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DEPARTMENT OF NEUROPHARMACOLOGY


STAFF

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.


RESEARCH FELLOWS

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.


VISITING SCIENTISTS

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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Chairman's Overview

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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Investigators' Reports


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.

NEUROZOOM

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.

PUBLICATIONS

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.

PUBLICATIONS

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.

PUBLICATIONS

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.

PUBLICATIONS

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.

PUBLICATIONS

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.

PUBLICATIONS

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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Behavioral Neuroscience

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.

PRIMATE NEUROPSYCHOLOGY

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.

PUBLICATIONS

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.

PUBLICATIONS

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).

PUBLICATIONS

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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Division of Psychopharmacology

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).

PUBLICATIONS

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.

Schulteis, G., Heyser, C.J., Koob, G.F. Opiate withdrawal signs precipitated by naloxone following a single exposure to morphine: Potentiation with a second morphine treatment. Psychopharmacology 129:56, 1997.

Schulteis, G., Koob, G.F. Reinforcement processes in opiate addiction: A homeostatic model. Neurochem. Res. 21:1437, 1996.

Sokoloff, P., Griffon, N., Sautel, F., Levesque, D., Pilon, C., Schwartz, J.-C., Ridray, S., Diaz, J., Simon, P., Costentin, J., Mann, A., Wermuth, C.G., Caine, S.B., Parsons, L., Koob, G.F. The dopamine D3 receptor: From cloning to function. In: Dopamine Receptors: From Basic Science to Clinic. Jenner, P., Demirdamar, R. (Eds.). IOS Press, Amsterdam, 1997, p. 1.

Solbrig, M.V., Koob, G.F. Fallon, J.H., Reid, S., Lipkin, W.I. Prefrontal cortex dysfunction in Borna disease virus (BDV)-infected rats. Biol. Psychiatry 40:629, 1996.

Solbrig, M.V., Koob, G.F., Joyce, J.N., Lipkin, W.I. A neural substrate of hyperactivity in borna disease: Changes in brain dopamine receptors. Virology 222:332, 1996.

Solbrig, M.V., Koob, G.F., Lipkin, W.I. Naloxone-induced seizures in rats infected with Borna disease virus. Neurology 46:1170, 1996.

Spina, M., Merlo-Pich, E., Chan, R.K.W., Basso, A.M., Rivier, J., Vale, W., Koob, G.F. Appetite-suppressing effects of urocortin, a CRF-related neuropeptide. Science 273:1561, 1996.

Vankova, M.E., Weinger, M.B., Chen, D.Y., Bronson, J.B., Motis, V., Koob, G.F., Negus, S.S. Role of central mu, delta-1, and kappa-1 opioid receptors in opioid-induced muscle rigidity in the rat. Anesthesiology 85:574, 1996.

Weissenborn, R., Deroche, V., Koob, G.F., Weiss, F. Effects of selective dopamine receptor subtype agonists and antagonists on the cocaine-induced increase in operant responding maintained by a drug-associated stimulus. Psychopharmacology 126:311, 1996.

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ErbB Receptors and Ligands in the Nervous System

C. Lai, J. Weber, N. Yu, A. Perez-Castillo, A. Santos-Montes<

Research in this laboratory focuses on understanding the signaling mechanisms that underlie the establishment and maintenance of mature neuronal phenotypes. We are studying the role played by one class of signal transduction molecules, the protein-tyrosine kinases. Although they are best known for their role in cell growth and proliferation, we wish to determine possible functions for these receptors in postmitotic neurons in the adult brain. We focus on the ErbB4 receptor and its ligands, neuregulin-1 and neuregulin-2.

ErbB4 is the fourth member of the epidermal growth factor receptor subfamily of molecules. Along with ErbB3, ErbB4 acts as a receptor for neuregulin-1, a family of molecules that arise via alternative splicing from a single gene. ErbB2 and the receptor for epidermal growth factor also participate in neuregulin signaling as a result of receptor heterodimerization, which is thought to occur in virtually all ErbB receptor combinations. Neuregulin-1 was first cloned as "heregulin" and "neu differentiation factor" and was initially thought to be a ligand for ErbB2. Neuregulin-1 has also been studied as "glial growth factor," which acts as a Schwann cell mitogen, and as "ARIA" (acetylcholine receptor inducing activity), a regulator of the expression of acetylcholine receptors.

In the peripheral nervous system, ErbB4 thus helps regulate glial proliferation and acts as a signaling molecule at the neuromuscular junction. In the mature CNS, ErbB4 is expressed in cerebellar granule cells and in a subset of cells that also express the neurotransmitter -aminobutyric acid. ErbB4 is critical in cardiac development; mice lacking ErbB4 die as a result of a failure in trabeculation in the developing heart ventricle. These mice also have neural defects, including apparent targeting errors of several cranial ganglia.

Recently, we identified neuregulin-2, a novel neuregulin-1--like molecule encoded by a distinct gene. Like neuregulin-1, neuregulin-2 also binds to ErbB3 and ErbB4, although it appears to signal distinctly, perhaps by activating different receptor heterodimers. Neuregulin-1 and neuregulin-2 are largely expressed in nonoverlapping sets of cells in the brain in adult rats. Neuregulin-2 is primarily found in granule cell populations in the cerebellum, hippocampus, and olfactory bulb. In the developing heart, neuregulin-2 is mainly found in the atrium, and neuregulin-1 is predominantly found in the ventricle. These patterns suggest that neuregulin-2 and neuregulin-1 have distinct functions. We are searching for additional neuregulin and ErbB molecules to better define the precise ligand-receptor relationships in the mature CNS.

PUBLICATIONS

Carraway, K.L. III, Weber, J.L., Unger, M.J., Ledesma, J., Yu, N., Gassmann, M., Lai, C. Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases. Nature 387:512, 1997.

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Neurobiology of Motivation and Emotion

A. Markou, G.F. Koob, F. Weiss, L.H. Parsons, D. Lin, A.A. Harrison, M. Epping-Jordan, B. Baldo, D. Macey, S.S. Watkins, M. Kreifeldt, R. Bianco, B.J. Everitt,* T.W. Robbins,* M. Arroyo,* T.R. Kosten**

* Cambridge University, Cambridge, England
** Yale University School of Medicine, West Haven, CT

Research efforts in our laboratory focus on the neurobiological mechanisms that mediate behavioral processes altered in psychiatric disorders, such as drug dependence and depression. Our studies on the neurobiology of drug dependence involve exploration of (1) the incentive-motivational processes (i.e., drug "craving") that lead to continued drug use, (2) the acute reinforcing effects of drugs, and (3) drug withdrawal.

We recently developed a rat model of the acute rewarding effects of nicotine. Rats, similar to monkeys and humans, readily self-administered stable amounts of intravenous nicotine during daily sessions. As the nicotine dose was changed systematically over days, an inverted U-shaped function was obtained, similar to that seen with other drugs of abuse, such as psychostimulants and opiates. Further, we found that the acute reinforcing effects of nicotine are probably mediated through activation of acetylcholine nicotinic receptors, because pretreatment with a noncompetitive nicotinic antagonist decreased nicotine self-administration.

In another series of studies, we have began to investigate nicotine withdrawal. Rats made dependent on nicotine through implantation of minipumps showed overt somatic signs of withdrawal when the pumps were removed (i.e., spontaneous nicotine withdrawal; Fig. 1B), or various nicotinic antagonists were administered (i.e., precipitated withdrawal). The most important finding was that both types of withdrawal were associated with elevations in brain reward thresholds, an effect that is interpreted as a blunting of the brain reward systems (Fig. 1A). We have observed similar effects on brain reward thresholds during withdrawal from cocaine, amphetamine, morphine, opiates, and ethanol. These results support the hypothesis that the affective signs of withdrawal are common to withdrawal from drugs from various pharmacologic classes and that these signs may be one of the defining aspects of drug dependence.

One of the core features of depression is also diminished reward. We are continuing to develop rat models of this indication of depression that are based on elevations in brain reward thresholds (i.e., diminished reward) observed during withdrawal from drugs of abuse, such as amphetamine, or after experiential manipulations, such as stress. We have also been working at a theoretical level on the neurobiological similarities that may explain the observed clinical comorbidity between depression and drug dependence. This work is based on a self-medication hypothesis.

PUBLICATIONS

Koob, G.F., Caine, S.B., Parsons, L., Markou, A., Weiss, F. Opponent process model and psychostimulant addiction. Pharmacol. Biochem. Behav. 57:513, 1997.

Koob, G.F., Carrera, R., Gold, L., Heyser, C., Maldonado-Irizarry, C., Markou, A., Parsons, L.H., Roberts, A., Schulteis, G., Stinus, L., Walker, J., Weissenborn, R., Weiss, F. Substance dependence as a compulsive disorder. J. Psychopharmacol., in press.

Koob, G.F., Markou, A., Parsons, L., Roberts, A., Schulteis, G., Caine, S.B., Weiss, F. Neurobiology of drug addiction. Am. Psychol., in press.

Markou, A., Kosten, T.R., Koob, G.F. Neurobiological similarities in depression and drug dependence: A self-medication hypothesis. Neuropsychopharmacology, in press.

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Sleep Research Group

M. Mitler, D. Darko, A. Dawson, M. Erman, R. Hayduk, S.J. Henriksen

NARCOLEPSY

As currently understood, narcolepsy is a CNS disorder characterized by disabling dysregulation of sleep and wakefulness. It affects about 200,000 persons in North America. Although inheritance of narcolepsy is not known, the disorder is strongly, but not always, associated with subtypes of the HLA region chromosome 6 [HLA-DR15(DRB1*1501) and HLA-DQ6(DQB1*0602)]. The presence of HLA- and non--HLA-associated forms indicates that narcolepsy may actually be at least two disorders, each with different etiologies.

Our work has shown five important findings: (1) The HLA antigen DQB1*0602 is associated with narcolepsy in only 70% of the patients in the case series, and the narcolepsy associated with this antigen occurs mainly in probands whose first-degree relatives are not affected. (2) The majority of multiplex cases (multiple patients in a single family who meet the clinical criteria for narcolepsy) do not have the associated HLA haplotype. (3) Monozygotic twins (zygosity confirmed by DNA typing) show differences with respect to clinical signs and symptoms; one has cataplexy and one does not. (4) Daytime and nighttime sleep studies of first-degree relatives of probands who meet full clinical diagnostic criteria for narcolepsy reveal subtle sleep disturbances. (5) In control subjects (persons without narcolepsy), certain indices of immune system function such as plasma levels of TNF- and IL-1ß fluctuate throughout the 24-hour day in a manner that is coordinated with fluctuations in electroencephalographic delta power; this phenomenon has not been studied in patients with narcolepsy.

SLEEP DISTURBANCES IN PATIENTS WITH HIV DISEASE

As part of research to prolong the functional period (free of disability) of patients with HIV infection, we are conducting a series of brief clinical trials designed to pharmacologically control disabling fatigue in patients infected with HIV and to test and generate hypotheses about underlying pathophysiologic changes. This work has general application to CNS inflammation; normal aging; and several pathologic CNS states, including Alzheimer's disease, syndromes that occur after stroke, chronic pain syndromes associated with stroke, and multiple sclerosis. On the basis of current hypotheses of the pathophysiology of HIV-related fatigue, we are determining potential therapeutic medications that reduce fatigue in early-stage HIV infection and are investigating further the pathophysiologic mechanisms that underlie this fatigue.

We are assessing fatigue as measured by objective, quantitative computer-based performance testing; CNS evoked potentials; blood levels of fatigue-promoting peptides; sleep disruption as measured by nocturnal polysomnography and quantitative electroencephalographic techniques; and a subjective, standard questionnaire. The medication trials are straightforward, early phase II--type, clinical trials that determine potential therapeutic medications, quantify the efficacy of the medications, and describe any toxic effects. Individual subjects take part in this project briefly (3 months), and only effects on signs and symptoms related to fatigue are under study.

We are using a parallel-groups, placebo-controlled, single-blind design involving 3 months of treatment to evaluate the TNF- antagonist pentoxifylline (Trental). We are determining its potential to reduce fatigue; lessen performance decrements; and normalize physiologic aspects that may be associated with functional disabilities, including poor sleep quality, altered sleep structure, elevated fatigue-promoting peptides, and abnormal evoked brain potentials, which are typically found in early-stage HIV infection. We are testing the hypothesis that the daytime fatigue and increased slow-wave sleep associated with early-stage HIV infection are mediated by TNF-, a fatigue-promoting peptide that is elevated in patients infected with HIV.

The dosage of pentoxifylline is 400--1600 mg/day. Pentoxifylline is tested against both active and inactive placebos. The active placebo is the nonspecific CNS stimulant dextroamphetamine (Dexedrine) in the dosages of 5--20 mg/day. We predict that pentoxifylline, because of its specificity for TNF-, will be at least as effective as dextroamphetamine in reducing fatigue and drowsiness and will have fewer side effects, such as insomnia. Both pentoxifylline and dextroamphetamine tested against inactive placebo.

EFFECTS OF ALCOHOL ON SLEEP

In our continuing work on the health effects of drinking alcohol before going to sleep at night, we measured inspiratory resistance, inspiratory occlusion pressure, and the ventilatory responses to hypercapnia and isocapnic hypoxia during waking and during stage 2 non-REM sleep in 9 young men who were habitual snorers. The subjects were studied on two nights during the 3 hours after receiving a bedtime drink containing either a placebo or 100 proof vodka (1.5 ml/kg) in orange juice. We compared the results with those obtained previously in 10 otherwise similar men who did not snore.

Waking inspiratory resistance was the same in nonsnorers and snorers and was not affected by ethanol. During sleep on the control night (no ethanol), inspiratory resistance increased by 70% in nonsnorers and by 280% in snorers. On the ethanol night, the increase from waking to sleeping was more than doubled in both nonsnorers and snorers. Inspiratory occlusion pressure and the responses to hypercapnia and hypoxia did not differ between nonsnorers and snorers, so the results from the two groups were pooled.

Minute ventilation and the hypercapnic response decreased from waking to sleeping, and inspiratory occlusion pressure was more negative during sleep, but ethanol had no significant effect. There was a significant correlation between the changes from waking to sleeping in inspiratory resistance and inspiratory occlusion pressure on the ethanol night, suggesting that inspiratory effort increased in response to the increased resistance. The response to isocapnic hypoxia showed no effect of either sleep state or drink. Inspiratory time did not change, but mean inspiratory flow was significantly reduced during sleep on both control and ethanol nights. The duty cycle ratio was significantly greater during sleep on the ethanol night.

Despite its great effect on inspiratory resistance, especially in snorers, ethanol, in the dose used in our study, does not augment the depression of minute ventilation or the depression of the hypercapnic response that occurs normally in stage 2 non-REM sleep. After ingesting ethanol, our subjects showed the decreased mean inspiratory flow and the increased duty cycle ratio that occur normally during sleep in response to an inspiratory resistive load. However, they also showed increased inspiratory effort.

The combination of increased inspiratory resistance and greater inspiratory effort would increase the tendency of an unstable upper airway to collapse and could account for the aggravation of obstructive sleep apnea by ethanol. Because of these findings, we are expanding this type of work to include measures of cerebral blood flow during sleep. Such studies will shed light on the possible exacerbating effects of drinking at bedtime on the well-known early morning increase in strokes.

SLEEP APNEA

We also published promising results on a nonsurgical, nonpharmacologic treatment for obstructive sleep apnea. The mandibular repositioning device is an oral appliance that is fitted by a dentist. In a study of 29 patients with sleep apnea, 23 used the device as directed and were assessed by using polysomnography a mean of 104 days after they had started using the device. The results showed a decrease in respiratory disturbance, from 37 ± 23 apneic events per hour of sleep to 18 ± 20 events per hour of sleep. Subjective and objective measures of daytime sleepiness, nocturnal oxygen desaturation, and snoring were all improved. We concluded that the device is useful in the long-term treatment of patients with mild to moderate sleep apnea.

SLEEP DEPRIVATION

We have concluded our studies of the sleep of long-haul truck drivers. The results have been published in a final report to the Federal Highways Administration and in an article in the New England Journal of Medicine. Sleep deprivation in association demanding work schedules is an important public safety concern. The work and sleep schedules of long-haul truck drivers are not clearly understood, and more data are needed on the role sleep deprivation may play in truck crashes.

In a federally mandated study, 80 long-haul truck drivers working sequential day shifts, night shifts, or irregular shifts in the United States and Canada had round-the-clock electrophysiologic monitoring of sleep and wakefulness. The drivers had an average of 4.78 hours of electrophysiologically verified sleep per day over the 5 days of the study, which was about 2 hours less than their reported ideal amount of sleep. No crashes or vehicle mishaps occurred. Two drivers had previously undiagnosed and untreated sleep apnea. Napping was detected in 35 drivers. Sleep during naps averaged 27.1 minutes and augmented the sleep obtained in principal sleep periods by 11%. While driving at night, some drivers had electrophysiologic recordings that occasionally showed sleeplike patterns (2 of 80 drivers on 2 of 360 trips). Such sleeplike patterns did not involve drivers with sleep apnea and were not associated with the presence of drugs as indicated by toxicologic screening of urine samples.

We concluded that sleep deprivation to a degree that is known to impair performance did occur, suggesting that sleep deprivation could be a contributory factor in accidents involving long-haul truck drivers. Napping can significantly augment the sleep obtained in principal sleep periods. Potential countermeasures include improvements in education and work scheduling to reflect the importance of obtaining adequate sleep.

PUBLICATIONS

Dawson, A., Bigby, B.G., Poceta, J.S., Mitler, M.M. Effect of bedtime alcohol on inspiratory resistance and respiratory drive in snoring and nonsnoring men. Alcohol. Clin. Exp. Res. 21:183, 1997.

Doghramji, K., Mitler, M., Sangal, R.B., Shapiro, C., Taylor, S., Walsleben, J., Belisle, C., Erman, M., Hayduk, R., Hosn, R., O'Malley, E., Sangal, J., Schutte S., Youakim, J. A normative study of the Maintenance of Wakefulness Test (MWT). Electroencephalogr. Clin. Neurophysiol., in press.

Doghramji, K., Mitler, M., Sangal, R.B., Shapiro, C., Taylor, S., Walsleben, J., the MWT Normative Study Group. A normative study of the Maintenance of Wakefulness Test (MWT): Preliminary report. Sleep Res. 25:233, 1996.

Hayduk, R., Flodman, P., Spence, M.A., Erman, M.K. Mitler, M. Monozygotic twins with narcolepsy: Preliminary report. Sleep Res. 25:252, 1996.

Hayduk, R., Flodman, P.M., Spence, M.A., Erman, M.K., Mitler, M.M. HLA haplotypes, polysomnography and pedigrees in a case series of patients with narcolepsy. Sleep, in press.

Kelly, T.I., Mitler, M.M., Bonnet, M.H. Sleep latency measures of caffeine effects during sleep deprivation. Electroencephalogr. Clin. Neurophysiol., in press.

Loube, D.I., Poceta, J.S., Morales, M.C., Peacock, M.D., Mitler, M.M. Self-reported snoring in pregnancy: Association with fetal outcome. Chest 109:885, 1996.

Menn, S.J., Loube, D.I., Morgan, T.D., Mitler, M.M., Berger, J.S., Erman, M.K. The mandibular repositioning device: Role in the treatment of obstructive sleep apnea. Sleep 19:794, 1996.

Mitler, M.M. Sleepiness and human behavior. Curr. Opin. Pulm. Med. 2:488, 1996.

Mitler, M.M., Miller, J.C., Lipsitz, J.J., Walsh, J.K., Wylie, C.D. The sleep of long-haul truck drivers. N. Engl. J. Med., in press.

Mitler, M.M., Poceta, J.S. Chronobiologic and medical aspects of alertness. In: Forensic Aspects of Sleep. Shapiro, C., Smith, A.M. (Eds.). Wiley, New York, 1997, p. 131.

Wylie, D., Miller, J.C., Shultz, T., Mitler, M.M., Mackie, R.R. Technical Report: Commercial Driver Fatigue, Loss of Alertness, and Countermeasures (FHWA-MC-97-001). Washington, DC: U.S. Department of Transportation, 1996.

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Division of Virology

Michael B.A. Oldstone, M.D., Division Head

The Division of Virology continues as a focus for intellectual enrichment and exchange with other scientists at TSRI who are studying animal and plant viruses and viral pathogenesis. Its areas of interest are (1) developing transgenic models of human disease; (2) studying the interactions between viruses and host cells; (3) analyzing interactions between viruses and immune cells that result in immunosuppression or autoimmunity; (4) mapping neural tracts and studying interactions between viruses and CNS cells; (5) defining the molecular basis of viral persistence; (6) analyzing mechanisms whereby viruses cause acute, chronic-degenerative, and demyelinating diseases; (7) developing antiviral therapies and vaccines; and (8) targeting drugs into the CNS by using viruses as carriers.


Viral-Immunobiology Laboratory

M.B.A. Oldstone, D. Berger, P. Borrow, W. Cao, J.C. de la Torre, C.F. Evans, J.E. Gairin, D. Gonzalez-Dunia, A. Holz, D. Homann, R. Kiessling, H. Lewicki, M. Manchester, D. Naniche, I. Novella, J. Patterson, C. Sauder, A. Tishon, M.G. von Herrath

The theme of the Viral-Immunobiology Laboratory is understanding the basic mechanisms by which viruses persist, escape immune recognition, and cause disease. An integral part of this goal is to correct the immunodeficiency that occurs in persistent infections so that viral persistence can be terminated. Another component is correcting virus-induced altered homeostasis, thereby restoring normal cell function and ending disease. Because different viruses have a number of unique life histories, the laboratory has focused its resources on three: lymphocytic choriomeningitis virus, measles virus, and Borna disease virus.


The Anatomy of Viral Persistence and the Essential Host Factors Involved in Terminating Persistent Viral Infection

A. Tishon, H. Lewicki, P. Borrow, D. Homann, D. Berger, N. Sevilla, W. Cao, I. Novella, J.C. de la Torre, M.B.A. Oldstone

Persistent viral infection has two basic ingredients: an immune response that is ineffectual in recognizing and clearing virus or virus-infected cells and a unique component or strategy of viral replication. Viral persistence occurs as an evasion of the host's immunologic surveillance system. Lymphocytic choriomeningitis virus (LCMV) persists in vivo by three distinct mechanisms. First, mice infected at birth with LCMV become persistently infected for life because the animals cannot mount an effective antiviral cytotoxic T-lymphocyte response as virus replicates in the thymus and specifically deletes (negative selection) LCMV-reactive T cells.

Second, in such persistently infected mice, LCMV variants are generated and selected for replication in immune cells. Unlike wild-type parental virus, several of these variants cause persistent infection when inoculated into immunocompetent adult mice and show tropism for interdigitating dendritic cells in the white matter of the lymph nodes and spleen. In contrast, similar inoculation of the parental virus generates LCMV-specific cytotoxic T lymphocytes that clear the virus so that neither persistent infection nor immunosuppression occurs. Such wild-type virus shows a preference for replicating in F4/80+ macrophages in the red pulp of the lymph nodes and spleen and not in the interdigitating dendritic cells. Hence, although titers of wild-type virus in the spleen and lymph nodes are equivalent to titers of immunosuppressive variants in these tissues, the cellular distribution of the two types of virus is different. Third, offspring from persistently infected mice are infected in utero, and such mice at or after birth cannot mount effective antiviral cytotoxic T-lymphocyte responses.

Adoptive transfer of virus-specific immune T-memory lymphocytes to any of these three categories of mice leads to clearance of the virus and of viral nucleic acid sequences from the animals' blood and infected cells, thereby terminating (curing) the persistent infection.

Although LCMV-specific MHC class I--restricted CD8 T cells can terminate an acute infection by themselves, curing of a persistent infection requires the participation of CD4 T cells. Interestingly, although CD8+ T cells and perforin are required for control and viral clearance during an acute infection, IFN-, L-selectin, TNF-, and B cells are not. In contrast, clearance of virus to terminate a persistent infection does not occur in mice whose genes are dysfunctional for IFN-, L-selectin (studied in collaboration with R. Flavell, Yale University), or TNF-.

The individual cells of the immune system are mobile and must interact in a specific and orderly fashion in primary and secondary lymphoid organs and then migrate to distal sites for effectiveness. We found that activated T lymphocytes from L-selectin knockout mice could enter the CNS, liver, and other organs, but their presence was not associated with viral clearance. Similarly, T lymphocytes from B-cell knockout mice were not inhibited in their trafficking to organs, but these lymphocytes also could not clear infected cells of viruses. Studies done in collaboration with W. Weigle, Department of Immunology, indicated that such B-cell knockout mice and, most likely, L-selectin knockout mice had defects in CD4 help and that this defect was the probable reason for the inability of the mice to terminate the persistent viral infection. Anti-LCMV antibodies per se did not play a significant role in clearance of virus from persistently infected mice.

Viruses often persist in neurons, indicating that these cells can preferentially evade immune surveillance. Previous studies showed that neurons have a specific defect in the expression of mRNA for the MHC heavy chain that does not affect transcription of MHC light chain or ß2-microglobulin mRNA or translation of ß2-microglobulin protein. Several sets of neurons also showed defects in transcription of the peptide transporters TAP-1 and TAP-2. Although these defects in antigen processing could be overcome in vitro by the addition of recombinant IFN-, in vivo the interferon was ineffective. For the in vivo experiments, IFN- was expressed solely in the CNS by using a CNS-specific promoter. High levels of IFN- occurred throughout the CNS, resulting in marked induction of MHC class I and class II molecules. However, immunocytochemical and confocal analyses clearly indicated that neither MHC class I nor MHC class II molecules were expressed on neurons even though the neurons were bathed in IFN-.


Virus-Induced Effects on the Cell Cycle

D. Naniche, J. Patterson, M.B.A. Oldstone

Despite an effective vaccine, infection with measles virus still kills more than 1 million persons per year. The main cause of the mortality and morbidity is the virus-induced immunosuppression of lymphocyte function that allows secondary bacterial, fungal, and parasitic infections. According to our work, in vitro infection of human T or B lymphocytes with measles virus results in immunosuppression associated with a block in the G1 phase of the cell cycle. Passage from G1 to S phase requires hyperphosphorylation of the retinoblastoma protein. Sequential complexes consisting of cyclin and cyclin-dependent protein kinase phosphorylate the retinoblastoma protein, and phosphorylation is tightly regulated by inhibitors of cyclin-dependent protein kinase. In collaboration with S. Reed, Department of Cell Biology, we found a significant decrease in the expression of G1-cyclins, including a 10-fold decrease in cyclin E. Phosphorylation of the retinoblastoma protein was also affected.

Recently, in collaboration with M. Billeter and his colleagues at the University of Zurich, we have begun to analyze the role of the various measles virus genes in aborting regulation of the cell cycle. Two additional gene products encoded within the measles virus phosphoprotein open reading frame are V and C. The functions of V and C are not known, but the molecules may be involved in regulation of the replication or transcription of measles virus. In preliminary studies, mutants of measles virus that lack the C protein inhibited cyclin E. In contrast, mutants in which V protein is dysfunctional did not block progression from G1 to S.


Transgenic Models of Human Disease

M. Manchester, J. Patterson, C.F. Evans, M.S. Horwitz, M.G. von Herrath, D. Homann, A. Holz,, M.B.A. Oldstone

In addition to causing profound immunosuppression, measles virus can invade the CNS and establish a persistent infection primarily in neurons and a disease called subacute sclerosing panencephalitis. The recent discovery that the human membrane glycoprotein CD46 is the measles virus receptor enabled us to establish transgenic mice in which the CD46 gene was transcriptionally regulated by a neuron-specific promoter. In collaboration with G. Rall, Fox Chase Cancer Center, Philadelphia, we found that expression of the measles virus receptor made neurons that expressed CD46 permissive to infection with both wild-type measles virus and a mutant strain of the virus. Virus spread throughout the neural axis, traveling along neuronal processes. Replication of virus in neurons was associated with infiltration of CD4 and CD8 T lymphocytes and activation of astrocytes and microglia. All mice died by 28 ± 2 days after viral inoculation. In contrast, no viral replication occurred in nontransgenic mice inoculated with virus. Measles virus infection of CD46+ transgenic mice lacking receptors for type 1 interferon resulted in accelerated death, with all mice dying by 7 days after viral inoculation.

Transgenic mice were generated that expressed the cDNA of murine IFN- in the CNS by using an oligodendrocyte-specific promoter. Expression of the transgene occurred after the mice were 6 weeks old, a time at which both the immune and the CNS systems were fully developed. In normal mice, MHC class I and class II molecules are only expressed on microglial and endothelial cells and not on neurons, astrocytes, or oligodendrocytes. In the transgenic mice, expression of IFN- in the CNS enhanced the constitutive expression of MHC class I and class II molecules on microglial and endothelial cells and induced novel expression of the molecules on oligodendrocytes. MHC molecules were not expressed by either neurons or astrocytes. These results indicate that in vivo in the CNS, only oligodendrocytes, microglial cells, and endothelial cells were capable of presenting antigens.

Express of the transgene was directly associated with primary demyelination, as shown by the uptake of myelin degradation products by activated microglial cells or macrophages and loss of the myelin sheath with morphologic preservation of both axons and oligodendrocytes (Fig. 1). Clinically, shaking, incoordination, and hind limb weakness developed in the transgenic mice, and 100% of the males and 50% of the females died within 8 months. Upregulation of both MHC class I and MHC class II molecules on microglial cells and oligodendrocytes, activation of microglial cells, T-lymphocyte infiltration, astrocytosis, elevated expression of inflammatory cytokines, and upregulation of intracellular adhesion molecule-1--findings associated with demyelination in humans with multiple sclerosis--were observed in transgenic mice after 8 weeks of age. These results provide evidence for a direct role of IFN- as an inducer of CNS demyelination and opportunities for dissecting the mechanisms by which demyelination occurs.

Specific peptides presented by MHC class I molecules on the surface of cells are crucial for the activation of cytotoxic T lymphocytes (CTLs) and subsequent killing of target cells. In collaboration with J. Gairin, CNRS, Toulouse, France, we designed a specific blocking-antagonistic peptide that bound with high affinity to the MHC class I Db allele but was engineered not to activate Db-restricted CTLs specific for lymphocytic choriomeningitis virus (LCMV). Ordinarily, transgenic mice that express an LCMV protein (nucleoprotein or glycoprotein) in ß-cells of the islets of Langerhans under the control of the rat insulin promoter live normal lives and show no indication of insulin-dependent diabetes mellitus (IDDM). However, when such mice are given replicating LCMV, IDDM develops in more than 90% of the animals.

When the engineered peptide was given to such mice, the CTL response was reduced severalfold, T lymphocytes were not found in the islets of Langerhans, and IDDM was prevented in more than 98% of the mice during an 8-month observation period. In these mice, the IDDM was Db-restricted, and control of the LCMV infection was the responsibility of primarily Db-restricted CTLs but also Kb-restricted CTLs. Therefore, when IDDM was aborted, virus was also cleared through the remaining Db-restricted CTLs and the Kb-restricted anti-LCMV CTL response, although viral clearance required 14--21 days rather than the normal 7--10 days. These findings indicate a novel approach for the treatment of an autoimmune disease that is linked to a specific MHC class I allele and initiated by a lymphocyte response to those molecules.

PUBLICATIONS

Borrow, P., Lewicki, H., Wei, X., Horwitz, M.S., Peffer, N., Meyers, H., Nelson, J.A., Gairin, J.E., Hahn, B.H., Oldstone, M.B.A., Shaw, G.M. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nature Med. 3:205, 1997.

Borrow, P., Oldstone, M.B.A. Lymphocytic choriomeningitis virus. In: Viral Pathogenesis. Nathanson, N. (Ed.). Lippincott-Raven, Philadelphia, 1997, p. 593.

Brot, M.D., Rall, G.F., Oldstone, M.B.A., Koob, G., Gold, L.H. Deficits in discriminated learning remain despite clearance of long-term persistent viral infection in mice. J. Neurovirol. 3:265, 1997.

Buesa-Gomez, J., Teng, M.N., Oldstone, C.E., Oldstone, M.B.A., de la Torre, J.C. Variants able to cause growth hormone deficiency syndrome are present within the disease-nil WE strain of lymphocytic choriomeningitis virus. J. Virol. 70:8988, 1996.

Cao, W., Oldstone, M.B.A., de la Torre, J.C. Viral persistent infection affects both transcriptional and post-transcriptional regulation of neuron-specific molecule GAP43. Virology 230:147, 1997.

Evans, C.F., Horwitz, M.S., Hobbs, M.V., Oldstone, M.B.A. Viral infection of transgenic mice expressing a viral protein in oligodendrocytes leads to chronic central nervous system autoimmune disease. J. Exp. Med. 184:2371, 1996.

Grewal, I.S., Borrow, P., Pamer, E.G., Oldstone, M.B.A., Flavell, R.A. The CD40/CD154 system in anti-infective host defense. Curr. Opin. Immunol., in press.

Horwitz, M.S., Evans, C.F., McGavern, D.B., Rodriguez, M., Oldstone, M.B.A. Primary demyelination in transgenic mice expressing interferon-gamma. Nature Med. 3:1037, 1997.

Hudrisier, D., Mazarguil, H., Oldstone, M.B.A., Gairin, J.E. Molecular dissection of MHC-peptide-TcR interaction: Use of chimeric viral epitopes provides evidence of the hierarchial role of peptide residues in T-cell receptor activation. J. Virol., in press.

Hudrisier, D., Oldstone, M.B.A., Gairin, J.E. The signal sequence of lymphocytic choriomeningitis virus contains an immunodominant cytotoxic T-cell epitope that is restricted by both H-2Db and H-2Kb molecules. Virology 234:62, 1997.

Lee, M.-S., Sawyer, S., Arnush, M., Krahl, T., von Herrath, M., Oldstone, M.B.A., Sarvetnick, N. Transforming growth factor-ß fails to inhibit allograft rejection or virus-induced autoimmune diabetes in transgenic mice. Transplantation 7:1, 1996.

Manchester, M., Gairin, J.E., Patterson, J.B., Alvarez, J., Liszewski, M.K., Eto, D.S., Atkinson, J.S., Oldstone, M.B.A. Measles virus recognizes its receptor, CD46, via two distinct binding domains within SCR1-2. Virology 233:174, 1997.

Oldstone, M.B.A. Commentary: Virus-lymphoid cell interactions. Proc. Natl. Acad. Sci. U.S.A. 93:12756, 1996.

Oldstone, M.B.A. How viruses escape from cytotoxic T lymphocytes: Molecular parameters and players. Virology 234:179, 1997.

Oldstone, M.B.A. Viruses and autoimmune diseases. Scand. J. Immunol. 46:320, 1997.

Rall, G.F., Manchester, M., Daniels, L.R., Callahan, E.M., Belman, A.R., Oldstone, M.B.A. A transgenic mouse model for measles virus infection of the brain. Proc. Natl. Acad. Sci. U.S.A. 94:4659, 1997.

Rall, G.F., Oldstone, M.B.A. Viral persistence in the central nervous system. In: In Defense of the Brain. Peterson, P.K., Remington, J.S. (Eds.). Blackwell, Cambridge, England, 1997, p.273.

Steinhauer, D.A., Martin, J., Lin, Y.P., Wharton, S.A., Oldstone, M.B.A., Skehel, J.J., Wiley, D.C. Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity. Proc. Natl. Acad. Sci. U.S.A. 93:12873, 1996.

Teng, M.N., Borrow, P., Oldstone, M.B.A., de la Torre, J.C. A single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome. J. Virol. 70:8438, 1996.

Teng, M.N., Oldstone, M.B.A., de la Torre, J.C. Suppression of lymphocytic choriomeningitis virus-induced growth hormone deficiency syndrome by disease-negative virus variants. Virology 223:113, 1996.

Tishon, A., Manchester, M., Scheiflinger, F., Oldstone, M.B.A. A model of measles virus-induced immunosuppression: Enhanced susceptibility of neonatal human PBLs. Nature Med. 2:1250, 1996.

von Herrath, M.G., Coon, B., Oldstone, M.B.A. Low-affinity cytotoxic T-lymphocytes require IFN- to clear an acute viral infection. Virology 229:349, 1997.

von Herrath, M.G., Dyrberg, T., Oldstone, M.B.A. Oral insulin treatment suppresses virus-induced antigen-specific destruction of ß cells and prevents autoimmune diabetes in transgenic mice. J. Clin. Invest. 98:1324, 1996.

von Herrath, M.G., Efrat, S., Oldstone, M.B.A., Horwitz, M.S. Expression of adenoviral E3 transgenes in ß-cells prevents autoimmune diabetes. Proc. Natl. Acad. Sci. U.S.A. 94:9808, 1997.

von Herrath, M.G., Evans, C.F., Horwitz, M.S., Oldstone, M.B.A. Using transgenic mouse models to dissect the pathogenesis of virus-induced autoimmune disorders of the islets of Langerhans and the central nervous system. Immunol. Rev. 152:111, 1996.

von Herrath, M., Homann, D., Oldstone, M.B.A. The role of viruses. In: Immunologically Mediated Endocrine Diseases. Gill, R., et al. (Eds.). Lippincott-Raven, Philadelphia, in press.

Waters, J.B., Oldstone, M.B.A., Hahn, K.M. Changes in the cytoplasmic structure of CTLs during target cell recognition and killing. J. Immunol. 157:3396, 1996.

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Mechanisms and Consequences of Viral Persistence in the CNS

D. Gonzalez-Dunia, C. Sauder, B. Cubitt, J.C. de la Torre

Epidemiologic and clinical data, together with virologic studies, have shown that viruses can establish chronic infections in the CNS. Viral persistence in the CNS can cause progressive neurologic disorders with diverse pathologic characteristics. These findings have led to the hypothesis that viruses can contribute to human mental diseases of unknown etiology. Therefore, uncovering the relevant infectious agents and elucidating the mechanisms and consequences of viral persistence in the CNS are of paramount importance to human health.

We are using infection with Borna disease virus (BDV) as a model to gain insights about these virus-host interactions. BDV causes CNS disease manifested by behavioral abnormalities in several vertebrate species. Infection with BDV is associated with diverse pathologic manifestations and a variable period of incubation, depending on both host and viral determinants. BDV infection is an important model for the study of viral persistence in the CNS. Studies on this viral system are contributing to the elucidation of virus-cell interactions in the CNS that can lead to immune-mediated damage, neurodevelopmental abnormalities, and alterations in cell-differentiated functions that affect brain homeostasis. In particular, we are investigating the mechanisms whereby BDV persistence in the brain in rats leads to disturbances in the postnatal maturation of the cerebellum and hippocampus. We are also assessing the consequences of the chronic and prominent astrocytosis associated with this persistent viral infection.

Seroepidemiologic data and recent molecular epidemiologic studies support the hypothesis that BDV infection is associated with certain human neuropsychiatric disorders, namely affective disorders and schizophrenia. Moreover, we recently, for the first time, detected BDV RNA and antigen in the CNS of patients who had a history of major depression.

We have cloned and sequenced the complete BDV genome and have shown that BDV has a nonsegmented, negative-stranded RNA genome with an organization characteristic of Mononegavirales. However, BDV differs from all other known negative-stranded RNA animal viruses in several aspects. For example, BDV has a nuclear site for the replication and transcription of its genome, uses RNA splicing to regulate expression of its genes, and has a novel way of processing the viral surface glycoprotein. Because of its unique features, BDV is considered the prototype of a new virus family: Bornaviridae.

Because of recently developed procedures, reverse genetic approaches can now be used to manipulate the genome of negative-stranded RNA viruses. We are using this experimental approach to generate an infectious cDNA clone for BDV. The ability to generate specific mutations within the BDV genome and analyze their phenotypic expression will enhance our knowledge of many aspects of BDV multiplication and its pathogenesis.

The possible role of BDV in human mental disorders and the prospect of finding other BDV-like emerging viruses, some of which may be clinically relevant, underscore the importance of developing reliable and sensitive diagnostic tools to evaluate the prevalence of BDV in humans and its association with specific mental disorders. We have now developed reagents and procedures that make feasible such studies. Moreover, progress in understanding the molecular biology of BDV should facilitate the design of effective antiviral therapies to combat BDV infection.

PUBLICATIONS

Cubitt, B., de la Torre, J.C. Amantadine does not have antiviral activity against Borna disease virus. Arch. Virol., in press.

Gonzalez-Dunia, D., Cubitt, B., Grässer, F.A., de la Torre, J.C. Characterization of Borna disease virus p56 protein, a surface glycoprotein involved in virus entry. J. Virol. 71:3208, 1997.

Gonzalez-Dunia, D., Sauder, C., de la Torre, J.C. Borna disease virus and the brain. Res. Bull., in press.

Sauder, C., Müller, A., Cubitt, B., Mayer, J., Steinmetz, J., Trabeit, W., Ziegler, B., Wanke, K., Mueller-Lantzsch, N., de la Torre, J.C., Grässer, F. Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: Evidence for high sequence conservation of human blood-derived BDV RNA. J. Virol. 70:7713, 1996.

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Pathogenesis of Virus-Induced Autoimmune Diabetes

M.G. von Herrath, A. Holz, M.B.A. Oldstone

To study the events leading to and to test novel therapeutic approaches for insulin-dependent diabetes mellitus (IDDM), we developed a transgenic mouse model of virus-induced autoimmune IDDM. These mice express proteins from lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter. IDDM does not occur spontaneously in these mice, because the transgene is normally not expressed or cross-presented by "professional" antigen-presenting cells and T cells are therefore unresponsive to the viral ß-cell (self) proteins. This unresponsiveness can be broken by infection with LCMV leading to activation of cytotoxic T lymphocytes (CTLs) that can destroy islets and induce development of IDDM. After LCMV infection, the occurrence of IDDM is slower in mice with thymic expression of the viral antigen (1--6 months) than in mice without thymic expression of the antigen (2 weeks), because fewer and only low-affinity antiself CTLs are generated in the mice with thymic expression.

In our recent studies, we detected an interesting regulatory role for cytokines. In mice with insulitis and islet destruction, the main cytokine found in islets is IFN-; in prediabetic mice without overt insulitis, the main cytokine is IL-4. IFN- is required for destruction of ß-cells; it upregulates MHC class I molecules on ß-cells above basal levels and thus enhances recognition of the cells by CTLs (Fig. 1). Further analyses showed that upregulation of MHC molecules associated with the attraction and activation of antigen-presenting cells to the islets occurred 2 days after inoculation with LCMV, clearly before CD4+ and CD8+ lymphocytes enter the islets (days 6 and 7 after LCMV infection). Thus, ß-cell destruction by virus-activated autoreactive lymphocytes is a multifactorial process that is regulated by cytokines and most likely requires pathologic changes within the islet milieu.

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Study on the Mechanism by Which Oral Antigen Administration Can Abrogate Autoimmune Disease

D. Homann, M.B.A. Oldstone, M.G. von Herrath

We used the transgenic mouse model of virus-induced autoimmune insulin-dependent diabetes mellitus (IDDM) to determine if oral administration of a potential self-antigen different from the initial triggering (viral) ß-cell antigen can be effective as a therapy for autoimmune diabetes. The mice in this model express proteins from lymphocytic choriomeningitis virus under control of the rat insulin promoter. When transgenic mice infected with lymphocytic choriomeningitis virus were given insulin orally twice per week until 6 weeks after infection, diabetes was prevented in 40--60% of the mice for up to 8 months (Fig. 1). Antiself (viral) cytotoxic T lymphocytes were generated at equal levels in insulin-treated mice with and without IDDM, but pancreas from insulin-treated mice without IDDM contained sixfold more lymphocytes producing IL-4, IL-10, or TGF-ß and twofold fewer lymphocytes producing IL-2 or IFN- than pancreas from insulin-treated mice with IDDM. Our findings suggest that a bystander effect in which regulatory T cells change the ratio of cytokines associated with type 1 and type 2 T-helper cells in the target tissue can be of value in preventing and treating IDDM.

The protective effect of insulin was completely abrogated by either a one (hormonally inactive) or a two (hormonally active) amino acid substitution in the ß-chain of the insulin molecule. These results suggest that the efficacy of insulin in preventing IDDM is not due to a direct hormonal effect and that antigens for oral administration must be evaluated carefully.

PUBLICATIONS

Lee, M.-S., Sawyer, S., Arnush, M., Krahl, T., von Herrath, M., Oldstone, M.B.A., Sarvetnick, N. Transforming growth factor-ß fails to inhibit allograft rejection or virus-induced autoimmune diabetes in transgenic mice. Transplantation 7:1, 1996.

Oldstone, M.B.A., von Herrath, M.G. Virus-induced autoimmune disease: Transgenic approach to mimic insulin-dependent diabetes mellitus and other autoimmune diseases. APMIS 104:689, 1996.

von Herrath, M.G., Coon, B., Oldstone, M.B.A. Low-affinity cytotoxic T-lymphocytes require IFN- to clear an acute viral infection. Virology 229:349, 1997.

von Herrath, M.G., Dyrberg, T., Oldstone, M.B.A. Oral insulin treatment suppresses virus-induced antigen-specific destruction of ß cells and prevents autoimmune diabetes in transgenic mice. J. Clin. Invest. 98:1324, 1996.

von Herrath, M.G., Efrat, S., Oldstone, M.B.A., Horwitz, M.S. Expression of adenoviral E3 transgenes in ß-cells prevents autoimmune diabetes. Proc. Natl. Acad. Sci. U.S.A. 94:9808, 1997.

von Herrath, M.G., Evans, C.F., Horwitz, M.S., Oldstone, M.B.A. Using transgenic mouse models to dissect the pathogenesis of virus-induced autoimmune disorders of the islets of Langerhans and the central nervous system. Immunol. Rev. 152:111, 1996.

von Herrath, M.G., Holz, A. Pathological changes in the islet milieu precede infiltration of islets and destruction of ß-cells by autoreactive lymphocytes in a transgenic model of virus-induced IDDM. J. Autoimm. 10:231, 1997.

von Herrath, M.G., Homann, D., Gairin, J.E., Oldstone, M.B.A. Pathogenesis and treatment of virus-induced autoimmune diabetes: Novel insights gained through the RIP-LCMV tg mouse model. Biochem. Soc. Trans. 25:630, 1997.

von Herrath, M.G., Homann, D., Oldstone, M.B.A. The role of viruses. In: Immunologically Mediated Endocrine Diseases. Gill, R., et al. (Eds.). Lippincott-Raven, Philadelphia, in press.

von Herrath, M.G., Oldstone, M.B.A. Interferon- is essential for destruction of ß-cells and development of IDDM. J. Exp. Med. 185:531, 1997.

von Herrath, M.G., Oldstone, M.B.A. Virus-induced autoimmune disease. Curr. Opin. Immunol. 8:878, 1996.

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Neurologic Effects of Feline Immunodeficiency Virus

T.R. Phillips, R.A. Hart, M.C. Barr, E. Mengesha, J.N. Billaud, K. Walker, O. Billet, D.R. Selway, S.J. Henriksen, V. Hampton, J.H. Elder

Feline immunodeficiency virus (FIV) is a lentivirus of domestic cats. In addition to being an important feline pathogen, FIV is closely related to HIV, the agent that causes AIDS. Like HIV, FIV causes a chronic progressive immunodeficiency that ultimately results in the development of opportunistic infections and the death of the host. Neurologic abnormalities are a frequent complication of both viral infections. Our objectives are to gain a better understanding of the neuropathogenesis associated with FIV infections and to investigate various therapeutic interventions.

Because low levels of virus are generally found in the brain concurrently with functional neurologic defects, researchers have speculated that neurologic dysfunction is the result of immune activation that results in increased levels of cytokines. In particular, the concentration of TNF- is increased within the CNS of patients infected with HIV type 1. Therefore, we are doing studies that specifically examine the effects of promising anti--TNF- agents (e.g., thalidomide and pentoxifylline) on FIV-induced neurologic disease. These studies will enable us to examine the effects of this therapeutic approach in a controlled experimental environment that only an animal model can provide.

We are also investigating the lentivirus regulatory protein FIV/Rev. To better understand Rev function, we constructed mutant FIV/Rev clones defective in either activation or binding. Interactions of potential cellular factors with FIV/Rev are also being examined. Our ultimate goals are to understand FIV/Rev function at the molecular level, to develop FIV/Rev as a therapeutic target, and to determine the possible involvement of this protein in the neuropathology of the virus.

In the United States, currently the most common way of acquiring HIV type 1 infection is intravenous drug abuse. Opioids have long been known to cause immunosuppression, and studies have shown that they enhance the replication of lentiviruses in vitro. Therefore, to determine if opioids can act as cofactors, we are doing studies with the FIV cat model of HIV infection to investigate the effects of morphine on the progression of FIV disease.

We are also investigating a cheetah lentivirus. Sera from some cheetahs cross-react with the capsid protein of FIV. Additionally, virus has been isolated from the blood of cheetahs that had an immunosuppressive-like disease. The information gained from these studies should help ensure the survival of this highly endangered species and provide insights into the disease mechanisms of another AIDS-like virus.

PUBLICATIONS

Billaud, J.-N., Phillips, T.R., FIV-A lentivirus model for opiate effects on disease. In: Drugs of Abuse, Immunomodulation, and AIDS. Friedman, H., Madden, J., Klein, T. (Eds.). Plenum, New York, in press.

Jacobson, S., Henriksen, S., Prospero-Garcia, O., Phillips, T., Elder, J., Bloom, F., Fox, H. Cortical neuronal cytoskeletal changes 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, FIV-PPR. J. Neurovirol. 2:388, 1996.

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Brain Waves in Southpaws

J. Polich, L. Hoffman

BRAIN WAVES AND HANDEDNESS

Electroencephalographic measures have shown consistent differences between the cerebral hemispheres in a wide variety of studies. In a similar fashion, P300 event-related brain potentials (ERPs) are asymmetric if task conditions encourage differential hemisphere-specific processing when stimuli are presented to the left or the right hemisphere of the brain. P300 amplitudes in healthy subjects are also greater over the frontal or central parts of the right hemisphere than over the left hemisphere when simple stimulus discrimination tasks or tests are used, and similar results are observed for split-brain patients when center-field presentations are used. Thus, hemispheric asymmetries in P300 ERPs can occur in the absence of lateralized stimulus or task variables.

Despite the general consistency of these findings, the hemispheric effects on P300 amplitudes in healthy subjects can vary in strength or location, often because the subjects are compromised by factors that affect differences in laterality. These differences in laterality are also an important consideration for clinical applications of ERPs, because such differences affect the accuracy of measurements in various groups of patients. A major factor in this context is the subject's handedness (i.e., preferential use of the right hand or the left hand), which is considered a strong index of hemispheric asymmetry.

Indeed, postmortem analysis of the size of the two cerebral hemispheres has indicated differences between left- and right-handed persons. In addition, magnetic resonance imaging studies have shown that hand preference is associated with the size of the corpus callosum, a brain structure that connects the two cerebral hemispheres: left-handed men have larger callosal areas than their right-handed counterparts do; exactly which callosal sections differ and how the sections may differ between sexes is less clear. However, a recent comprehensive review of the pertinent data concluded that (1) persons who are left-handed have significantly larger corpus callosal areas than do persons who are right-handed and (2) when brain size is taken into account, women have larger callosal areas than men do. Handedness therefore appears to originate from neuroanatomic differences related to the size of the corpus callosum, and these differences can produce the neuropsychologic effects observed for these groups of subjects.

BRAIN WAVES AND THE CORPUS CALLOSUM

Because the corpus callosum is critical for the effectiveness of transmissions between the left and right hemispheres of the brain, it is reasonable to suppose that the P300 ERPs would be influenced by this anatomic structure. Studies of patients with brain lesions have suggested that generation of P300 ERPs involves interhemispheric integration of sensory information. Because corpus callosum transmission is involved with ERP attentional and mnestic processing, the relationship between handedness and relative callosal size could readily affect measurement of P300 ERPs at the scalp. New findings from this laboratory support this hypothesis. The study sample consisted of 20 female and 20 male subjects who were left-handed and 20 female and 20 male subjects who were right-handed. The subjects' P300 ERPs to auditory and visual stimulus discrimination tasks with centrally presented stimuli were compared.

As shown in Figure 1, P300 amplitude was appreciably larger for left-handed subjects than for right-handed subjects. These findings are consistent with neuroanatomic studies of differences in callosal size and handedness and suggest that the corpus callosum is an important neural generator of the P300 ERP component observed at the scalp. The implication for clinical applications is that the P300 ERP clearly reflects the integration of sensory information in a much more profound manner than previously understood. These differences between the brain waves of left- and right-handed subjects point to new ways of using P300 ERPs in clinical populations to assess differences in sensory and cognitive hemispheric processing.

PUBLICATIONS

Alexander, J.E., Kuperman, S., Rohrbaugh, J., Morzorati, S., O'Connor, S.J., Porjesz, B., Begleiter, H., Polich, J. Hemispheric differences for P300 amplitude from an auditory oddball task. Int. J. Psychophysiol. 21:189, 1996.

Alexander, J.E., Polich, J. Handedness and P300 from auditory stimuli. Brain Cogn., in press.

Cass, M., Polich, J. P300 from an auditory single-stimulus paradigm: Intensity and tone frequency effects. Biol. Psychol. 46:51, 1997.

Cohen, J., Polich, J. On the number of trials needed for P300. Int. J. Psychophysiol. 25:249, 1997.

Covington, J.W., Polich, J. P300, stimulus intensity, and modality. Electroencephalogr. Clin. Neurophysiol. 100:579, 1996.

Katayama, J., Polich, J. P300 from one-, two-, and three-stimulus auditory paradigms. Int. J. Psychophysiol. 23:33, 1996.

Katayama, J., Polich, J. P300, probability, and the three-tone paradigm. Electroencephalogr. Clin. Neurophysiol. 100:555, 1996.

Katayama, J., Polich, J. Stimulus context determines P3a and P3b. Psychophysiology, in press.

Koziol, J., Alexander, J.E., Bauer, L., Kuperman, S., Morzorati, S., O'Connor, S.J., Rohrbaugh, J., Porjesz, B., Begleiter, H., Polich, J. A new method for displaying correlational arrays. Am. Statistician, in press.

Lardon, M.T., Polich, J. EEG changes from long-term physical fitness. Biol. Psychol. 44:19, 1996.

Mertens, R., Polich, J. P300 from a single stimulus: Modality and response effects. Electroencephalogr. Clin. Neurophysiol., in press.

Morgan, C.D., Covington, J.W., Geisler, M.W., Polich, J., Murphy, C. Olfactory event-related potentials: Older males demonstrate the greatest deficits. Electroencephalogr. Clin. Neurophysiol. 104:351, 1997.

Polich, J. EEG and ERP assessment of normal aging. Electroencephalogr. Clin. Neurophysiol. 104:228, 1997.

Polich, J. On the relationship between EEG and P300: Individual differences, aging, and ultradian rhythms. Int. J. Psychophysiol. 26:299, 1997.

Polich, J. P300 in clinical applications. In: Niedermeyer, E., Lopes da Silva, F. (Eds.). Electroencephalography: Basic Principles, Clinical Applications and Related Fields, 4th ed. Urban & Schwarzenberg, Baltimore, in press.

Polich, J. P300 clinical utility and control of variability. J. Clin. Neurophysiol., in press.

Polich, J., Alexander, J.E., Bauer, L.O., Kuperman, S., Rohrbaugh, J., Morzorati, S., O'Connor, S.J., Porjesz, B., Begleiter, H. P300 topography of amplitude/latency correlations. Brain Topogr. 9:275, 1997.

Polich, J., Bondurant, T. P300 stimulus sequences: Probability and inter-stimulus interval effects. Physiol. Behav. 61:843, 1997.

Polich, J., Heine, M.R.D. P300 topography and modality effects from a single stimulus paradigm. Psychophysiology 33:747, 1996.

Polich, J., Lardon, M.T. P300 and long-term physical exercise. Electroencephalogr. Clin. Neurophysiol., in press.

Polich, J., Margala, C. P300 and probability: Comparison of oddball and single-stimulus paradigms. Int. J. Psychophysiol. 25:169, 1997.

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Characterization of the Protein-Tyrosine Kinase Tyro-3 and Its Ligand Gas6

A.L. Prieto

Receptor tyrosine kinases play important roles in differentiation and survival of neurons and glia by transducing a variety of extracellular signals that result in activation of the receptors' intracellular tyrosine kinase domain. We are interested in determining the function of tyro-3, a tyrosine kinase that belongs to a subfamily of receptors with similar extracellular structures but diverse cellular localizations.

Tyro-3 is of special interest because of its predominant localization in the nervous system, time course of expression, and extracellular structure. Although it is abundant throughout the neocortex, preliminary evidence suggests that tyro-3 is expressed mainly in the dendrites of the CA1 pyramidal neurons of the hippocampus. It reaches its maximal levels of expression during early postnatal development, a time coinciding with the formation and elaboration of synapses. We are exploring the subcellular localization of tyro-3 in the hippocampus to determine the presence of the kinase at synaptic or extrasynaptic sites. The results will provide initial cues for elucidating cellular functions of tyro-3 and will clarify its involvement in synaptic or extrasynaptic processes.

The extracellular domain of tyro-3 has two immunoglobulin-like domains and two fibronectin type-III repeats that have strong structural similarities to such domains found in neural cell adhesion molecules. This finding suggests that tyro-3 may have a role in adhesive cell-cell interactions. Two closely related proteins, gas6 and protein S, induce phosphorylation of the catalytic domain of tyro-3 when they bind to the extracellular region of the kinase. We are analyzing the interaction between the carboxy-terminal region of gas6 and its binding site on the extracellular region of tyro-3 and are identifying potential new ligands that affect the tyrosine kinase activity of tyro-3.

So far, no molecules have been detected that interact with tyro-3 in the neuronal intracellular compartment. We are searching for intracellular targets of tyro-3 that associate with its cytoplasmic domain in an activity-dependent or an activity-independent manner. Identification of these molecules is important, because it could provide clues to the cellular functions regulated by this protein kinase in neurons. These functions could include roles in local signaling events, influx or mobilization of calcium, postsynaptic mechanisms of plasticity, and modulatory interactions with structural or cytoskeletal components. We hope our results will help define the role of tyro-3 in the molecular mechanisms that underlie important functional processes of the developing and mature nervous system.

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Pharmacology of Brain Cannabinoids

P. Schweitzer, G.R. Siggins, S.G. Madamba, D. Piomelli*

* The Neurosciences Institute, La Jolla, CA

Research in our laboratory focuses on the mechanisms of action of cannabinoids in the brain. Cannabinoid substances have powerful psychoactive properties and alter many physiologic processes, including memory and movement. A primary challenge is to determine the endogenous signaling system and the mechanisms that underlie the effects of cannabinoids. Anandamide is currently the prevalent endogenous cannabinoid ligand, but the existence of additional endogenous molecules has been suggested.

The compound 2-arachidonylglycerol (2-AG) was recently isolated from intestine and binds to cannabinoid receptors. In collaboration with D. Piomelli, we investigated the occurrence of 2-AG in the brain and its possible role in the hippocampus. Analyses of lipid extracts from rat brains showed that 2-AG is present in the brain and is about 170 times more abundant than anandamide. We then used hippocampal slice preparations to determine if neural activity increases the production of 2-AG. We electrically stimulated the Schaffer collaterals, a fiber tract that projects to CA1, and then immersed the slices in methanol for later analysis. High-frequency stimulation caused a fourfold increase in the level of 2-AG but had no effect on the formation of anandamide. Further experiments with enzyme inhibitors and cortical neurons in culture indicated that phosphatidylinositol-specific phospholipase C and diacylglycerol lipase activities were needed for formation of 2-AG. Thus, increases in calcium concentration may stimulate phosphatidylinositol-specific phospholipase C activity to produce diacylglycerol, which in turn is hydrolyzed to form 2-AG.

Cannabinoid agonists inhibit long-term potentiation (LTP), an electrophysiologic model for learning and memory. Because 2-AG levels are markedly increased by neural activity, we investigated the effect of 2-AG on LTP in CA1 hippocampus. The presence of 2-AG in the superfusate had little effect on basal synaptic transmission but completely prevented LTP. The presence of 2-AG did not affect preestablished LTP, indicating that the lipid altered the induction rather than the maintenance of LTP. This effect appeared to be receptor mediated, because prior addition of a cannabinoid receptor antagonist in the superfusate prevented the LTP blockade by 2-AG.

Our results indicate that 2-AG is present in the brain and that neural activity increases the biosynthesis of 2-AG in a depolarization- and calcium-dependent manner. In addition, 2-AG is physiologically active on brain neurons and may modulate synaptic plasticity in the hippocampus through activation of cannabinoid receptors. Our findings suggest that 2-AG, like anandamide, may act as an endogenous cannabinoid ligand in the brain. It appears, however, that 2-AG and anandamide may be produced under different physiologic conditions.

PUBLICATIONS

Stella, N., Schweitzer, P., Piomelli, D. A second endogenous cannabinoid that modulates long-term potentiation. Nature 388:773, 1997.

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Central Neuronal Communication and Mechanisms of Drugs of Abuse

G.R. Siggins, P. Schweitzer, S. Madamba, Z. Nie, G. Martin, M. Tallent, T. Krucker, T. Nuñez, W. Zieglgänsberger,* F. Berton,** W. Francesconi,** R. Przewlocki,*** D. Piomelli****

* Max Planck Institut für Psychiatrie, Munich, Germany
** University of Pisa, Pisa, Italy
*** Polish National Academy of Sciences, Krakow, Poland
**** The Neurosciences Institute, La Jolla, CA

Our physiology group studies mechanisms of neuronal communication in the CNS and the effects of abused drugs and pathologic insult on neuronal function. We use extracellular and intracellular recording of neurons in brain slices in vitro and apply drugs, transmitters, cytokines, and viral products by superfusion or from micropipettes. We also activate inhibitory and excitatory synaptic events by using stimulating electrodes.

We previously reported that the neuropeptide somatostatin, abundant in the hippocampus, inhibits neuronal firing and hyperpolarizes hippocampal pyramidal neurons (HPNs) by opening two potassium channels, the M-channel and the so-called leak channel. These actions are mediated by a leukotriene and by arachidonic acid, respectively, as second messengers. Because it is colocalized in interneurons with the inhibitory neurotransmitter -aminobutyric acid (GABA), somatostatin also may modulate neurotransmission. Therefore, we assessed the effects of somatostatin on pharmacologically isolated synaptic components in HPNs.

Somatostatin inhibited excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptors but had no effect on inhibitory postsynaptic potentials (IPSPs) mediated by GABAA or GABAB receptors. After pharmacologic block of inhibitory synapses, somatostatin had a particularly pronounced depressant effect on the resulting polysynaptic (hyperexcitable) events. This attenuation of excitatory input suggests that the net effect of somatostatin on HPNs is, like the effect of GABA, inhibitory, especially under near-epileptiform conditions. Our other pharmacologic studies suggest that this depressant synaptic effect occurs presynaptically via reduction of glutamate release, mediated by activation of sst2 receptors linked to a Gi/Go GTP-binding protein.

We continue study of abused drugs in the hippocampus and nucleus accumbens, regions thought to play major roles in drug abuse. As previously reported, because of their structural and metabolic similarities to arachidonic acid, we tested the action of anandamide, a putative endogenous ligand for brain cannabinoid receptors, and other cannabinoids on hippocampal neurons (see also the report by P. Schweitzer). These agonists had no effect on leak potassium channels but decreased M-channel activity, an effect surprisingly opposite to that of arachidonic acid. Other results showed the formation and release of anandamide in the hippocampus.

We also have continued to evaluate the role of endogenous opiates in the hippocampus, where dynorphin, enkephalins, and orphanin FQ (nociceptin) exist in interneurons and in various input pathways. Recent ultrastructural studies have revealed opiate and "orphan" receptors on HPN dendrites. However, we found no significant effect of several selective or µ receptor agonists on membrane currents (including the M current) in HPNs. Surprisingly, the agonists dynorphin A and U50,488h increased M-channel activity. Nociceptin mimicked this effect but also opened leak potassium channels. The opiate antagonist naloxone and a selective receptor antagonist reversed the M-channel effect of all three ligands but not the leak-channel effect of nociceptin. These findings suggest an important inhibitory role for receptor agonists in the hippocampus and suggest that nociceptin can postsynaptically modulate the excitability of HPNs through both opioid and nonopioid receptors linked to potassium channels.

However, the functional implication of these opiate effects in the hippocampus remains unclear. Therefore, we stimulated rat hippocampal slices with high-intensity tetanic stimulation to induce long-term depression (a cellular model of synaptic plasticity) of population EPSPs. Naloxone blocked this depression, suggesting that the strong stimulation used for the induction of long-term depression may release opioid peptides that could play a role in long-term plasticity.

We previously described studies of ethanol effects in the hippocampus, in which we pharmacologically isolated IPSPs to reevaluate a controversy over ethanol enhancement of GABA effects. We found that ethanol had no effect on isolated IPSPs unless GABAB receptors were blocked, whereupon ethanol significantly augmented the remaining GABAA component. To determine the generality of this complex interaction between ethanol and GABA receptors, we have repeated these studies in the nucleus accumbens, with essentially identical results. However, in the nucleus accumbens, unlike in the hippocampus, ethanol enhanced the currents evoked by exogenous GABA, even without block of GABAB receptors. Interestingly, superfusion of a metabotropic glutamate receptor antagonist inhibited this ethanol enhancement of GABA currents. These data suggest that ethanol enhancement of GABAergic neurotransmission in the nucleus accumbens is regulated by multiple metabotropic influences.

The nucleus accumbens also contains abundant opiate peptides. Because opiates reduced glutamatergic EPSPs, these synapses could be major sites underlying the rewarding effects of heroin. Ethanol also markedly decreased these EPSPs, and naloxone reversed this effect. These findings suggest that the addictive properties of alcohol could involve reduction of synaptic transmission via release of endogenous opiates. Very low concentrations of ethanol also antagonized responses to exogenous NMDA; thus, ethanol may act postsynaptically to reduce NMDA receptor activity as well as presynaptically (via opioid receptors) to reduce glutamate release. Further, GABAB (metabotropic) receptor antagonists blocked the ethanol reduction of EPSPs mediated by NMDA receptors. Because the nucleus accumbens probably plays a role in alcoholism, these multiple actions could represent major elements in alcohol craving.

Hypothesizing that long-term exposure to ethanol also alters NMDA receptors, we assessed the sensitivity of NMDA receptors in neurons taken from the nucleus accumbens of rats withdrawn from several weeks of ethanol treatment. Whereas short-term exposure to ethanol blocked NMDA receptors, long-term exposure shifted the dose-response curve for exogenous NMDA to the left of control curves, showing enhanced NMDA potency but not efficacy. Thus, long-term exposure to ethanol upregulates the sensitivity of NMDA receptors in nucleus accumbens neurons, perhaps accounting for the increased behavioral excitability of humans and animals during withdrawal from long-term use of ethanol.

Acamprosate is a new drug that reduces relapse in weaned alcoholics. Because its mechanisms of action are unknown, we applied acamprosate to neurons in slices obtained from the hippocampus and the nucleus accumbens of rats. Acamprosate significantly increased NMDA receptor--mediated EPSPs in both regions but had no effect on non-NMDA EPSPs, suggesting that the clinical efficacy of acamprosate may be due to modulation of NMDA receptor--mediated glutamatergic neurotransmission. More recent studies of nucleus accumbens neurons showed that acamprosate also inhibits presynaptic GABAB receptors, which also could play a role in the clinical effects of the drug.

It is becoming clear that NMDA receptors play some role in opiate tolerance and dependence. In our studies on pharmacologically isolated EPSPs in the nucleus accumbens, activation of µ opiate receptors significantly decreased amplitudes of glutamatergic EPSPs yet markedly enhanced currents evoked by exogenous NMDA. Further, metabotropic glutamate receptor agonists dramatically reduced both NMDA EPSPs and postsynaptic responses to exogenous NMDA. Our pharmacologic data suggest that NMDA receptor--mediated neurotransmission in the nucleus accumbens is under dual inhibitory regulation by different subtypes of metabotropic receptors: group 3 receptors located postsynaptically and group 2 receptors located presynaptically.

We also evaluated the effects of long-term opiate treatment on the interactions of these glutamate receptor subtypes by using rats subcutaneously implanted with morphine pellets for 5 days. We found that the inhibitory effect of a group 2 metabotropic agonist (but not group 3 agonists) on the size of NMDA EPSPs was significantly increased in slices obtained from the nucleus accumbens of these treated rats (Fig. 1). These data suggest that both metabotropic and NMDA glutamate receptors could be involved in morphine tolerance and dependence.

We have continued studies on viral infection and brain function. Evidence suggests that AIDS-related dementia may be due to neurotoxic effects of the HIV coat protein gp120. Therefore, we studied synaptic transmission and plasticity in hippocampal slices from transgenic mice that express gp120. These slices showed greater paired-pulse facilitation and short-term potentiation than slices obtained from littermate controls, but the magnitude of long-term potentiation (a hippocampal model of memory and learning) was significantly reduced. Topically applied gp120 also decreased long-term potentiation, suggesting that effects of the transgene were not merely compensatory. Moreover, the isolated NMDA component of the population EPSP was increased in gp120 transgenic mice. These neuronal alterations in gp120 transgenic animals model memory impairment in AIDS patients and support the HIV coat-protein hypotheses of AIDS-related dementia.

PUBLICATIONS

Berton, F., Francesconi, W., Madamba, S., Siggins, G.R. Acamprosate enhances the N-methyl-D-aspartate receptor-mediated neurotransmission but inhibits presynaptic GABAB receptors in nucleus accumbens neurons. Alcohol. Clin. Exp. Res., in press.

Campbell, I.L., Stalder, A.K., Chiang, C.-S., Bellinger, R., Heyser, C.J., Steffensen, S., Masliah, E., Powell, H., 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.

Francesconi, W., Berton, F., Demuro, A., Madamba, S.G., Siggins, G.R. Naloxone blocks long-term depression of excitatory transmission in rat CA1 hippocampus in vitro. Arch. Ital. Biol. 135:274, 1997.

Krucker, T., Toggas, S.M., Mucke, L., Siggins, G.R. Dissociation of short-term and long-term potentiation in CA1 hippocampus of transgenic mice with cerebral expression of the HIV-1 coat protein. Neuroscience, in press.

Martin, G., Nie, Z., Siggins, G.R. Mu opioid receptors modulate N-methyl-D-aspartate-receptor-mediated responses in nucleus accumbens neurons. J. Neurosci. 17:11, 1997.

Martin, G., Siggins, G.R. Metabotropic glutamate receptors regulate N-methyl-D-aspartate-receptor-mediated responses in nucleus accumbens neurons in vitro. J. Neurophysiol., in press.

Tallent, M., Siggins, G.R. Somatostatin depresses excitatory but not inhibitory neurotransmission in rat CA1 hippocampus. J. Neurophysiol., in press.

Zieglgänsberger, W., Hauser, Ch., Wetzel, Ch., Putzke, J., Siggins, G.R., Spanagel, R. Actions of acamprosate on neurons of the central nervous system. In: Acamprosate in Relapse Prevention of Alcoholism. Soyka, M. (Ed.). Springer-Verlag, New York, 1996, p. 65.

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Neurophysiology of Learning and Memory

S.C. Steffensen, S.J. Henriksen, J.R. Criado, R.-S. Lee, T. Krucker, R.H. Purdy, V.M. Pickel,* A.L. Svingos*

* Cornell University Medical College, New York, NY

In order to further understand the role of the hippocampus in the acquisition of rewarding behaviors, work in this laboratory is dedicated to the neurophysiologic and neurochemical characterization of (1) subcortical inputs that project to the hippocampus, a brain structure implicated in learning and memory, and (2) the neuronal targets of these inputs.

EFFECTS OF DRUGS ON THE MESOLIMBIC SYSTEM

We found that antagonists selective for subtypes of dopamine receptors block ethanol suppression of long-term potentiation, a candidate neurophysiologic mechanism that subserves the formation of memory in the hippocampus. In situ application of ethanol to the ventral tegmental area (VTA), a structure that contains mesolimbic dopamine neurons, disrupts hippocampal function in a manner similar to that produced by systemic ethanol. Although dopamine neurons in the VTA are only slightly affected, nondopamine neurons are markedly inhibited by in situ application or systemic administration of ethanol or heroin.

Using intracellular electrophysiologic and immunocytochemical techniques in vivo, we recently determined that nondopamine neurons in the VTA are indeed neurons that contain -aminobutryic acid (GABA) (Fig. 1). However, they are not merely local circuit neurons subserving inhibition of dopamine neurons, because they project to limbic structures implicated in drug reinforcement behavior. We postulate that nondopamine neurons in the VTA may, by virtue of their wideband activity and widespread mesolimbic connectivity, be critical neuronal effectors of drug reinforcement behavior.

NEUROSTEROIDS, NEUROACTIVE STEROIDS AND HIPPOCAMPAL EXCITABILITY

We found that the neurosteroid dehydroisoandrosterone sulfate (DHEAS) markedly decreases GABA-mediated inhibitory processes in the hippocampus and entrains hippocampal cells to the rhythmic activity (theta) generated by subcortical inputs to the hippocampus. Several studies have established a role for estrogens in ameliorating specific neurodegenerative disorders, mainly disorders associated with the cholinergic neurons of the basal forebrain and the targets of these neurons in the cortex and hippocampus. We evaluated the effects of the estrogens estrone 3-sulfate, estriol 3-sulfate, and estradiol 3-sulfate, DHEAS (positive control), and 17-deoxyestrone 3-sulfate (negative control) on monosynaptic field potential responses, facilitation of excitatory postsynaptic potentials, and recurrent inhibition in the dentate and CA1 hippocampus of male rats and of control (sham ovariectomy) and ovariectomized female rats in vivo and in vitro.

All three estrogen sulfates and DHEAS markedly decreased GABA-mediated recurrent inhibition in the CA1 hippocampus in female control rats. The order of potency was DHEAS > estrone 3-sulfate > estriol 3-sulfate > estradiol 3-sulfate. The lack of effect of 17-deoxyestrone 3-sulfate indicated the selective effect of the estrogens. The effects of the estrogens on recurrent inhibition in CA1 were similar to the effect of DHEAS; however, unlike DHEAS, the estrogens had no effect on recurrent inhibition in this subfield of the hippocampus. The magnitude of the disinhibition of CA1 responses was considerably reduced in male rats and increased in ovariectomized female rats, suggesting sex-related bias and possible sensitization of estrogen-reactive elements.

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Neurobiology of Drug and Alcohol Dependence

F. Weiss, L.H. Parsons, G.F. Koob, F.R. De Fonseca,* S.N. Katner, A.D. Smith, M.R.A. Carrera, T.M. Kerr, B.A. Nadeau, D.L. Smith

* Universidad Complutense, Madrid, Spain

Work in our laboratory continues to focus on neurochemical and neuroendocrine mechanisms in drug addiction. Using in vivo neurochemical and pharmacologic approaches in conjunction with analysis of drug- and alcohol-seeking behavior, we have examined the role of forebrain monoamine and neuropeptide systems in cocaine, cannabinoid, and alcohol abuse.

ACTIVATION OF CORTICOTROPIN RELEASING FACTOR DURING CANNABINOID WITHDRAWAL

Previously, our studies implicated enhanced release of the neuropeptide corticotropin releasing factor (CRF) from neurons in the central nucleus of the amygdala in the stresslike and negative affective consequences of cocaine and ethanol withdrawal. In the past year, we investigated whether CRF release in the central nucleus of the amygdala also has a role in cannabinoid dependence. Short-term administration of a potent synthetic cannabinoid suppressed CRF release in this area of the brain. In contrast, withdrawal induced by a cannabinoid antagonist after long-term cannabinoid treatment was accompanied by a marked elevation in extracellular CRF that was closely time-locked to behavioral signs of cannabinoids withdrawal. In addition, a distinct pattern of Fos activation in the central nucleus of the amygdala and in other stress-responsive limbic brain regions occurred. These data suggest that long-term administration of cannabinoids alters CRF function in the brain limbic system in a manner similar to that observed with other drugs of abuse and induces neuroadaptive processes that may result in vulnerability to future drug dependence.

SEROTONIN RECEPTORS AND THE REINFORCING ACTIONS OF COCAINE

The reinforcing effects of cocaine depend on the functional integrity of the mesocorticolimbic dopamine system of the brain. However, a growing body of literature now also implicates brain serotonin (5-HT) neurotransmission as a substrate for certain aspects of the reinforcing and dependence-inducing properties of cocaine. In particular, recent evidence implicates 5-HT1B receptors in the interoceptive effects of cocaine.

We found that drugs that selectively stimulate 5-HT1B receptors reliably enhanced the reinforcing actions of cocaine in rats. This effect appeared to be the result of a potentiation of the increase in mesolimbic dopamine transmission induced by cocaine. Specifically, 5-HT1B agonists decreased the release of the inhibitory neurotransmitter -aminobutyric acid in the ventral tegmental area, resulting in disinhibition of mesolimbic dopamine neurons and amplification of the cocaine-induced elevation of extracellular levels of dopamine in the nucleus accumbens. We also found that the function of 5-HT1B receptors is altered biphasically during withdrawal after long-term cocaine self-administration. The behavioral response to a 5-HT1B agonist was diminished during the first 2 days of cocaine withdrawal, and a persistent rebound supersensitivity of 5-HT1B receptors to activation emerged 1 week after withdrawal. These findings confirm a role of 5-HT1B receptors in cocaine abuse and suggest that
5-HT1B receptor dysfunction may play a role in the cocaine withdrawal syndrome. Because 5-HT1B receptors are the rodent homolog of 5-HT1Dß receptors in humans, these results may have direct clinical implications and suggest that the 5-HT1Dß receptor is a potentially promising target for interventions used to treat drug abuse.

MESOLIMBIC DOPAMINE FUNCTION AND ETHANOL PREFERENCE

Data from genetic models of ethanol preference suggest that predisposition to ethanol abuse may be related to abnormalities in the functional activity of the mesolimbic--nucleus accumbens dopamine pathway. We therefore examined more precisely the neurochemical characteristics that predict ethanol preference. The results revealed a significant relationship between basal dopamine release in the nucleus accumbens, as measured by quantitative microdialysis, and the degree of preference for ethanol over water. Rats with higher basal synaptic overflow of dopamine had greater ethanol preference in later behavioral tests than rats with low basal dopamine output did. In addition, we found a significant positive correlation between the increase in dopamine release induced by an ethanol challenge and later ethanol preference. These data suggest that both heightened basal dopaminergic tone and high sensitivity to the dopaminergic effects of ethanol may be factors in ethanol preference.

PUBLICATIONS

de Fonseca, R.F., Carrera, M.R.A., Navarro, M., Koob, G.F., Weiss, F. Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276:2050, 1997.

Katner, S.N., Kerr, T.M., Weiss, F. Ethanol anticipation enhances dopamine efflux in the nucleus accumbens of alcohol-preferring (P) but not Wistar rats. Behav. Pharmacol. 7:669, 1996.

Koob, G.F., Parsons, L.H. Caine, S.B., Weiss, F., Sokoloff, P, Schwartz, J.C. Dopamine receptor subtype profiles in cocaine reward. In: Beninger, R.J., Palermo, T., Archer, T. (Eds.). Dopamine Disease States. Editorial CYM, Madrid, Spain, 1996, p. 433.

Raber, J., Prashant, P.M., Kreifelt, M., Parsons, L.H., Weiss, F., Bloom, F.E., Wilson, M.C. Coloboma hyperactive mutant mice exhibit regional and transmitter-specific deficits in neurotransmission. J. Neurochem. 68:176, 1997.

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Viral Pathogenesis, DNA Vaccines, and Ribozymes

J.L. Whitton, L.L. An, S. Harkins, I. Mena, D.E. Hassett, F. Rodriguez, J. Zhang

The ongoing studies in this laboratory are concerned with making life as difficult for viruses as viruses do for us. To this end, we focus on three aspects of the interaction between viruses and their hosts. First, we study antiviral vaccination to understand how vaccines work and how they might be improved. Second, we study viral pathogenesis to determine how these agents cause disease. Third, we are evaluating ribozymes as a therapeutic measure to intervene in ongoing infection.

VACCINES

Antiviral vaccines are a landmark in medical care and have led to the eradication of smallpox; the approaching eradication of poliomyelitis; and the diminution, at least in developed countries, of a variety of microbial diseases. Nevertheless, new challenges (e.g., HIV disease) have arisen, and old foes (e.g., tuberculosis, measles) threaten to reappear. A recent approach to immunization is the direct inoculation of plasmid DNA.

This approach shows great promise, but, surprisingly, the precise mechanism that underlies this technology remains unclear. We showed that inoculation of DNA into an immune host results in protective immunity and a florid local inflammatory response, rich in CD4+ and CD8+ T cells. We also found that DNA immunization can be improved by covalently attaching the viral protein to ubiquitin, a cellular protein that "marks" other proteins for degradation. The ubiquitinated protein is much better than the unmodified viral protein at inducing protective cytotoxic T lymphocytes, but it cannot induce antibodies. In other ongoing studies, we are evaluating the use of minigenes--isolated T- and B-cell epitopes--to induce protective immunity; the capacity of soluble proteins to induce cytotoxic T lymphocytes; and, in collaboration with M. von Herrath, Department of Neuropharmacology, the role of CD4+ T cells in maintenance of immunologic memory.

VIRAL PATHOGENESIS

Coxsackievirus B3 is an important human pathogen that causes a variety of clinical syndromes, including myocarditis. It is strongly implicated as an underlying cause of idiopathic dilated cardiomyopathy, the most common indicator for heart transplantation in young males. We previously showed the importance of CD4+ and CD8+ T cells in the control of virus-induced myocarditis and have begun to study the role of perforin in this model system.

Furthermore, in mice that have intact T-cell responses but no B-cell responses, coxsackievirus B3 persists for at least 45 days at high titers in a variety of organs. Infection with coxsackievirus B3 in these mice may be useful as a model of the chronic infections caused by a related picornavirus, echovirus, in humans with agammaglobulinemia. Echoviral infections in these patients cause morbidity and mortality through long-term persistence, often in the CNS.

ANTIVIRAL RIBOZYMES

Antibiotic therapy for bacterial infections is conceptually simple, because these organisms in general replicate extracellularly. In contrast, viruses replicate intracellularly, subverting the machinery of host cells. The intimate interaction between viruses and host proteins in part explains the difficulty in producing specific antiviral agents. Antisense approaches, which rely on the uniqueness of the viral nucleic acid sequence, offer a tantalizing alternative approach to antiviral therapy.

Previously, we showed that antiviral ribozymes expressed in tissue culture can diminish viral titers up to 100-fold. To determine whether ribozymes expressed in the right place, at the right time, and in a good concentration can interfere with viral infection and disease in vivo, we are attempting to express antiviral ribozymes in the CNS of transgenic mice.

PUBLICATIONS

An, L.L., Pamer, E.G., Whitton, J.L. A recombinant minigene vaccine containing a nonameric cytotoxic-T-lymphocyte epitope confers limited protection against Listeria monocytogenes Infection. Infect. Immun. 64:1685, 1996.

An, L.L., Whitton, J.L. A multivalent minigene vaccine, containing B cell, CTL, and Th epitopes from several microbes, induces appropriate responses in vivo, and confers protection against more than one pathogen. J. Virol. 71:2292, 1997.

Gebhard, J.R., Perry, C.M., Mahadeviah, S., Whitton, J.L. Use of a non-viral vector to express a chimeric tRNA-ribozyme against lymphocytic choriomeningitis virus: Cytoplasmic accumulation of a catalytically-competent transcript, but minimal antiviral effect. Antisense Nucleic Acid Drug Dev. 7:3, 1997.

Hildeman, D., Salvato, M.S., Whitton, J.L., Muller, D. Vaccination protects ß2-microglobulin deficient mice from immune mediated mortality but not from persisting viral infection. Vaccine 14:1223, 1996.

Prasad, Y., Whitton, J.L. CNS-specific ribozyme expression. Methods 10:365, 1996.

Whitton, J.L., Gebhard, J.R., Mena, I. 1997. Antisense and ribozyme approaches to the control of virus infection. In: Persistent Viral Infections. Ahmed, R., Chen, I. (Eds.). Wiley, New York, in press.

Whitton, J.L., Yokoyama, M. Proteins expressed by DNA vaccines induce both local and systemic immune responses. Ann. N.Y. Acad. Sci. 797:196, 1996.

Whitton, J.L., Yokoyama, M., Zhang, J. DNA immunization in an arenavirus model. In: Symposium in Immunology. V. Antiviral Immunity. Eibl, M., et al. (Eds.). Springer-Verlag, New York, 1996, p. 151.

Yokoyama, M., Hassett, D.E., Zhang, J., Whitton, J.L. DNA immunization can stimulate florid local inflammation, and the antiviral immunity induced varies depending on injection site. Vaccine 15:553, 1997.

Yokoyama, M., Zhang, J., Whitton, J.L. DNA immunization: Effects of vehicle and route of administration on the induction of protective antiviral immunity. FEMS Immunol. Med. Microbiol. 14:221, 1996.

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