, and amygdala. The interneurons that contained both 5-HT3R and cholecystokinin accounted for 35--66% of the total population of cholecystokinin-containing cells in the neocortex.
To further characterize the neurons that contain both 5-HT3R and GABA, we determined the immunoreactivity of the cells to calcium-binding protein. The results showed that these neurons lack parvalbumin and are a subpopulation of interneurons containing calbindin that are preferentially present in the CA1--CA3 subfield of the hippocampus. Some neurons that contained 5-HT3R, GABA, and calretinin were found in the neocortex, olfactory cortex, hippocampus, and amygdala, but these neurons were more often present in the agranular insular and piriform cortices.
We conclude that the neuronal expression of 5-HT3R is selective within the GABA neuron population in the rat telencephalon. These 5-HT3R--expressing interneurons might contain cholecystokinin, calbindin, and calretinin. We suggest that via the 5-HT3R, serotonin may regulate GABA and cholecystokinin neurotransmission in the telencephalon.
Recent developments in unbiased quantitative techniques have had a dramatic impact on the way neuroanatomic data are quantified and have created an opportunity and a demand for computerized techniques that can simplify the acquisition, analysis, and organization of these data. To address this need, we have developed NeuroZoom, a software package with full-scale mapping capacities, three-dimensional cell reconstruction, a stereology package, and capabilities for the development of databases and atlases.
Currently, NeuroZoom has the following stereologic tools: optical dissector and optical fractionator for particle counts, calculation of surface area, and estimation of volume based on the Cavalieri principle; the Rotator; and the Nucleator. These last two newer tools have been validated against each other and against the Cavalieri technique by using a confocal microscope, and their usefulness for the quantification of changes in cell volume at the population level has been shown. All tools are designed to be used in conjunction with a light microscope equipped with a motorized stage, but they can be applied to imported images, including electron micrographs and confocal images, and to any scanned photograph of known scale.
This software package has been used to assess neuronal loss in visual pathways in a monkey model of glaucoma, study brain aging in macaque monkeys, and analyze vulnerable spinal cord neurons in a mouse model of amyotrophic lateral sclerosis. Ongoing applications include the development of quantitative databases of strains of mice commonly used to generate transgenic and knockout animals to provide normative data for the analysis of models of neurodegenerative diseases.
Bloom, F.E. Science and technology policy: A scientist's view. Calif. West. Law Rev. 33:63, 1996.
Bloom, F.E., Trembleau, A., Morales, M., Battenberg, E.F., Young, W.G. The gains in brain through stain remain germane. In: Molecular Mechanisms of Neuronal Communication: A Tribute to Nils-Ake Hillarp. Fuxe, K., et al. (Eds.). Pergamon, New York, 1997, p. 3.
Bloom, F., Young, W.G., Nimchinsky, E.A., Hof, P.R., Morrison, J.H. Neuronal vulnerability and informatics in human disease. In: Neuroinformatics: An Overview of the Human Brain Project. Koslow, S.H., Huerta, M.F. (Eds.). Lawrence Erlbaum, Mahwah, NJ, 1997, p. 83.
Cooper, J.R., Bloom, F.E., Roth, R.H. The Biochemical Basis of Neuropharmacology, 7th ed. Oxford University Press, New York, 1996.
Cunningham, E.T., Jr., Stalder, A., Sanna, P.P., Liu, S., Bloom, F.E., Howes, E.L., Jr., Campbell, I.L., Margolis, T.P. Localization of tumor necrosis factor receptor messenger RNA in normal and herpes simplex virus infected mouse eye. Invest. Ophthalmol. Vis. Sci. 38:9, 1997.
Gallen, C.C., Tecoma, E., Iragui, V., Sobel, D.F., Schwartz, B., Bloom F.E. Magnetic source imaging of abnormal low frequency magnetic activity (ALFMA) in pre-surgical evaluations of epilepsy. Epilepsia, in press.
Gautvik, K.M., de Lecea, L., Gautvik, V.T., Danielson, P.E., Tranque, P., Dopazo, A., Bloom, F.E., Sutcliffe, J.G. Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction. Proc. Natl. Acad. Sci. U.S.A. 93:8733, 1996.
Heinrichs, S.C., Stenzel-Poore, M.P., Gold, L.H., Battenberg, E., Bloom, F.E., Koob, G.F., Vale, W.W., Pich, E.M. Learning impairment in transgenic mice with central overexpression of corticotropin-releasing factor. Neuroscience 74:303, 1996.
Jacobson, S., Henriksen, S.J., Prospero-Garcia, O., Phillips, T.R., Elder, J.H., Young, W.G., Bloom, F.E., Fox, H.S. Cortical neuronal cytoskeletal changes associated with FIV infection. J. Neurovirol., in press.
Mathiasen, J.R., Maciejewski-Lenoir, D., Bloom, F.E., Sladek, C.D. Increased vasopressin secretion from hypothalamic cultures following administration of exogenous vasopressin mRNA. Exp. Neurol. 141:165, 1996.
Mehta, P.P., Battenberg, E., Wilson, M.C. SNAP-25 and synaptotagmin involvement in the final Ca2+-dependent triggering of neurotransmitter exocytosis. Proc. Natl. Acad. Sci. U.S.A. 93:10471, 1996.
Nelson, C.A., Bloom, F.E. Child development and neuroscience. Child Dev., in press.
Phillips, T.R., Prospero-Garcia, O., Wheeler, D.W., Wagaman, P.C., Lerner, D.L., Fox, H.S., Whalen, L.R., Bloom, F.E., Elder, J.H., Henriksen, S.J. Neurologic dysfunctions caused by a molecular clone of feline immunodeficiency virus, FIV-PPR. J. Neurovirol. 2:388, 1996.
Prospero-Garcia, O., Gold, L.H., Fox, H.S., Polis, I., Koob, G.F., Bloom, F.E., Henriksen, S.J. Microglia-passaged simian immunodeficiency virus induces neurophysiological abnormalities in monkeys. Proc. Natl. Acad. Sci. U.S.A. 93:14158, 1996.
Raber, J., Koob, G.F., Bloom, F.E. Interferon- and transforming growth factor-ß1 regulate corticotropin-releasing factor release from the amygdala; comparison with the hypothalamic response. Neurochem. Int. 30:455, 1997.
Ryabinin, A.E., Criado, J.R., Henriksen, S.J., Bloom, F.E., Wilson, M.C. Differential sensitivity of c-FOS expression in hippocampus and other brain regions to moderate and low doses of alcohol. Mol. Psychiatry 2:32, 1997.
Sanna, P.P., De Logu, A., Williamson, R.A., Hom, Y.-L., Straus, S.E., Bloom, F.E., Burton, D.R. Protection of nude mice by passive immunization with a type-common human recombinant monoclonal antibody again HSV. Virology 215:101, 1996.
Weiss, F., Parsons, L.H., Schulteis, G., Hyytia, P., Lorang, M.T., Bloom, F.E., Koob, G.F. Ethanol self-administration restores withdrawal-associated deficiencies in accumbal dopamine and 5-hydroxytryptamine release in dependent rats. J. Neurosci. 16:3474, 1996.
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Viral Structure, Function, and Pathogenesis
M.J. Buchmeier, T.E. Lane, T. Nash, A. Paoletti, K.L. Gaarder, H. McClary, R. Marteijn, J.R. Baldridge,* T. McGraw**
* RIBI Immunochemical Research, Hamilton, MT
** University of Florida, Gainesville, FL
Current hypotheses to explain the etiology and pathogenesis of demyelinating diseases in humans include the evidence that an infectious agent encountered early in life may trigger a disease process that is manifested later in life as demyelination of white matter in the CNS. In our laboratory, we use mouse hepatitis virus (MHV) infection of mice as a model for virus-induced demyelination. Because it causes a chronic demyelinating disease with many clinical and pathologic correlates with the human demyelinating disease multiple sclerosis, MHV infection is used as an animal model for this human disease. We have focused our attention on the mechanisms by which demyelination occurs after infection with the virus.
Maintenance of a chronic inflammatory response appears to be critical in demyelination. A prominent feature of MHV-induced demyelination is the presence of infiltrates composed of inflammatory cells, including T lymphocytes and monocytes associated with demyelinating lesions. We have new evidence that CD4 T cells are required for demyelination; little to no demyelination develops in CD4 knockout mice infected with MHV.
In addition, we have investigated the signals that may recruit cells into the CNS during chronic disease. The chemokines are members of a family of low molecular weight, proinflammatory cytokines that function as important chemoattractant signals for distinct leukocyte populations during periods of inflammation. In collaboration with I. Campbell, Department of Neuropharmacology, we used a multichemokine probe set to evaluate chemokine transcripts that are expressed during acute and chronic disease. Transcripts for the chemokines CRG-2, RANTES, MCP-1, MCP-3, and MIP-1ß were detected during acute viral encephalomyelitis, whereas only the ones for CRG-2 and RANTES were expressed during chronic demyelination. Further analysis revealed that CRG-2 mRNA colocalized with MHV RNA during all stages of infection and was associated with demyelinating plaque lesions in the spinal cords of infected mice (Fig. 1).
Astrocytes were the predominant cell type expressing CRG-2 transcripts, although expression was also observed in microglial cells and macrophages during the acute phase of disease. MHV infection of neonatal mouse astrocytes in vitro revealed a chemokine transcript profile similar to that observed in vivo. Astrocytes expressed transcripts for CRG-2, MCP-1, MCP-3, MIP-1ß, and MIP-2 and the cytokines IL-6 and transforming growth factor-ß1 after 24 hours' exposure to MHV. Expression of chemokines and cytokines appeared to depend on both the concentration of virus and viral replication.
Our studies clearly show that viral infection of the CNS results in an orchestrated cascade of chemokine expression that directs the recruitment of T cells and macrophages into the CNS to sites of MHV persistence. The mechanism by which this chronic inflammatory response damages myelin is under investigation.
The route and mechanism by which viruses enter and infect cells may have a profound influence on the pathogenesis of disease. We have studied a unique fusion mutant of MHV-JHM (OBLV60) to establish its route of entry into cells of neuronal and nonneuronal origin.
OBLV60 is an acid-dependent syncytium-forming variant isolated from OBL21 cells persistently infected with the pH-independent MHV-4 strain. The fusion activity of OBLV60 can be strictly regulated by controlling pH and thus provides the means to definitively examine the entry of MHV into cells by endosomal and nonendosomal pathways. Shortly after high-multiplicity infection, both MHV-4 and OBLV60 were detected by electron microscopy in endosomal vesicles and were recovered from lysates of cells treated with proteinase K to remove extracellular virus. For OBLV60, but not MHV-4, exposure to lysosomotropic compounds early in infection prevented viral penetration and significantly reduced viral yields. These results suggest that both MHV-4 and OBLV60 used the endosomal route of entry into cells but that MHV-4 did not require acidification of endosomal vesicles.
For studies on the entry of virus by fusion at the cell surface, we briefly exposed surface-bound OBLV60 to a fusion-permissive pH under conditions that prevent endocytic entry. Acid treatment of surface-bound OBLV60 caused a significant increase in the yields of virus produced in cultures of fusion-sensitive Sac or DBT cells, showing entry of virus by fusion at the cell surface. No measurable increase in the production of virus was detected with acid treatment of OBLV60 bound to OBL21 cells, suggesting that entry at the cell surface does not occur in these cells, which are resistant to MHV-induced formation of syncytia. These results raise interesting questions about how mechanisms of MHV entry influence the selection of fusion variants and how the fusion phenotype affects neuronotropic effects of the virus.
Emerging Virus Research Center
M.J. Buchmeier, D.R. Burton, S. St. Jeor,* K. McGwire,* A. Sette,** S. Southwood,** C.J. Peters***
* University of Nevada, Reno, NV
** Cytel Corporation, San Diego, CA
*** Centers for Disease Control and Prevention, Atlanta, GA
The TSRI-UNR Emerging Virus Research Center was established to address several fundamental problems related to epidemiology of and immune responses to emerging viruses of the arenavirus, filovirus, and hantavirus groups. The center supports work in four specific project areas.
Lassa virus is an important pathogen that infects large numbers of people in West Africa and causes substantial morbidity and mortality. In a project directed by M. Buchmeier, TSRI, with the collaboration of A. Sette and S. Southwood, Cytel Corp., peptide epitopes for Lassa-specific cytotoxic T-lymphocyte responses in humans are being determined. Predictive computer algorithms are used to select 9- and 10-mer epitopes from the sequences of the Lassa virus nucleoprotein and glycoprotein genes. Peptides selected for synthesis have been assessed for the capacity to bind at high affinity to MHC class 1 molecules.
To date, we have selected and synthesized 84 peptides from Lassa nucleoprotein and glycoprotein and have completed binding-assay determinations of A11, A31, A33, and A68 alleles. Fifteen of these peptides bound to one or more alleles at high affinity (<50 nM). Eight of these 15 bound to three or more A alleles. These data will provide a clear view of the diversity and specificity of the human cytotoxic T-lymphocyte response to Lassa virus and a guide for efforts to develop an effective vaccination strategy against this important arenavirus.
Hantavirus and Ebola virus epidemics in the United States and Africa have heightened public awareness to the threat posed by emerging viruses. In research directed toward developing effective therapeutic and vaccine approaches to infections caused by these viruses, D. Burton, Department of Immunology, is using recombinant phage-display techniques to generate panels of human antibodies to Ebola virus and hantavirus. RNA derived from the bone marrow of two patients who recovered from infection with Ebola virus during the 1995 outbreak in Kikwit, Zaire, were used to develop antibody phage-display libraries. The libraries were panned against purified virions and inactivated by -irradiation, and antibodies that bind well to the envelope glycoprotein on the surface of Ebola-infected cells and neutralize the virus were selected. These antibodies will be evaluated in animal models.
The center includes two investigators at the University of Nevada at Reno. One of these, S. St. Jeor, is investigating why Sin Nombre virus has a relatively high rate of endemic infection in Peromyscus spp. but a low incidence of infection in high-risk human populations exposed to infected rodents. Factors to be investigated include variations in viral strains, genetic differences within the genus Peromyscus, pathogenesis of the virus in Peromyscus, and ecologic factors that affect the incidence of infection. Biological and immunologic properties of variants of Sin Nombre virus isolated from humans and Peromyscus spp. will be compared. Antibodies from rodents, humans, and a human antibody expression library will be tested for viral neutralization (plaque reduction) of the variants. Pathogenesis studies will use in situ hybridization and immunochemistry to determine the distribution of virus in tissues of naturally infected Peromyscus.
K. McGwire, University of Nevada, Reno, is studying the ecology and epidemiology of hantavirus infection in populations of wild deer mice in collaboration with S. St. Jeor. To establish a map of the codistribution of the virus and its rodent host, extensive sampling in a variety of habitats, longitudinal (repeated) sampling of several sites, and examination of remote sensing data were combined. Hantavirus was present in deer mice at about 80% of the sites within the Walker River basin of California and Nevada. Sites at lower elevations, with relatively low densities of vegetation, were significantly less likely than higher elevations to harbor hantaviruses, although deer mice were present at these sites in moderate densities. The prevalence of the virus cycled in populations, with a period of 6--12 months and an amplitude of 0--60%. No evidence was found for a relationship between density of deer mice and local viral prevalence. In study areas where deer mice were banded and recaptured multiple times, it has been possible to obtain estimates of the length of time virus remains in the blood and of the time required after the detection of antibody for viral clearance. Convincing evidence has been found for the coevolution of hantaviruses with their rodent hosts in the field.
Baldridge, J., McGraw, T., Paoletti, A., Buchmeier, M.J. Antibody prevents establishment of persistent arenavirus infection in synergy with endogenous T cells. J. Virol. 71:755, 1997.
Geiger, K.D., Nash, T.C., Sawyer, S., Krahl, T., Patstone, G., Reed, J.C., Krajewski, S., Dalton, D., Buchmeier, M.J., Sarvetnick, N. Interferon-gamma protects neurons against herpes simplex virus-1 mediated neuronal death. Virology, in press.
Lane, T.E., Asensio, V., Paoletti, A., Yu, N., Campbell, I., Buchmeier, M.J. Dynamic regulation of and ß chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease. J. Immunol, in press.
Lane, T.E., Buchmeier, M.J. Murine coronavirus infection: A paradigm for virus-induced demyelinating disease. Trends Microbiol. 5:9, 1997.
Lane, T.E., Buchmeier, M.J. Response to Letters, T. Christensen. Trends Microbiol. 5:139, 1997.
Lane, T.E., Paoletti, A., Buchmeier, M.J. Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus. J. Virol. 71:2202, 1997.
Nash, T.C., Buchmeier, M.J. Entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways. Virology 233:1, 1997.
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Mechanisms of Inflammation in the CNS
I.L. Campbell, Y. Akwa, V. Asensio, M. Benedict, P. Castelnau, C. Kincaid, A. Pagenstecher, A.K. Stalder, E. Masliah,* H.C. Powell*
* University of California, San Diego, CA
Inflammation in the CNS is involved in the pathogenesis of a variety of important neurologic disorders such as multiple sclerosis, HIV encephalopathy, stroke, Alzheimer's disease, scrapie (spongiform encephalopathy), other infectious diseases, and trauma. Knowledge of the mechanisms of inflammation in the CNS therefore is an important research objective. We continue to focus on the cytokines, because members of this diverse family of soluble factors are the principal regulators of inflammatory processes. We have developed transgenic approaches that enable us to target the constitutive expression of a variety of cytokines to astrocytes in the intact CNS in mice.
Using glial fibrillary acidic protein (GFAP) fusion gene constructs, we have generated transgenic mice that express the murine cytokines IL-6, IL-3, IFN-, TNF-, or IL-12 in astrocytes. Transgenic expression of each cytokine produces a unique spectrum of neuropathologic and functional alterations (Fig. 1),
thereby directly implicating these mediators in the pathogenesis of inflammatory CNS disease. Many of the neurologic features observed in the CNS of the GFAP-cytokine transgenic mice are similar to those reported in human neurodegenerative and demyelinating diseases such as HIV encephalopathy, Alzheimer's disease, and multiple sclerosis and further underscore the likely importance of cytokine-mediated inflammation in the pathogenesis of the human disorders. Moreover, these models are valuable tools that we can use in multilevel analysis to link molecular and cellular alterations to specific electrophysiologic, neuroendocrine, and behavioral outcomes. Such integrative studies in the GFAP-cytokine transgenic mice are providing a more thorough understanding of the actions of cytokines in the CNS and bridge the gap between structural and functional neuropathologic changes.
We are exploiting these unique transgenic models, together with other experimental models of neuroinflammation such as experimental autoimmune encephalomyelitis, lipopolysaccharide endotoxemia, and lymphocytic choriomeningitis, to better understand the key regulatory steps involved in the neuroinflammatory processes associated with CNS expression of different cytokines. We have focused particularly on the mechanisms involved in the recruitment and trafficking of mononuclear cells to the brain and on the events that influence the function of these cells after entry into the brain. Key targets for investigation are the chemoattractant cytokines or chemokines, matrix metalloproteinases (MMPs), and the tissue inhibitors of the MMPs (TIMPs).
Chemokines play a crucial role in the tissue trafficking of leukocytes during inflammation. Our results suggest that distinct patterns of expression of chemokine genes occur in the brain in different neuroinflammatory states (Fig. 2)
and that these patterns may ultimately determine the repertoire and activity of the infiltrating leukocytes. Important topics under investigation are (1) the precise role of individual chemokines in the trafficking of specific leukocytes and other functions of these mediators and (2) the possible use of chemokines or their receptors as potential therapeutic targets to ameliorate inflammation in the CNS.
MMPs are enzymes involved in the degradation of the extracellular matrix during processes such as organogenesis and inflammation. TIMPs are important inhibitors of MMP activity and protect cells and tissues against potentially damaging actions of the MMPs. Examination of MMP and TIMP gene regulation in the CNS revealed distinct temporospatial expression of these genes in experimental autoimmune encephalomyelitis and in GFAP-cytokine transgenic mice. The MMPs are expressed predominantly by infiltrating mononuclear cells; the TIMPS, by resident CNS cells. An imbalance in the MMP:TIMP ratio may facilitate trafficking of the immune cells through the brain parenchyma and may contribute to tissue destruction. These possibilities and the potential for therapeutic intervention via inhibition of MMP activity will be examined in ongoing studies.
Asensio, V.C., Campbell, I.L. Chemokine gene expression in the brain in lymphocytic choriomeningitis. J. Virol. 71:7832, 1997.
Barnum, S.R., Jones, J.L., Samimi, A., Campbell, I.L. Chronic complement C3 expression in the CNS of transgenic mice with astrocyte-targeted interleukin-6 expression. Glia 18:107, 1996.
Campbell, I.L. Editorial. Methods 10:331, 1996.
Campbell, I.L. Exacerbation of lymphocytic choriomeningitis in mice treated with the inducible nitric oxide synthase inhibitor aminoguanidine. J. Neuroimmunol. 71:31, 1996.
Campbell, I.L. Structural and functional impact of transgenic expression of cytokines in the CNS. Ann. N.Y. Acad. Sci., in press.
Campbell, I.L. Transgenic mice and cytokine actions in the brain: Bridging the gap between structural and functional neuropathology. Brain Res. Rev., in press.
Campbell, I.L., Powell, H.C. Role of cytokines in demyelinating disease studied in transgenic mice. Methods 10:462, 1996.
Campbell, I.L., Stalder, A.K., Chiang, C.-S., Bellinger, R., Heyser, C.J., Steffensen, S.C., Masliah, E., Powell, H.C., Gold, L.H., Henriksen, S.J., Siggins, G.R. Transgenic models to assess the pathogenic actions of cytokines in the central nervous system. Mol. Psychiatry 2:125, 1997.
Cunningham, E.T., Jr., Stalder, A., Sanna, P.P., Liu, S., Bloom, F.E., Howes, E.L., Jr., Campbell, I.L., Margolis, T.P. Localization of tumor necrosis factor receptor messenger RNA in normal and herpes simplex virus infected mouse eyes. Invest. Ophthalmol. Vis. Sci. 38:9, 1997.
Cunningham, E.T., Jr., Stalder, A.K., Sanna, P.P., Liu, S.S., Bloom, F.E., Howes, E.L., Jr., Campbell, I.L., Margolis, T.P. Distribution of tumor necrosis factor receptor messenger RNA in normal and herpes simplex virus infected trigeminal ganglia in the mouse. Brain Res. 758:99, 1997.
Hernandez, J., Molinero, A., Campbell, I.L., Hidalgo, J. Transgenic expression of interleukin 6 in the central nervous system regulates brain metallothionein-I and -III expression in mice. Mol. Brain Res. 48:125, 1997.
Heyser, C.J., Masliah, E., Samimi, A., Campbell, I.L., Gold, L.H. Progressive learning impairment paralleled by inflammatory neurodegeneration in CNS-interleukin-6 transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 94:1500, 1997.
Pagenstecher, A., Stalder, A.S., Campbell, I.L. RNase protection assays for the simultaneous and semiquantitative analysis of multiple murine matrix metalloproteinase (MMP) and MMP inhibitor mRNAs. J. Immunol. Methods 206:1, 1997.
Stalder, A., Pagenstecher, A., Yu, N.C., Kincaid, C., Chiang, C.-S., Hobbs, M.V., Bloom, F.E., Campbell, I.L. Lipopolysaccharide-induced interleukin-12 expression in the CNS and cultured astrocytes and microglia. J. Immunol. 159:1344, 1997.
Wood, L.C., Stalder, A.S., Liou, A., Campbell, I.L., Grunfeld, C., Elias, P.M., Feingold, K.R. Barrier disruption increases gene expression of cytokines and the 55Kd TNF receptor in murine skin. J. Invest. Dermatol. 6:98, 1997.
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Laboratory of Clinical Neurophysiology and San Diego Native American Project
C.L. Ehlers, S. Lopez, S. Robeck, E. Phillips, C. Somes, D. Cloutier, J. Havstad, T. Wall, E. Riley, V. Wong
Our research program is dedicated to understanding the genetic factors involved in the development of alcoholism and the toxic consequences of alcohol abuse. Alcoholism is a disorder in which genetic factors interact with environmental variables to produce patterns of abuse and dependence. The importance of genetics in the vulnerability to alcoholism is supported by evidence from family, twin, and adoption studies. These studies have shown that sons of alcoholics have a fourfold to fivefold greater risk than sons of nonalcoholics for the development of alcoholism. Individual sensitivity to alcohol has been proposed as one of the best understood inherited factors that affect the likelihood of drinking and mediate disposition for the development of alcoholism.
Our findings and those of our collaborators predict that persons at higher genetic risk for alcoholism are less sensitive to the effects of alcohol. Support for this theory is provided by studies that compared responses in subjects with and without a family history of alcoholism. At moderate doses of alcohol, the two groups attained equivalent blood alcohol concentrations, but subjects with a family history of alcoholism rated themselves as significantly less intoxicated than the other subjects did and had CNS signs that indicated a less depressant effect of alcohol. Additional support for this theory is provided by our studies in Asians, who, as a whole, have significantly lower rates of alcoholism than do non-Asians. Asian subjects with mutations in the gene for aldehyde dehydrogenase, an enzyme that breaks down alcohol and that is deficient in 40% of Asians, had more intense, although not necessarily more adverse, responses to alcohol than Asians without this mutation.
In parallel studies, we have been investigating the response to alcohol in Native American subjects. Compared with other ethnic groups, some tribes of Native Americans have higher rates of alcohol abuse and alcohol dependence. Thus, Native Americans are an especially important group to study because of their extremely high risk for alcoholism. Most hypotheses advanced to explain the problem drinking that can occur in some Native American tribes are sociocultural explanations. A once popular hypothesis, the firewater myth, proposes that Native Americans are constitutionally unable to handle alcohol.
Although the idea that Native Americans are more sensitive to the effects of alcohol has been propagated for more than three centuries, it has not been previously directly investigated in the laboratory. We found that young adult Native American men may have a qualitatively different response to alcohol, although not what would be expected on the basis of the firewater myth, as well as genetic variations in alcohol dehydrogenase. Both of these factors can contribute to the development of alcoholism or alcohol-related disease.
Native American men as a group had markedly diminished objective and subjective responses to ingestion of alcohol, a finding similar to what we found in Caucasian subjects at high risk for alcoholism. In response to a challenge dose of alcohol, Native American men had no changes in cardiovascular status and no increases in stress hormones, findings commonly seen in Asian and Caucasian subjects. In addition, the Native American subjects had fewer CNS signs of the depressant effects of alcohol. Finally, we found that a small proportion of Native American men have variations in the gene for one isozyme of alcohol dehydrogenase. Although this enzymatic difference most likely has no affect on the development of alcoholism, it could make the subjects at greater risk for liver disease if they choose to drink. These findings clearly show that biological factors play a role in the increased risk for the development of alcoholism in Native American men.
These findings, of probable genetic mediation of differences in response to drinking, led us to investigate what mechanisms in the CNS might regulate alcohol intoxication and dependence. Therefore, we have studied both animal models of alcohol dependence and rats genetically selected and bred for alcohol-preferring and alcohol-nonpreferring drinking behaviors. Using electrophysiologic measures, we found that, like sons of alcoholics, rats that prefer to drink alcohol have differences in electroencephalographic patterns and show fewer intoxicating and more activating effects of alcohol than the alcohol-nonpreferring rats do.
The alcohol-preferring rats also have significantly lower levels of the neuromodulators corticotropin releasing factor and neuropeptide Y in several areas of the brain. Because these neurohormones are intimately involved in the stress response, these studies suggest that the genetic mediation of alcohol-seeking behavior and the response to alcohol in these animals may involve physiologic differences in responses to "stressors." We also found that neuropeptide Y, when injected into the brain, is a powerful anxiety-reducing compound and can mimic many of the effects of alcohol, as well as block the effects of corticotropin releasing factor.
Persons with a drinking problem who wish to quit drinking must learn to change their habits and thoughts about drinking behaviors. We have used an animal model to investigate how the brain responds and adapts to changes in stimuli previously associated with "rewards." In this study, a form of conditioning is used so that an animal (in this case, a rat) anticipates the delivery of a highly palatable food with the presentation of certain sounds.
We showed that a series of brain waves called event-related potentials are elicited by the sounds associated with food rewards. If over a period of a week or so, the sound previously associated with reward is presented to the animal but is now not rewarded by food, the animal's brain responses to the sound change dramatically and predictably but only in certain key areas. In anterior areas of the cerebral cortex and in a deep brain structure called the amygdala, processing of these sounds is now done differently. A brain wave component of the event-related potential called the N1-P2 that has been associated with attention is now dramatically reduced, whereas other components associated with memory and the behavioral relevance of the stimuli remain unchanged.
These data suggest that animals react to changes in reward by reducing their attention to or ignoring previously important stimuli. Such findings may have important implications for the treatment of drug abuse disorders; they imply that previously rewarding stimuli (e.g., drugs) do not lose their importance by a process of passive habituation or loss of salience but rather through an active process of not paying attention to "triggering" stimuli. In addition, these results suggest that changes in information processing in the frontal cortex and amygdala may underlie behavioral changes associated with reward-related processes.
One of the tragic consequences of alcohol abuse during pregnancy can be development of the fetal alcohol syndrome (FAS). FAS is characterized by the presence of several birth defects, including growth retardation, craniofacial abnormalities, and CNS disorders. Delineation of the mechanisms underlying the development of FAS has been hampered by the lack of an objective clinical test. We have recently developed a clinical battery of tests to characterize the behavior, temperament, and neurophysiology in children with FAS.
This battery has been used to compare children known to have FAS from birth with matched control subjects with no abnormalities and with other children with mental retardation such as Down syndrome. Whereas children with Down syndrome had significantly lower IQ scores, children with FAS had markedly more behavior problems such as aggression, sexual problems, and low task orientation. Electrophysiologic findings also differed in these two groups. In children with Down syndrome, electrophysiologic abnormalities were more widespread over cortical areas; in children with FAS, they were mild and limited to the left hemisphere and posterior cortical regions.
This study also provides support for a set of objective clinical tests that can be used to distinguish children with FAS from control subjects (children without FAS) and from other children with cognitive disorders. These studies not only will facilitate the detection of children with the syndrome but also may help in understanding what brain regions may be particularly vulnerable to alcohol abuse and dependence during pregnancy.
Ehlers, C.L., Havstad, J.W., Kupfer, D.J. Estimation of the time course of slow-wave sleep over the night in depressed patients: Effects of clomipramine and clinical response. Biol. Psychiatry 39:171, 1996.
Ehlers C.L., Kupfer, D.J. Slow wave sleep: Do young adult men and women age differently? J. Sleep Res. 6:211, 1997.
Ehlers, C.L., Phillips, E., Parry, B.S. Electrophysiological findings during the menstrual cycle in women with and without late luteal phase dysphoric disorder: Relationship to risk for alcoholism? Biol. Psychiatry 39:720, 1996.
Ehlers, C.L., Somes, C., Lopez, A., Kirby, D., Rivier, J.E. Electrophysiological actions of neuropeptide Y and its analogs: New measures for anxiolytic therapy? Neuropsychopharmacology 17:31, 1997.
Ehlers, C.L., Somes, C., Seifritz, E., Rivier, J.E. CRF/NPY interactions: A potential role in sleep dysregulation in depression and anxiety. Depress. Anxiety 6:1, 1997.
Ehlers, C.L., Somes, C., Thomas, J., Riley, E.P. Effects of neonatal exposure to nicotine on electrophysiological parameters in adult rats. Pharmacol. Biochem. Behav. 58:713, 1997.
Garcia-Andrade, C., Wall, T.L., Ehlers, C.L. Alcohol expectancies in a Native American population. Alcohol. Clin. Exp. Res. 20:1438, 1997.
Garcia-Andrade, C., Wall, T.L., Ehlers, C.L. The firewater myth and response to alcohol in Mission indians. Am. J. Psychiatry 154:7, 1997.
Kaneko, W.M., Ehlers, C.L., Phillips, E.L., Riley, E.P. Auditory event-related potentials in fetal alcohol syndrome and Down syndrome children. Alcohol. Clin. Exp. Res. 20:35, 1996.
Kaneko, W.M., Phillips, E.L., Riley, E.P., Ehlers, C.L. EEG findings in fetal alcohol syndrome and Down syndrome children. EEG Clin. Neurophysiol. 98:20, 1996.
Kaneko, W.M., Riley, E.P., Ehlers, C.L. Effects of artificial rearing on electrophysiology and behavior in adult rats. Depression 4:279, 1997.
Orozco, S., Ehlers, C.L. Gender differences in electrophysiological responses to facial stimuli. Biol. Psychiatry, in press.
Parry, B.L., Ehlers, C.L., Mostofi, N., Phillips, E. Personality traits in LLPDD and normal controls during follicular and luteal menstrual cycle phases. Psychol. Med. 26:197, 1996.
Wall, T.L., Garcia-Andrade, C., Thomasson, H.R., Carr, L.G., Ehlers, C.L. Alcohol dehydrogenase polymorphisms in Native Americans: Identification of the ADH2*3 allele. Alcohol Alcohol. 32:129, 1997.
Wall, T.L., Garcia-Andrade, C., Thomasson, H.R., Cole, M., Ehlers, C.L. Alcohol elimination in Native American Mission indians: An investigation of interindividual variation. Alcohol. Clin. Exp. Res. 20:1159, 1996.
Wall, T.L., Peterson, C.M., Peterson, K.P., Johnson, M.L., Thomasson, H.R., Cole, M.D., Ehlers, C.L. Alcohol metabolism in Asian American men with genetic polymorphisms of aldehyde dehydrogenase. Ann. Intern. Med. 127:376, 1997.
Wall, T.L., Thomasson, H., Ehlers, C.L. Investigator-observed flushing but not self-report of flushing is a valid predictor of ALDH2 genotype. J. Stud. Alcohol 57:267, 1996.
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Pathogenesis of AIDS-Related CNS Dysfunction
H.S. Fox, S. Browne, Z. Chen, T. Horn, T.E. Lane, C. Phillipson, D.D. Watry, M. Zandonatti
Among the many problems affecting those with HIV infection is CNS dysfunction, ranging from a mild cognitive or motor disorder to severe dementia. We use two models, infection with simian immunodeficiency virus (SIV) in rhesus macaques and infection with feline immunodeficiency virus (FIV) in cats, to understand the roles of both the virus and the host response in inducing CNS disease.
We have selected a neurovirulent SIV strain by serially transferring infected microglia in vivo. The passage resulted in enrichment of a unique env quasi species in the infected brains. Serial passage also expanded the tropism of the virus to allow infection of cerebrovascular endothelial cells. A number of infectious molecular clones of SIV constructed from virus after the third serial passage of microglia are being analyzed in vitro. The goal is to use them for in vivo molecular virology studies of neuropathogenesis. We are also examining the genetic loci responsible for cerebrovascular endothelial cell tropism.
Although the factors leading to CNS dysfunction are still obscure, we have now detected a number of distinct pathologic changes in the brains of infected animals. SIV-specific cytotoxic T cells have been found in both the cerebrospinal fluid and the brain parenchyma. We also found a number of potentially deleterious molecules produced in the CNS by macrophages and T cells, including IFN-, IL-1ß, TNF-, and the inducible form of nitric oxide. Furthermore, SIV infection can induce leakage across the blood-brain barrier. To further investigate the role of this barrier in disease, we have started an in-depth study. We have detected novel molecules that are expressed by the specialized endothelial cells of the blood-brain barrier. Further characterization of these cells should lead to a better understanding of the role of the blood-brain barrier.
The neurovirulent SIV also induces functional changes in the CNS, as shown by behavioral and electrophysiologic testing by our colleagues in the department. We are currently using a cell-free stock of virus derived from infected microglia. With this stock, we have detected electrophysiologic abnormalities as early as 1 month after infection. Radiotelemetric methods have been developed to enable the remote monitoring of body temperature, movement, and electroencephalographic changes in the animals. Before infection, monkeys have stable circadian rhythms. Acute SIV infection induces an increase in body temperature, overlapped by a decrease in movement, that causes a disruption of the circadian rhythm for 3--4 days. Electroencephalographic recordings show a reduction in slow-wave sleep frequencies during the first month of infection. Intermittently during the course of infection, and during the final stage of disease, loss of circadian rhythm also occurs. Both the neurophysiologic abnormalities and the pathologic changes described earlier can play a role in decreased cognitive performance.
Neuronal damage and loss have been found in the brain in patients with AIDS. We have detected an alteration in the neuronal cytoskeleton in the FIV model that may precede these degenerative changes. Normally less than 3% of pyramidal cells in the frontal lobe are immunoreactive with an antibody that recognizes a nonphosphorylated epitope on neurofilament proteins. However, in cats infected with FIV, the number of immunoreactive neurons increased up to sixfold before immunodeficiency developed. These changes were similar to those seen in an animal with a noninfectious injury, suggesting that the increase in reactivity represents a deleterious effect of FIV on the CNS.
These results of early detectable neurophysiologic abnormalities after infection make the SIV and FIV models ideal for testing new therapies aimed at preventing or arresting cognitive decline induced by HIV. In conjunction with our collaborators on the SIV and FIV systems here at TSRI--F. Bloom, M. Buchmeier, J. Elder, L. Gold, S. Henriksen, and T. Phillips--and elsewhere--J. Nelson, Oregon Health Sciences University, and S. Jacobson, Tufts University--we are planning treatment studies to both test hypotheses about pathogenesis and develop therapeutics to treat HIV disease.
Jacobsen, S., Henriksen, S.J., Prospero-Garcia, O., Phillips, T.R., Elder, J.H., Bloom, F.E., Fox, H.S. Cortical neuropathology associated with FIV infection. J. Neurovirol. 3:283, 1997.
Phillips, T.R., Prospero-Garcia, O., Wheeler, D.W., Wagaman, P.C., Lerner, D.L., Fox, H.S., Whalen, L.R., Bloom, F.E., Elder, J.H., Henriksen, S.J. Neurologic dysfunctions caused by a molecular clone of feline immunodeficiency virus. J. Neurovirol. 2:388, 1996.
Prospero-Garcia, O., Gold, L.H., Fox, H.S., Polis, I., Koob, G., Bloom, F.E., Henriksen, S.J. Microglia-derived simian immunodeficiency virus induces neurophysiological abnormalities in monkeys. Proc. Natl. Acad. Sci. U.S.A. 93:14158, 1996.
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In Vivo Neurophysiology: Functional Correlates of CNS Disease
J.L. Giacchino, S.J. Henriksen, G.I. Berg, S. Casalman
Investigation of the neural basis of cognition has historically included evaluation of higher order mental functions such as motivation, anticipation, comparison, and organization. Available clinical data indicate that these and other aspects of executive cognitive control depend on an intact prefrontal cortex. Therefore, neurophysiologic and neuropharmacologic changes in the prefrontal cortex may contribute to abnormalities such as addiction and dementia. Recent evidence suggests a major contribution of the prefrontal cortex to the etiology of substance abuse and the development of age-related alterations in memory. To elucidate the neuronal correlates of drug-seeking behavior in the prefrontal cortex, we have investigated the effects of opiates and nicotine and their interactions with pertinent endogenous neurochemicals. We also have evaluated the function of prefrontal cortical neuronal circuits to clarify age-associated changes in synaptic plasticity that may contribute to cognitive impairment.
Using single-cell electrophysiologic techniques and anesthetized rats, we examined spontaneous and evoked activity of medial prefrontal cortical neurons during both systemic and local application of opioids and nicotine. Systemic morphine inhibited neuronal firing, whereas systemic nicotine increased firing. Local application of morphine or nicotine caused similar effects in part of the population of neurons. To elucidate the mechanisms of these drug actions, we investigated interaction of opiates and nicotine with excitatory neurochemicals. Acetylcholine and glutamate drive activity in both spontaneously firing and silent neurons. Systemic and locally applied opioids had little effect on the actions of acetylcholine but attenuated the response to glutamate. Morphine attenuated the excitatory response of prefrontal cortical cells to stimulation of major (putatively glutamatergic) afferents from the mediodorsal thalamus, CA1/subiculum, and basolateral amygdala. Systemic nicotine potentiated afferent-evoked excitation in aged rats.
We have observed significant age-related changes in evoked prefrontal cortical neuronal activity. The induction of long-term potentiation, a reputed neurophysiologic model of memory, is significantly impaired in aged rats. If any potentiation of evoked response occurs, a marked decrease occurs within minutes. This attenuated potentiation of prefrontal cortical response in aged rats has many possible explanations. Preliminary neuropathologic data obtained by E. Masliah, University of California, San Diego, suggest that aged rats have fewer synapses in the prefrontal cortex than young adult rats do. Studies on short-term potentiation, long-term depression, and alterations in the function of glutamatergic and cholinergic receptors in aged animals will elucidate how these changes might contribute to deficient synaptic potentiation in the prefrontal cortex.
Giacchino, J.L., Henriksen, S.J. Opioid effects on prefrontal cortical neuronal activity and excitatory response. NIDA Res. Monogr. 174:150, 1997.
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L.H. Gold, G.F. Koob, M.R. Weed, M.A. Taffe, F. Dellu, V. David, A. Contarino, C.J. Heyser, A.J. Roberts, I. Polis, J.S. McDonald, S. Davis
GENETIC DIFFERENCES IN MOUSE BEHAVIORAL PERFORMANCE
The genetic background of an organism can influence the acquisition and maintenance of learned behavior. Because transfer of information is a critical component of information processing, we used a discriminated Y-maze task to examine serial reversal learning in three mouse strains. The Y-maze task measures the ability of the test animal to discriminate between right and left arms of the maze and can be used to study more complex learning by reversing the designation of the correct arm. All mice acquired the initial discrimination at an equivalent rate and level of performance, but strain differences were observed during the serial reversals (Fig. 1).
C57 x SJL mice showed enhanced acquisition with each reversal. This type of "savings" was observed in Balb/cByJ mice to a lesser degree and was absent in DBA mice. Because the reversals were conducted after a set number of sessions (i.e., were history based), animals within a strain might have learned the task at different points during training. Therefore we used criterion-based testing to evaluate the earlier findings. With this procedure, we found significant reversal learning in DBA mice.
The availability of genetically homogeneous mouse strains allows the analysis not only of genetic influences on behavior but also of behavioral sensitivity to drugs of abuse. We have characterized the effect of cocaine on discriminated operant responding for food in six mouse strains. All strains increased operant responding as the schedule of reinforcement was raised from a continuous reinforcement schedule to one that required 15 responses to obtain one reinforcer. However, significant differences in response rate and discrimination learning were observed among the various strains of mice. Two strains were more resistant than the others to the reduction in operant responding for food produced by cocaine. Collaborative studies with L. Parsons, Department of Neuropharmacology, showed that this behavioral sensitivity was not accounted for by the amount of cocaine or the metabolites of cocaine in brain tissue. Therefore, flexibility in learning and the effect of cocaine on behavior is influenced by the genetic background of the organism. The demonstrated sensitivity of these procedures to genetic differences in mouse strains will permit use of the procedures in further studies of performance in transgenic and knockout mice for investigations of candidate genes involved in drug abuse and viral pathogenesis.
A computerized behavioral battery based on neuropsychologic tests used in humans has been developed to assess changes in cognitive performance produced by neuropharmacologic manipulations in rhesus monkeys. The battery includes tests of memory, attention, motivation, reaction time, and motor skill. Because dysfunction in brain cholinergic systems is thought to underlie many cognitive disorders, the effect of a cholinergic antagonist, scopolamine, on performance in this battery was evaluated. The results suggest that scopolamine impairs fine motor control, sustained attention, motivation to respond, and accuracy of spatial working memory and show that this test battery is useful for distinguishing the effects of neuropharmacologic manipulation on various aspects of cognitive performance in monkeys.
Other studies with the same computerized test battery involve the longitudinal analysis of rhesus monkeys infected with simian immunodeficiency virus (SIV). In collaboration with H. Fox, Department of Neuropharmacology, we are monitoring immunologic and virologic parameters and assessing behavior and function. The time course of disease progression is characterized by using the measures of cognition and motor skill described previously and neurophysiologic recording conducted in the laboratory of S. Henriksen, Department of Neuropharmacology. One to two months after monkeys were infected with SIV, neurophysiologic testing showed slowing in the brain stem auditory evoked potential peak latencies, a profile similar to that seen in humans with HIV infection. Infected animals also had performance decrements in one or more neuropsychologic domains, including spatial working memory, attentional set-shifting, reaction time, and fine motor abilities, during the course of SIV disease (Fig. 2),
similar to findings observed in a subset of humans infected with HIV. These data indicate a relationship between the neurobehavioral sequelae of SIV infection and pathologic changes in CNS and immune functions. The SIV model will aid in the further study of candidate viral and host factors important for CNS dysfunction and in testing novel therapeutic agents.
Brot, M.D., Rall, G.F., Koob, G.F., Oldstone, M.B.A., Gold, L.H. Cognitive deficit remains despite clearance of long-term persistent viral infection in mice. J. Neurovirol., in press.
Campbell, I.L., Stalder, A.K., Chiang, C.-S., Bellinger, R., Heyser, C.J., Steffensen, S., Masliah, E., Powell, H.C., Gold, L.H., Henriksen, S.J., Siggins, G.R. Transgenic models to assess the pathogenic actions of cytokines in the central nervous system. Mol. Psychiatry 2:125, 1997.
Deroche, V., Caine, S.B., Heyser, C.J., Polis, I., Koob, G.F., Gold, L.H. Differences in the liability to self-administer intravenous cocaine between C57BL/6 x SJL and Balb/cByJ mice. Pharmacol. Biochem. Behav., in press.
Gold, L.H. Integration of molecular biological techniques and behavioural pharmacology. Behav. Pharmacol. 7:589, 1996.
Heyser, C.J., Masliah, E., Samimi, A., Campbell, I.L., Gold, L.H. Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin-6 in the brain. Proc. Natl. Acad. Sci. U.S.A. 94:1500, 1997.
Heyser, C.J., McDonald, J.S., Beauchamp, V., Koob, G.F., Gold, L.H. The effects of cocaine on operant responding for food in several strains of mice. Psychopharmacology, in press.
Prospero-Garcia, O., Gold, L.H., Fox, H.S., Polis, I., Koob, G.F., Bloom, F.E., Henriksen, S.J. Microglia-passaged simian immunodeficiency virus (SIV) induces neurophysiological abnormalities in monkeys. Proc. Natl. Acad. Sci. U.S.A. 93:14158, 1996.
Roberts, A.J., Polis, I., Gold, L.H. Intravenous self-administration of heroin, cocaine and the combination in Balb/c mice. Eur. J. Pharmacol., in press.
Schulteis, G., Gold, L.H., Koob, G.F. Preclinical behavioral models for addressing unmet needs in opiate addiction. Semin. Neurosci., in press.
Uhl, G., Gold, L.H., Risch, N. Genetic analyses of complex behavioral disorders. Proc. Natl. Acad. Sci. U.S.A. 94:2785, 1997.
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Molecular Mechanisms of Neuronal Signaling and Pathologic Changes
D. Gruol, J. Netzeband, Z. Qiu, T. Nelson, K. Parsons, C. Trotter, C. Ur, J. Caguioa, S. Conroy, C. Ly, D. Sweeney
OPIATE REGULATION OF CALCIUM SIGNALING
Opiate receptors mediate cell-to-cell communication between neurons of the CNS and are involved in the CNS actions of therapeutic agents and of drugs of abuse. The mechanisms by which opiates or their endogenous counterpart, opioid peptides, influence neuronal function via actions at opiate receptors have not been fully elucidated. Recent studies with a neuronal-like cell line indicate that opiate receptor agonists can activate an intracellular calcium signaling pathway. Because intracellular calcium is an important second messenger, we have begun studies to determine if this pathway is expressed in native CNS neurons.
A variety of opiate receptor agonists (e.g., morphine) were tested for their ability to induce calcium signals in cultured rat hippocampal neurons. The opiate agonists did not alter resting calcium levels, as had been observed in the neuronal cell line, and had no effect on the calcium signals elicited by exogenous application of the neurotransmitter glutamate. However, spontaneous calcium oscillations induced by spontaneous synaptic input to the hippocampal neurons and mediated by glutamate receptors were significantly altered (Fig. 1).
The opiates enhanced the frequency and amplitude of the calcium oscillations and synchronized the oscillatory activity of a group of neurons within a microscopic field. Surprisingly, although opiates had a pronounced effect on the calcium signals, electrophysiologic recordings showed only minor effects on the synaptic potentials that initiate the calcium signals. Thus, alterations in calcium signaling may be an important mechanism by which opiates and endogenous opioid peptides influence the function of CNS neurons.
CYTOKINE-INDUCED NEUROTOXIC EFFECTS
The concentration of the cytokine IL-6 is elevated in the brains of patients with HIV disease, and this increase is thought to play a role in the CNS dysfunction and neuropathologic changes characteristic of the neurologic manifestations of AIDS. To determine actions of IL-6 that could contribute to the CNS effects of HIV infection, we are investigating the actions of IL-6 in primary cultures of CNS neurons.
Previously, we showed that treatment of the cultured neurons with IL-6 alters resting calcium levels and the calcium signal elicited by N-methyl-d-aspartate (NMDA), an agonist at the NMDA subtype of glutamate receptors. Glutamate is the main excitatory transmitter in the brain and an important neurotrophic factor in the developing CNS. Glutamate receptors of the NMDA subtype mediate many of the actions of glutamate and are one of the main pathways for calcium entry into CNS neurons. Calcium is an important second messenger and is essential to normal neuronal function. However, excess intracellular calcium produced by entry of calcium via glutamate receptors of the NMDA subtype can cause neurotoxic effects, which may contribute to the neuropathologic changes in AIDS.
During the past year, we examined the ability of IL-6 to induce neurotoxic effects alone or in combination with NMDA in a culture model system. The cultured CNS neurons were treated with pathophysiologic concentrations of IL-6, and standard assays for neurotoxic effects were done after 7 days of treatment. Long-term treatment with IL-6 induced cytotoxic effects and decreased cell survival. Moreover, the size of neuronal clusters formed during neuronal development in culture were smaller in the IL-6--treated cultures than in control cultures not treated with IL-6. All effects were dose dependent. Thus, several different assays showed a neurotoxic effect of long-term treatment with IL-6. Brief treatment (1 hour) with NMDA also induced significant toxic effects in the neuronal cultures, and these effects were significantly higher in IL-6--treated cultures than in cultures not treated with IL-6. These results show that pathophysiologic concentrations of IL-6 can be neurotoxic and that exposure to IL-6 predisposes CNS neurons to the neurotoxic effects of NMDA, perhaps by affecting intracellular calcium homeostasis.
Gruol, D.L., Netzeband, J.G., Parsons, K.L. Calcium signaling pathways linked to glutamate receptor activation in the somatic and dendritic regions of cultured cerebellar Purkinje neurons. J. Neurophysiol. 76:3325, 1996.
Gruol, D.L., Parsons, K.L. Chronic alcohol reduces calcium signaling elicited by glutamate receptor stimulation in developing cerebellar neurons. Brain Res. 728:166, 1996.
Gruol, D.L., Parsons, K.L.,DiJulio, N. Acute alcohol alters calcium signaling elicited by glutamate receptor agonists in cultured cerebellar Purkinje neurons. Brain Res., in press.
Netzeband, J.G., Parsons, K.L., Sweeney, D.D., Gruol, D.L. Metabotropic glutamate receptor agonists alter neuronal excitability and Ca2+ levels via the phospholipase C transduction pathway in cultured Purkinje neurons. J. Neurophysiol. 78:63, 1997.
Sorg, O., Horn, T.F.W., Yu, N., Gruol, D.L., Bloom, F.E. Inhibition of astrocyte glutamate uptake by reactive oxygen species: Role of antioxidant enzymes. Mol. Med. 3:431, 1997.
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In Vivo Neuropharmacology
S.J. Henriksen, S. Steffensen, J. Giacchino, O. Prospéro-García,* R.-S. Lee, J. Criado, S. Huitron-Resendez, G. Berg, R. Cole, N. Herold, S. Casalman, P. Griffin
* University Nacional Autonoma de Mexico, Mexico City, Mexico
Research in this laboratory continues to focus on brain neural circuits that underlie complex cognitive functions such as drug seeking, motivated behaviors, learning, and sleep. We use experimental animals in which the CNS is intact or has been compromised by viral infection, genetic manipulations, or molecular engineering. By examining the intact nervous system in whole animals, we can determine the consequences of specific experimental manipulations on brain circuitry thought to be important for normal as well as pathologic behaviors.
BRAIN CIRCUITS UNDERLYING DRUG SELF-ADMINISTRATION
We have expanded our studies of the CNS actions of drugs of abuse to include prefrontal cortical regions of the brain (see J. Giacchino's report) and brain stem nuclei, including the ventral tegmental area. We record data from specific neurons in these areas in both anesthetized and unanesthetized freely moving rats and correlate neuronal discharge with drug administration, spontaneous behaviors, and changes in arousal and sleep. Systemic and locally applied drugs of abuse, including ethanol and nicotine, and ingestion of preferred foods generally reduce the spontaneous and evoked discharge of these cells, indicating that specific circuits of the brain may be registering the rewarding properties of a variety of environmental stimuli. This effort, including our earlier and continuing studies of the nucleus accumbens, reflects our growing belief that these distributed brain circuits are critical components of a wider brain system that underlies motivation and reward.
This year we have been exploring the relative sensitivity of specific cellular components of this circuit to these drugs and natural behaviors. Medial prefrontal cortical neurons are extremely sensitive to systemic opioids but have quite selective inhibitory actions on the effects of classical neurotransmitters on these neurons. In addition, nicotine, another highly addictive drug, has direct and indirect actions on these neurons. Dr. Giacchino is beginning to look at age-related differences in these responses in an attempt to discern why nicotine may be so highly addictive in naive, young subjects. In addition, recent progress in our laboratory has shown for the first time that specific neurons containing -aminobutyric acid in the region of the ventral tegmental nucleus are quite sensitive to opioids and ethanol and may be a new critical component of the reinforcement circuit (Fig. 1).
ANIMAL MODELS OF AIDS
In collaboration with T. Phillips, Department of Neuropharmacology, and J. Elder, Department of Molecular Biology, we have continued to investigate the physiopathology of various strains of feline immunodeficiency virus (FIV), including cloned variants, as a small-animal model of infection with HIV type 1. Specific strains of FIV, including a microglia-passaged strain and virus obtained from cultured glial cells are highly CNS-tropic and cause early CNS functional changes in infected cats. These new findings provide us with the opportunity to evaluate newer treatment modalities in a timely fashion.
In studies with H. Fox, Department of Neuropharmacology, in collaboration with S. Jacobson, Tufts University, we found that neurons from cats infected with FIV had a greater and more intense neuronal staining with SMI-32, a nonphosphorylated neurofilament protein, than neurons from uninfected cats did. This finding suggests that infection with FIV causes early neuropathologic changes in large neurons in the brain. Finally, in new parallel studies in Rhesus monkeys infected with simian immunodeficiency virus, in collaboration with L. Gold, Department of Neuropharmacology, we found that changes in sensory evoked potentials are progressively delayed in association with neurocognitive measures. Therefore, our physiologic studies have shown important cross-species similarities in the functional pathologic changes associated with lentiviral infection.
CIRCADIAN RHYTHMS IN A POIKILOTHERMIC MAMMAL
All mammals are homeothermic, that is, they are able to maintain their body temperature in the face of changing environmental temperatures. However, a unique mammal that is an exception to this rule is the naked mole rat (Heterocephalus glaber).
This fossorial, blind, molelike creature, found in Northeast Africa, is poikilothermic (reptilelike) and has little or no ability to maintain its body temperature in changing temperature environments. As a part of our studies to understand the relationship between sleep and temperature-control mechanisms, we studied the temperature and circadian activity of naked mole rats in a colony of mole rats we have raised. Using telemetric data acquisition, surprisingly, we observed circadian fluctuations in both temperature and motor activity in these animals when temperature and light conditions were kept constant. Sleep-wake investigations of these interesting animals are now being done (Fig. 2).
Campbell, I.L., Stalder, A.K., Chiang, C.-S., Bellinger, R., Heyser, C.J., Steffensen, S.C., Masliah, E., Powell, H.C., Gold, L.H., Henriksen, S.J., Siggins, G.R. Transgenic models to assess the pathogenic actions of cytokines in the central nervous system. Mol. Psychiatry 2:125, 1997.
Criado, J.R., Berg, G.I., Mayer, J.H., Henriksen, S. Electrophoretic effects of DAMGO on glutamate-driven and fimbria-driven neurons in NAcc shell. NIDA Monogr. 174:150, 1997.
Criado, J.R., Lee, R.-S., Berg, G.I., Henriksen, S.J. Ethanol inhibits single-unit responses in the nucleus accumbens evoked by stimulation of the basolateral nucleus of the amygdala. Alcohol. Clin. Exp. Res. 21:368, 1997.
Gold, L.H., Heyser, C.J., Roberts, A.J., Henriksen, S., Steffensen, S.C., Siggins, G.R., Bellinger, F.P., Chiang, C.-S., Powell, H.C., Masliah, E., Campbell, I.L. Behavioral and neurophysiological effects of CNS expression of cytokines in transgenic mice. Adv. Exp. Med. Biol. 402:199, 1996.
Phillips, T.R., Prospéro-García, O., Wheeler, D., Wagaman, P., Lerner, D., Fox, H.S., Whalen, L., Bloom, F., Elder, J., Henriksen, S. Neurologic dysfunctions caused by a molecular clone of feline immunodeficiency virus, FIV-PPR. J. Neurovirol. 2:388, 1996.
Prospéro-García, O., Gold, L.H., Fox, H.S., Polis, I., Koob, G., Bloom, F., Henriksen, S. Microglia-passaged simian immunodeficiency virus induces neurophysiological abnormalities in monkeys. Proc. Natl. Acad. Sci., U.S.A. 93:14158, 1996.
Ryabinin, A.E., Criado, J.R., Henriksen, S., Bloom, F.E., Wilson, M.C. Differential sensitivity of c-Fos expression in hippocampus and other brain regions to moderate and low doses of alcohol. Mol. Psychiatry 2:32, 1997.
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George F. Koob, Ph.D., Director
Research in the Division of Psychopharmacology continues to focus on the brain substrates that subserve emotional behavior and learning and on how these systems change with drug dependence, stress, and mental disease. These studies are critical for understanding the vulnerability associated with acquiring and maintaining such chronic relapsing disorders. Neuropharmacologic advances in drug dependence and stress are covered in the following report. Neurochemical studies, neuromolecular and neuroviral studies, and studies on the neuropharmacology of reward systems are covered in the reports of F. Weiss, L. Gold, and A. Markou, respectively.
Neurobiology of Stress, Dependence, and Disease
G.F. Koob, F. Weiss, L. Gold, A. Markou, L. Parsons, K.T. Britton,* M.B. Weinger,* M. Geyer,* M. Le Moal,** E. Riley,*** L. Stinus,** M. Cador,** L. Pulvirenti,**** F. De Fonseca,***** G. Schulteis,* S. Heinrichs,+ C. Heyser, S. Ahmed, S. Katner, A. Smith, Y. Akwa, E. Zorrilla, R. Martin-Fardon, M. Spina, A. Roberts, D. Lin, A. Harrison, J. Walker, M. Weed, A. Contarino, M. Taffe, N. Gracy, V. David, A. Morse, E. Izzo, B. Baldo, D. Macey, S. Watkins, M. Arends, R. Lintz, J. McDonald, I. Polis, B. Nadeau, S. Hoffman, R. Schroeder, D. Smith, T. Kerr, R. Bianco, S. Davis, M. Beschen
* University of California, San Diego, CA
** INSERM U. 259 and Universite Bordeaux II, Bordeaux, France
*** San Diego State University, San Diego, CA
**** Universita di Roma "TorVergata," Rome, Italy
***** Universidad Complutense de Madrid, Madrid, Spain
+ Neurocrine Biosciences, Inc., San Diego, CA
NEUROBIOLOGY OF DRUG DEPENDENCE
Studies on the neurobiology of drug dependence have detected multiple neurochemical systems within the basal forebrain that are important substrates for the acute reinforcing actions of drugs of abuse. Much is known about the roles of dopamine and opioid peptide receptors in the reinforcing actions of cocaine and heroin, respectively. During the past year, we have begun to focus on the neuropharmacologic mechanisms of action of tetrahydrocannabinol (THC) and nicotine. Intravenous self-administration of nicotine and quantifiable measures of nicotine withdrawal (see A. Markou's report) have been established. Similarly, neurobiological and behavioral measures of THC withdrawal have been described. Studies indicate that an antagonist of corticotropin releasing factor (CRF) can block the "anxiogenic-like" effects of short-term administration of THC. Perhaps more important, recent evidence suggests a role for CRF in cannabinoid withdrawal in rats made dependent on THC. These findings suggest that THC induces neuroadaptation in stress systems in common with major drugs of abuse (see report of F. Weiss).
Studies in the past year have also explored animal models of relapse and the neurochemical mechanisms involved. Signs of opiate withdrawal can be precipitated by the opiate antagonist naltrexone after a single injection of morphine, and this effect is potentiated by a second treatment with morphine. These results suggest that a history of opiate use can induce a prolonged vulnerability to opiate withdrawal. In addition, rats with a history of alcohol self-administration showed increased responding for alcohol after enforced abstinence, and this alcohol-deprivation effect increased with the duration of the enforced abstinence. The alcohol-deprivation effect was reversed by repeated treatment with low doses of naltrexone and acamprosate, drugs that are clinically effective in blocking relapse in detoxified alcoholic patients. These animal models show promise as models of prolonged abstinence and relapse, and current studies will be directed at exploring the neurobiological bases of these effects.
NEUROBIOLOGY OF STRESS: NEUROPEPTIDES
CRF in the brain is involved in behavioral responses to stress. Exogenous administration of CRF produces a dose-dependent behavioral activation, enhances behavioral responses to stress, and suppresses appetite. Recently, urocortin, a peptide structurally similar to CRF, has been isolated from the brain. Urocortin can depress food intake up to 6 hours at doses lower than those of CRF that produce a similar effect, but it has less behavioral activating effects than CRF does.
To further characterize the nature of the appetite-suppressing effects of urocortin, we studied the effects of the peptide in free-feeding rats, animals not submitted to food deprivation. Intracerebroventricular urocortin significantly decreased meal size at doses of 0.01--1.0 µg and decreased the number of meals at a dose of 1.0 µg. Fenfluramine, a known serotonin appetite suppressant, produced an identical behavioral profile. These results suggest that urocortin has powerful appetite-suppressing effects that resemble those of the serotonin appetite suppressants.
The effects of three different CRF-receptor antagonists, astressin, -helical CRF (9--41), and D-Phe CRF (12--41), on the urocortin-induced anorectic response in food-deprived animals were determined. Only D-Phe CRF (12--41) completely reversed the decrease in food intake caused by intracerebroventricular injection of urocortin. Astressin and -helical CRF attenuated the feeding effect only during the first 30--60 minutes after injection. These results suggest that antagonists of the CRF 1 and 2 receptors block the feeding suppression induced by urocortin and that the effectiveness of the antagonists may differ over time after injection.
Knockout mice lacking the CRF 1 receptor have provided a unique opportunity to test the behavioral phenotype of these animals to help elucidate the function of endogenous CRF systems. A dark-light emergence task was used to expose mice deficient in the CRF 1 receptor and wild-type control mice to an anxiogenic environment. The knockout mice showed an anti-stresslike response, whereas the wild-type mice did not. These results are consistent with a reduced behavioral response to stress in the mutant mice. In addition, the mutant mice showed a blunting of the diurnal rhythm of locomotor activity, suggesting that CRF may play a role in changes in arousal associated with daily activity cycles (see L. Gold's report).
Koob, G.F. Neurochemical explanations for addiction. Hosp. Pract., April 1997, p. 12.
Koob, G.F., Parsons, L.H., Caine, S.B., Weiss, F., Sokoloff, P., Schwartz, J.-C. Dopamine receptor subtype profiles in cocaine reward. In: Dopamine Disease States. Beninger, R.J., Palomo, T., Archer, T. (Eds.). Editorial CYM, Madrid, 1996, p. 433.
Korte, S.M., Korte-Bouws, G.A.H., Koob, G.F., De Kloet, E.R., Bohus, B. Mineralocorticoid and glucocorticoid receptor antagonists in animal models of anxiety. Pharmacol. Biochem. Behav. 54:261, 1996.
Menzaghi, F., Heinrichs, S.C., Vargas-Cortes, M., Goldstein, G., Koob, G.F. IRI-514, a synthetic peptide analogue of thymopentin, reduces the behavioral response to social stress in rats. Physiol. Behav. 60:397, 1996.
Parsons, L.H., Caine, S.B., Sokoloff, P., Schwartz, J.-C., Koob, G.F., Weiss, F. Neurochemical evidence that postsynaptic nucleus accumbens D3 receptor stimulation enhances cocaine reinforcement. J. Neurochem. 67:1078, 1996.
Parsons, L.H., Weiss, F., Koob, G.F. Serotonin 1b receptor stimulation enhances dopamine-mediated reinforcement. Psychopharmacology 128:150, 1996.
Prospero-Garcia, O., Gold, L.H., Fox, H.S., Polis, I., Koob, G.F., Bloom, F.E., Henriksen, S.J. Microglia-passaged simian immunodeficiency virus (SIV) induces neurophysiological abnormalities in monkeys. Proc. Natl. Acad. Sci. U.S.A. 93:14158, 1996.
Pulvirenti, L., Balducci, C., Koob, G.F. Dextromethorphan reduces intravenous cocaine self-administration in the rat. Eur. J. Pharmacol. 321:279, 1997.
Pulvirenti, L., Balducci, C., Koob, G.F. Inhibition of nitric oxide synthesis reduces intravenous cocaine self-administration in the rat. Neuropharmacology 35:1811, 1996.
Pulvirenti, L., Koob, G.F. Bases neurologicas de la adiccíon a la cocaina. Invest. Ciencia, July 1996, p. 48.
Raber, J., Koob, G.F., Bloom, F.E. Interferon- (IFN-) and transforming growth factor (TGF-ß1) regulate corticotropin-releasing hormone (CRH) release from the amygdala: Comparison with the hypothalamic response. Neurochem. Int. 67:1078, 1996.
Roberts, A.J., Cole, M., Koob, G.F. Intra-amygdala muscimol decreases operant ethanol self-administration in dependent rats. Alcohol. Clin. Exp. Res. 20:1289, 1996.
Rodriguez de Fonseca, F., Rubio, P., Menzaghi, F., Merlo-Pich, E., Rivier, J., Koob, G.F., Navarro, M. Corticotropin-releasing factor (CRF) antagonist [D-Phe12,Nle21,38,CaMeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J. Pharmacol. Exp. Ther. 276:56, 1996.