Molecular and
Integrative Neurosciences
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Cecilia Marcondes, Staff Scientist, Howard S. Fox, M.D., Ph.D., Associate Professor,
and Peter Gaskill, Graduate Student,
Molecular Integrative Neurosciences Department
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005 291
MOLECULAR AND
I N T E G R AT I V E
Paul L. Herrling, Ph.D.
Adjunct Professor
Benjamin Neuman, Ph.D.
Assistant Professor
Tammy Wall, Ph.D.
Adjunct Associate Professor
Tomas Hokfelt, M.D., Ph.D.
Adjunct Professor
Michael B.A. Oldstone, M.D.
Professor
Friedbert Weiss, Ph.D.
Professor
Danny Hoyer, Ph.D.
Adjunct Professor
Shirley M. Otis, M.D.
Adjunct Professor
J. Lindsay Whitton, M.B.,
Ch.B., Ph.D.
Professor
George F. Koob, Ph.D.
Professor
Loren Parsons, Ph.D.
Associate Professor
Henri Korn, M.D., Ph.D.
Adjunct Professor
Tommy Phillips, Ph.D.
Adjunct Assistant Professor
Thomas Krucker, Ph.D.
Adjunct Assistant Professor
John Polich, Ph.D.
Associate Professor
Stefan Kunz, Ph.D.
Assistant Professor
Luigi Pulvirenti, M.D.
Adjunct Associate Professor
Cary Lai, Ph.D.
Associate Professor
Marisa Roberto, Ph.D.
Assistant Professor
Michel Le Moal, M.D., Ph.D.
Adjunct Professor
Amanda Roberts, Ph.D.
Assistant Professor
Jan O. Lundstrom, Ph.D.
Adjunct Professor
Michael G. Rosenfeld, M.D.
Adjunct Professor
Athina Markou, Ph.D.
Associate Professor
Pietro P. Sanna, M.D.
Associate Professor
NEUROSCIENCES
DEPAR TMENT
S TA F F
Tamas Bartfai, Ph.D.
Professor and Chairman
M. Margarita Behrens, Ph.D.
Assistant Professor
Floyd E. Bloom, M.D.
Professor Emeritus
Jason Botten, Ph.D.
Assistant Professor
Karen T. Britton, M.D., Ph.D.
Adjunct Associate Professor
Michael Buchmeier, Ph.D.
Professor
Iain L. Campbell, Ph.D.
Adjunct Professor
Zhen Chai, Ph.D.
Adjunct Assistant Professor
Bruno Conti, Ph.D.
Assistant Professor
Jose Criado, Ph.D.
Adjunct Assistant Professor
Juan Carlos de la Torre, Ph.D.
Associate Professor
Cindy L. Ehlers, Ph.D.
Associate Professor
Howard S. Fox, M.D., Ph.D.
Associate Professor
Eric Zorilla, Ph.D.
Assistant Professor
S TA F F S C I E N T I S T S
Salvador Huitrón-Reséndiz,
Ph.D.
M. Cecilia Marcondes, Ph.D.
Remi Martin-Fardon, Ph.D.
Zhiguo Nie, Ph.D.
Robert Purdy, Ph.D.
Mitra Rebek, Ph.D.
Heather Richardson, Ph.D.
Svetlana Semenova, Ph.D.
Barbara J. Mason, Ph.D.
Professor
Paul Schweitzer, Ph.D.
Associate Professor
Dorian McGavern, Ph.D.
Assistant Professor
George R. Siggins, Ph.D.
Professor
Madis Metsis, Ph.D.
Adjunct Associate Professor
Craig Slawecki, Ph.D.
Assistant Professor
Thomas Nelson, Ph.D.
Assistant Professor
Michael A. Taffe, Ph.D.
Assistant Professor
S C I E N C E A S S O C I AT E S
Xiaoying Lu, Ph.D.
Elena Crawford
Rong-Sheng Lee, Ph.D.
Hanna Lewicki
Walter Francesconi, Ph.D.
Assistant Professor
S E C T I O N C O V E R F O R T H E M O L E C U L A R A N D I N T E G R AT I V E N E U R O S C I E N C E S
Hermann H. Gram, Ph.D.
Adjunct Associate Professor
Donna L. Gruol, Ph.D.
Associate Professor
D E P A R T M E N T : Background: Positive immunoreactivity for corticotropin-releasing factor (CRF) in
cell bodies and neuronal processes in the central nucleus of the amygdala (CeA) in a rat. Overlays:
Typical records of in vitro intracellular and patch-clamp recordings of membrane (A) and γ-aminobutyric acid (GABA) synaptic properties (B–D) of CeA neurons. CRF increases the GABAergic transmission through an increase of GABA release. Work done by Marisa Roberto, Ph.D., in the laboratory of
Steven J. Henriksen, Ph.D.
Adjunct Professor
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
George R. Siggins, Ph.D.
292 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Sam Madamba
Ralph Feuer, Ph.D.
Matthew Trifilo, Ph.D.
Antoinette Tishon
Cindy Funk, Ph.D.
Roman Vlkolinsky, Ph.D.
Kazutaka Ikeda, M.D.
Tokyo Institute of Psychiatry
Tokyo, Japan
SENIOR RESEARCH
Lucile Garidou, Ph.D.
Karen Wager-Smith, Ph.D.
Shinchi Iwasaki, M.D., Ph.D.
Sandy Ghozland, Ph.D.
Brendan Walker, Ph.D.
Thomas Greenwell, Ph.D.
Sunmee Wee, Ph.D.
Rolf Kiessling, Ph.D.
Karolinska Institutet
Stockholm, Sweden
Isabelle Hunziker, Ph.D.
Jason Whitmire, Ph.D.
Jefferson W. Kinney, Ph.D.
R E S E A R C H A S S O C I AT E S
Peter James, Ph.D.
Manisha Yadav, Ph.D.
Mehrdad Alirezaei, Ph.D.
Paul John Kenny, Ph.D.
Yu Zhao, Ph.D.
Denise Naniche, Ph.D., M.P.H.
Universitat de Barcelona
Barcelona, Spain
Lily Alvarez, Ph.D.
Henning Lauterbach, Ph.D.
Han-E Zhou, Ph.D.
Harinder Aujla, Ph.D.
Dusan Lekic, M.D., Ph.D.
Elina Zuniga, Ph.D.
Michal Bajo, M.D., Ph.D.
Matthias Liechti, Ph.D.
A S S O C I AT E S
Bumsuk Hahm, Ph.D.
Simon Katner, Ph.D.
VISITING
Hilda Bajova, D.V.M.
Lee Martin, Ph.D.
Marco A. Baptista, Ph.D.
Monica Mendez-Diaz, Ph.D.
Fluvia Berton, Ph.D.
Victor Mendoza-Fernandez,
Ph.D.
I N V E S T I G AT O R S
Hedieh Badie, Ph.D.
Genomics Institute of the
Novartis Research Foundation
San Diego, California
David Brooks, Ph.D.
Covadonga Paneda, Ph.D.
Adriaan Bruijnzeel, Ph.D.
Neil Paterson, M.D.
Roberto Ciccioppo, Ph.D.
University of Camerino
Camerino, Italy
Tricia Burdo, Ph.D.
Renaud Jean Burrer, Ph.D.
Vez Repunte Canonigo, Ph.D.
Roberto Cervera, Ph.D.
Zhifeng Chen, Ph.D.
Christopher Cornell, Ph.D.
Rebecca Crean, Ph.D.
Andrew Ray, Ph.D.
Maria Rodriguez-Carreno,
Ph.D.
Jilla Sabeti, Ph.D.
Valentina Sabino, Ph.D.
Ana Sanchez, Ph.D.
Stephen J. Crocker, Ph.D.
Manuel Sanchez-Alavez,
M.D., Ph.D.
Chris Davis, Ph.D.
Karin Sandoval, Ph.D.
Christopher Dayas, Ph.D.
Lisa Sharkey, Ph.D.
Kurt Edelmann, Ph.D.
Nimish Sidhpura, Ph.D.
Toby Escher, Ph.D.
Iustin Tabarean, Ph.D.
Eva Fekete, Ph.D.
David Thurbon, Ph.D.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Urs Christen, Ph.D.
Johann Wolfgang Göthe
Universität
Frankfurt, Germany
Jean E. Gairin, Ph.D.
CNRS
Toulouse, France
Gustavo Cuevas Gonzales,
Ph.D.
Universidad Complutense de
Madrid
Madrid, Spain
Katsuro Hagiwara, Ph.D.
Rakuno Gakuen University
Hokkaido, Japan
Dirk Homann, M.D., Ph.D.
University of Colorado
Health Sciences Center
Denver, Colorado
Laura O’Dell, Ph.D.
University of Texas
El Paso, Texas
Noemi Sevilla, Ph.D.
Universidad Autonoma de
Madrid
Madrid, Spain
Christina Spiropoulou, Ph.D.
Centers for Disease Control
and Prevention
Atlanta, Georgia
Persephone Tough, M.D.
Edward Jenner Institute for
Vaccine Research
Compton, England
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Tamas Bartfai. Ph.D.
Chairman’s Overview
he Department of Neuropharmacology, now called
the Molecular and Integrative Neurosciences Department, was successfully led for more than 2 decades
by Floyd Bloom, the outstanding researcher and organizer
of neurosciences at Scripps Research, in the United States,
and around the world. Under his leadership, the department became one of the key sites for research on drug
and alcohol abuse and on neurovirology, nationally and
internationally. Dr. Bloom became an emeritus professor
March 1, 2005. We thank him for his contributions in
building this department.
Neuroscience is an area that benefits most from the
human genome project, because many hereditary diseases are neurologic and psychiatric disorders. The ability
of modern molecular biologists to manipulate the protein composition of individual selected neurons is opening up possibilities to delineate neuronal networks that
underlie specific behaviors. In order to tap into these
new possibilities, the coming years will see an addition
of a new research branch to the department to complement neurovirology and addiction research. Researchers
in this new branch will study in molecular detail those
ion channels, receptors, and other proteins that contribute
to changes at the levels of cells, networks, and whole
organisms that are manifested as different behaviors. The
explicit goal of the department is to link its successful
T
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293
in vivo work in rodents, primates, and humans to the
outstanding chemistry and structural biology of other
departments at Scripps Research via a strengthened
molecular neuroscience effort, based on the work of
several new faculty members. The recruitment of these
scientists is in progress.
The research reviewed in the following paragraphs
describes findings in 2 main areas of activity this past
year. It showcases the work of 2 young scientists who
already have attracted ample attention with the originality of their approaches to important basic science
problems that also carry serious weight in addressing
diseases such as alcohol addiction and viral infections
of the brain.
Marisa Roberto, who has received the prestigious
Research Society on Alcoholism Young Investigator Award
for 2005, has addressed the cellular and in vivo correlates of acute and chronic alcohol exposure. The focus
of most research on the cellular modifications caused
by ethanol intake has been γ-aminobutyric acid, which
is crucially involved in the anxiolytic effects of alcohol.
George Siggins has played a key role in these studies.
The stress hormone corticotropin-releasing factor and
the antistress and antipain sensation hormone nociceptin
compete for regulation of the effects of ethanol on the
release of γ-aminobutyric acid, and thus their competition decides if the intake of ethanol will be associated
with lessened anxiety and reward or not. During chronic
ethanol exposure, the relative strength of these interactions is changing, as Dr. Roberto has shown. The strength
of the research done by Dr. Roberto and Loren Parsons
is that the results with cells in vitro are similar to the
results in free-moving control (no previous exposure to
ethanol) and ethanol-addicted animals. Thus, the findings form a much closer link to the human disease than
did those of many earlier in vitro studies. Drs. Roberto
and Parsons have also placed the site of interaction
between γ-aminobutyric acid, ethanol, and corticotropin-releasing factor at the releasing presynaptic site of
the communication place: synapses between neurons.
Dorian McGavern’s research joins questions pertaining to virology-immunology and brain research. He studies
how the brain can rid itself of a persistent viral infection
when the most common mechanism by which the body
fights viral infections in the periphery (i.e., the mobilization of cytotoxic T lymphocytes) is not easily applicable.
This situation occurs because the blood-brain barrier
restricts entry of the lymphocytes into the brain and
regulates interactions of the cytotoxic cells with infected
294 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
target cells, such as neurons. Dr. McGavern studies viral
clearance by using infection with a neurotropic virus,
lymphocytic choriomeningitis virus (LCMV), that has
been studied in the department for more than 3 decades
by Michael Oldstone and colleagues.
When mice are infected at birth or in utero with
LCMV, the virus establishes lifelong persistence in every
tissue compartment (e.g., spleen, thymus, lymph nodes,
liver, lung, heart, kidney, CNS; Fig. 1A). As a further
complication, neurons are the sole population of LCMVinfected cells in the CNS parenchyma (Figs. 1B and 1C).
Adoptive transfer of LCMV-specific memory T lymphocytes (both CD8 + and CD4+) obtained from syngeneic
mice immune to LCMV can completely eliminate virus
from all tissue compartments (including the CNS) of
LCMV carrier mice. During this therapeutic process,
peripheral tissues are purged of virus within 15 days; CNS
viral clearance requires 100 days. The reason for this
considerable delay in viral clearance from the brain and
the mechanism by which it occurs are not entirely understood. Dr. McGavern is now conducting studies to determine how cytotoxic T lymphocytes accomplish this mission
within the brain. Understanding these mechanisms will
enable us to manufacture therapeutic T cells to resolve
CNS viral infections.
F i g . 1 . A, Whole-body reconstruction of an adult mouse persis-
tently infected from birth with LCMV. The distribution of LCMV is
shown in white. B, A 2-color sagittal brain reconstruction of a mouse
persistently infected from birth with LCMV (white). Cell nuclei are
shown in gray. Note the even distribution of LCMV throughout the
brain parenchyma. C, Neurons (gray) are the primary LCMV-infected
(white) cell population within the CNS of persistently infected mice.
This high-resolution 2-dimensional image was obtained by using
confocal microscopy. D, LCMV-specific cytotoxic T lymphocytes
labeled with green fluorescent protein engaging infected meningeal
cells (gray) in the CNS. Note that the adhesion molecule lymphocyte
function–associated antigen 1 (white) is polarized to the interface
(or immunologic synapse) between the 2 cells. This image is a maximal projection of a 3-dimensional image obtained by using deconvolution microscopy.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
INVESTIGATORS’ R EPORTS
Studies of Severe Acute
Respiratory Syndrome Virus
and Other Coronaviruses
B. Neuman, R. Burrer, J. Ting, J. Abma, A. Paulino,
P. Iversen,* D. Stein,* B. Adair, J. Rempel, D.L. Gruol,
M. Yeager, P. Kuhn, R. Milligan, M.J. Buchmeier
* AVI BioPharma Inc., Corvallis, Oregon
I N H I B I T I O N O F S E V E R E A C U T E R E S P I R AT O R Y
S Y N D R O M E – A S S O C I AT E D C O R O N AV I R U S B Y P E P T I D E C O N J U G AT E D A N T I S E N S E M O R P H O L I N O O L I G O M E R S
evere acute respiratory syndrome–associated coronavirus (SARS-CoV) is a potent pathogen of
humans and is capable of rapid global spread.
Currently, no antiviral agents effective against this virus
are available for clinical use. To address this problem,
we designed peptide-conjugated antisense morpholino
oligomers (P-PMOs) to bind by base pairing to specific
sequences in the SARS-CoV (Tor2 strain) genome. We
determined the capacity of the P-PMOs to inhibit production of infectious virus and used the oligomers to
probe the function of conserved viral RNA motifs and
secondary structures.
Several virally targeted P-PMOs and a randomsequence control P-PMO had low inhibitory activity
against SARS-CoV. Certain other virally targeted P-PMOs
reduced virus-induced cytopathologic changes and cellto-cell spread as a consequence of decreasing viral amplification. Active P-PMOs were effective when administered
at any time before peak viral synthesis and had sustained antiviral effects while present in culture medium.
P-PMOs had low nonspecific activity against nontarget
RNA or an unrelated arenavirus. Two P-PMOs targeting the viral transcription regulatory sequence region
in the 5′ untranslated region were the most effective
inhibitors. After several passages of virus in the presence of a P-PMO targeted to the transcription regulatory
sequence, partially drug-resistant SARS-CoV mutants
arose, which contained 3 contiguous base point mutations at the binding site of a P-PMO target at the transcription regulatory sequence. The partially resistant
viruses grew more slowly and formed smaller plaques
than did wild-type SARS-CoV.
These results suggest that PMO compounds have
powerful therapeutic and investigative potential in coro-
S
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295
navirus infections. In confirmation, we have shown that
similarly targeted PMOs can lower viral titers by up to
10,000-fold in mice acutely infected with a hepatogenic
strain of mouse hepatitis virus.
P R O P O S E D S T R U C T U R E O F C O R O N AV I R U S E S
We have proposed a model based on electron cryomicroscopy and image analysis for the structure of
SARS-CoV, feline infectious peritonitis virus, and mouse
hepatitis virus. All 3 of these coronaviruses are enveloped and pleomorphic; the diameters of membranebound particles have a gaussian distribution around a
mean of about 80.0 nm. We investigated the structure
and organization of the surface glycoprotein spike, a
triple-pass integral membrane or matrix glycoprotein,
and the viral nucleoprotein.
Electron cryomicroscopy and image analysis revealed
concentric paracrystalline shells of density near the
virion surface (Fig. 1). Orthorhombic lattices of viral
spikes and nucleoproteins were organized in alignment
on the viral envelope. The nucleoprotein lattice may
provide a scaffold that guides assembly of viral parti-
F i g . 1 . Supramolecular design of SARS-CoV. A, Electron cyromi-
crographs show frozen-hydrated pleomorphic SARS-CoV particles
550–1450 Å in diameter. The images are shown in reversed contrast, with density displayed in white. B, Membrane-proximal inner
track 1 (IT-1) and inner track 2 (IT-2) densities are visible in the
averaged radial density plot from 10 semicircular half-virions.
C, Class averages from the edge of particle projections show spike
(S) oligomers alone (left) and in the context of punctate nucleoprotein (N) molecules appended to the inner face of the virion
membrane. D, Linear transmembrane densities ascribed to matrix
(M) protein molecules are visible perpendicular to the viral membrane. E, Schematic side view of the viral membrane shows the
structural proteins. Scale bars = 100 Å.
296 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
cles on compartment membranes of an endoplasmic
reticulum–Golgi complex intermediate in a process
mediated by interaction with the viral matrix protein.
Homotrimeric spike proteins and individual integral
membrane proteins and nucleoproteins were identified
in refined images on the basis of position, predicted size,
and copy number.
We also characterized the structure of the 300-Å
transmembrane protein complex that surrounds the viral
spike protein oligomer, and we proposed the first model
to depict the relationships among these proteins for any
coronavirus. These results provide a structural model
of the spatial relationships between the spike proteins,
the nucleoproteins and the matrix proteins; this model
is essential for understanding the assembly pathway of
SARS-CoV.
I N N AT E I M M U N E R E S P O N S E S I N T H E C N S
We investigated the ability of the CNS to generate
innate immune responses in an in vitro model of CNS
infection. Cultures containing CNS cells were infected
with mouse hepatitis virus strain JHM, which causes
fatal encephalitis in mice. Results from gene array analysis, assessed on RNA isolated from virally infected and
sham-infected cultures, were compared with the results
of parallel protein assays for cytokine, chemokine, and
cell-surface markers.
Of the 126 transcripts differentially expressed
between viral and sham infections, the majority were
related to immunologic responses. Virally induced
increases in IL-6 and TNF-α mRNA and protein expression correlated with the genomic induction of acutephase proteins. Genomic and protein analysis indicated
that viral infection resulted in prominent expression of
neutrophil and macrophage chemotactic proteins. In
addition, mRNA expression of nonclassical class I MHC
molecules H2-T10, H2-T17, H2-M2, and H2-Q10 was
3 to 5 times higher in virally infected cells than in shaminfected cells. Thus, upon infection, resident brain cells
induced a breadth of innate immune responses that
could be vital in directing the outcome of the infection
and in vivo would provide signals that would induce a
response in the peripheral immune system.
Published by TSRI Press®. © Copyright 2005,
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Arenavirus Structure, Function,
and Immunology
J. Botten, B. Neuman, A. Saunders, B. Adair, J. Ting,
J. Abma, A. Paulino, M. Yeager, A. Sette,* M.J. Buchmeier
* La Jolla Institute for Allergy and Immunology, San Diego, California
ecovery from Lassa virus infection usually precedes the appearance of neutralizing antibodies,
indicating that cellular immunity plays a primary
role in clearance of the virus. To date, the role of Lassa
virus–specific CD8+ T lymphocytes has not been evaluated in humans. To facilitate such studies, we sought
to identify human CD8+ T-lymphocyte epitopes, peptide sequences that in association with proteins on
antigen-presenting cells are required for antigen recognition by specific cytotoxic T cells. We searched for
peptides encoded by the genes for the glycoprotein
precursor and the nucleoprotein of 2 genetically distinct strains of Lassa virus. Potential HLA-A2 supertype epitopes were identified by using an HLA epitope
prediction algorithm and were assayed for binding
affinity against a panel of 5 purified HLA-A2 supertype alleles. Of 84 peptides, 33 were able to bind 1
or more alleles with high affinity.
Immunogenicity of high-affinity peptides was evaluated by screening splenic CD8+ T lymphocytes from
peptide-immunized HLA-A*0201/Kbxd transgenic mice
for the ability to produce IFN-γ in response to peptidepulsed HLA-A*0201–restricted target cells in enzymelinked immunospot assays. Among 33 peptides, we
found 19 that were immunogenic in the transgenic
mouse system (Fig. 1). Among the 19 peptides, 17
induced CD8 + T lymphocytes that recognized HLAA*0201–restricted antigen-presenting cells pulsed with
low concentrations of peptide (high avidity).
Infection of human HLA-A*0201–restricted antigenpresenting cells with recombinant vaccinia constructs
that expressed either Lassa virus glycoprotein precursor or nucleoprotein revealed that 4 of the 19 peptides
were naturally processed from native Lassa virus antigen and were recognized by CD8+ T lymphocytes primed
by exposure to Lassa virus peptides. CD8+ T lymphocytes
primed by immunization with one of the glycoprotein precursor peptides also recognized antigen-presenting cells
pulsed with variant peptides derived from the corresponding regions of 2 other Old World and 5 New World
arenaviruses (Fig. 2). The epitopes identified in this
study will aid in the characterization of T-lymphocyte
R
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
297
Arenavirus Reverse Genetics:
Implications for Novel
Antiviral Therapies
M. Perez, A.B. Sánchez, B. Cubitt, J.C. de la Torree
F i g . 1 . Identification of immunogenic peptides in HLA-A*0201
mice after immunization with pools of candidate peptides from
Lassa virus. Immunospot analysis of mice immunized with pools of
peptides from the sequence of the Lassa glycoprotein precursor or
nucleoprotein sequences reveals that significant responses against
approximately 20 peptides are restricted by human HLA-A02.
F i g . 2 . Conservation of a human CD8 + T-cell epitope among
diverse arenaviruses. T cells specific for residues 441–449 of the
Lassa virus glycoprotein precursor can recognize the corresponding
peptide in several other pathogenic arenaviruses, thus providing a
potential basis for a single vaccine against multiple arenaviruses.
responses against Lassa virus and the design of multiepitope vaccines.
PUBLICATIONS
Neuman, B.W., Adair, B.D., Burns, J.M., Milligan, R.A., Buchmeier, M.J., Yeager, M.
Complementarity in the supramolecular design of arenaviruses and retroviruses
revealed by electron cryomicroscopy and image analysis. J. Virol. 79:3822, 2005.
Neuman, B.W., Stein, D.A., Kroeker, A.D., Churchill, M.J., Kim, A.M., Dawson, P., Moulton, H.M., Bestwick, R.K., Iversen, P.L., Buchmeier, M.J. Inhibition, escape and attenuation of SARS coronavirus treated with antisense morpholino oligomers. J. Virol., in press.
Rempel, J.D., Quina, L.A., Blakely-Gonzales, P.K., Buchmeier, M.J., Gruol, D.L.
Viral induction of central nervous system innate immune responses. J. Virol.
79:4369, 2005.
Rodriguez-Carreno, M.P., Nelson, M.S., Botten, J., Smith-Nixon, K., Buchmeier,
M.J., Whitton, J.L. Evaluating the immunogenicity and protective efficacy of a
DNA vaccine encoding Lassa virus nucleoprotein. Virology 335:87, 2005.
Sampath, R., Hofstadler, S.A., Blyn, L.B., Eshoo, M.W., Hall, T.A., Massire, C., Levene,
H.M., Hannis, J.C., Harrel, P.M., Neuman, B., Buchmeier, M.J., Jiang, Y., Ranken, R.,
Drader, J.J., Samant, V., Griffey, R.H., McNeil, J.A., Crook, S.T., Ecker, D.J. Rapid identification of emerging pathogens: coronavirus. Emerg. Infect. Dis. 11:373, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
renaviruses that cause severe hemorrhagic fever
in humans are also a threat as potential agents
of bioterrorism. We developed a reverse genetics system for the prototypic arenavirus lymphocytic
choriomeningitis virus (LCMV) that provides a novel
and powerful approach for investigating the mechanisms that control arenavirus replication, gene expression, assembly, and budding.
Using this system, we found that the viral nucleoprotein and L protein are the minimal trans-acting factors required for RNA synthesis. Notably, genetic and
biochemical evidence indicates that oligomerization of
the L protein is required for arenavirus polymerase function. We also found that both sequence specificity within
the highly conserved 3′-terminal 19 nucleotides of arenavirus genomes and integrity of the predicted panhandle
structure formed between the 5′ and 3′ termini of viral
genome RNAs are required for the viral promoter activity.
In addition, we discovered that the arenavirus small
RING finger Z protein is the driving force of arenavirus
budding. This process is mediated by proline-rich late
domain motifs, similar to motifs that control budding
of several other viruses, including HIV and Ebola virus,
via interaction with specific host-cell proteins. Targeting of Z to the plasma membrane, the location of arenavirus budding, strictly required myristoylation of the
protein. We are using genetic and proteomic approaches
to identify cellular proteins that interact with Z and are
required for arenavirus budding.
The established LCMV reverse genetics system is
an excellent platform for the investigation of novel antiviral strategies against highly pathogenic arenaviruses.
Thus, the sequence and structural constrains of the
arenavirus genome promoter provide new potential targets for aminoglycoside-based drugs for treatment of
arenavirus infections. We have already identified several aminoglycoside-based structures that can potently
inhibit LCMV replication in cell-based assays. Likewise,
the use of short interfering RNA to specifically decrease
expression levels of Z-interacting host-cell proteins led
to inhibition of virus budding, opening new targeting
strategies against arenaviruses.
A
298 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
LCMV is a Rosetta stone for the investigation of
virus-host interactions. We can now generate predetermined specific mutations within the LCMV genome and
analyze their phenotypic expression in vivo, providing
a novel and powerful approach for elucidating the molecular mechanisms that underlie interactions between
arenaviruses and host cells and associated disease.
PUBLICATIONS
de la Torre, J.C. Arenavirus extinction through lethal mutagenesis. Virus Res.
107:207, 2005.
Perez, M., Greenwald, D.L., de la Torre, J.C. Myristoylation of the RING finger Z
protein is essential for arenavirus budding. J. Virol. 78:11443, 2004.
Pinschewer, D.D., Perez, M., de la Torre, J.C. Dual role of the lymphocytic choriomeningitis virus intergenic region in transcription termination and virus propagation. J. Virol. 79:4519, 2005.
Pinschewer, D.D., Perez, M., Jeetendra, E., Bachi, T., Horvath, E., Hengartner, H.,
Whitt, M.A., de la Torre, J.C., Zinkernagel, R.M. Kinetics of protective antibodies
are determined by the viral surface antigen. J. Clin. Invest. 114:988, 2004.
Ritchie, K.J., Hahn, C.S., Kim, K.I., Yan, M., Rosario, D., Li, L., de la Torre, J.C.,
Zhang, D.-E. Role of ISG15 protease UBP43 (USP18) in innate immunity to viral
infection. Nat. Med. 10:1374, 2004.
Sánchez, A.B., de la Torre, J.C. Genetic and biochemical evidence for an oligomeric structure of the functional L polymerase of the prototypic arenavirus lymphocytic choriomeningitis virus. J. Virol. 79:7262, 2005.
cells. BDV has strong tropism for neurons in the limbic system. We showed that the ectodomain of the
BDV p56 glycoprotein is solely responsible for recognition of the virus receptor and cell entry, and we developed reagents to identify cellular receptors of BDV.
Neonatal infection of rats with BDV causes distinct
CNS neurodevelopmental and behavioral abnormalities
that parallel those reported in certain neuropsychiatric
disorders in humans. We compared global gene expression profiles in the CNS of BDV-infected rats and mockinfected control rats. We identified changes in host
gene expression associated with BDV persistence. We
are using a variety of culture cell systems and animal
models to examine the contribution of identified targets to BDV-induced CNS disturbances.
PUBLICATIONS
Perez, M., de la Torre, J.C. Identification of the Borna disease virus (BDV) proteins
required for the formation of BDV-like particles. J. Gen. Virol., in press.
Rosario, D., Perez, M., de la Torre, J.C. Functional characterization of the genomic
promoter of Borna disease virus (BDV): implications of the 3′-terminal sequence
heterogeneity for BDV persistence. J. Virol. 79:6544, 2005.
Sánchez, A.B., Perez, M., Cornu, T., de la Torre, J.C. RNA interference mediated
virus clearance from cells both acutely and chronically infected with the prototypic
arenavirus LCMV. J. Virol., in press.
NeuroAIDS: Chronic Virus-Host
Interaction in the CNS
Virus-Cell Interactions in
Persistently Infected Brains
H.S. Fox, T. Burdo, C. Flynn, P.J. Gaskill, C. Marcondes,
M. Perez, B. Cubitt, K. Hagiwara, D. Rosario,
J.C. de la Torre
ersistent viral infections of the CNS can cause
progressive neurologic disorders associated with
diverse abnormalities. These findings led to the
hypothesis that viruses can contribute to human mental disorders of unknown etiology. We use infection
with Borna disease virus (BDV) as a model system to
investigate cellular and molecular mechanisms that
underlie nonlytic persistent viral infections of the CNS
and associated diseases.
BDV is an enveloped virus with a nonsegmented
negative-stranded RNA genome and is the prototypic
member of the virus family, Bornaviridae, within the
group Mononegaviruses. We established a reverse genetics system for BDV that enables us to investigate the
mechanisms that control BDV RNA replication and gene
expression and interactions between the virus and host
P
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
R. Ojakian, E. Roberts, D. Watry, M. Yadav, M. Zandonatti
he HIV pandemic continues worldwide. Although,
in at least some countries, antiviral treatment is
available, leading to greatly prolonged survival,
the CNS disorders due to HIV (neuroAIDS) continue to
afflict a significant proportion of those who are infected
with the virus. Using infection of rhesus monkeys with
simian immunodeficiency virus (SIV) as a model of
neuroAIDS in humans, we are studying the virology,
immunology, pathology, and neurobiology of the resulting CNS disease.
We have defined the different stages of SIV disease in the CNS. Initial brain invasion by virus occurs
early, by the second week after infection. At this time,
an innate immune response occurs in the brain, with
an upregulation of genes induced by interferon and IL-6.
The adaptive immune response is beginning, with a
switch in the brain CD8+ T-cell phenotype from a surveillance mode to an effector mode, and SIV-specific
cytotoxic CD8+ T cells can be detected in the brain.
By 3 months after infection, the level of virus in the
T
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
brain has decreased by 100-fold, but the number of
CD8 + T cells has increased greatly, and this number
is preserved in the brain during this chronic, relatively
stable phase. Late in the disease, the adaptive immune
response wanes, and the level of virus can again increase
in the brain. This increase is accompanied by a number of measures of CNS dysfunction and an influx of
macrophages, leading to SIV encephalitis. Molecularly,
an upregulation of genes associated with innate immune
responses occurs again, as the macrophages and brain
glial cells are activated.
This molecular profiling of SIV encephalitis led to
2 new areas of investigation. First, we found that several genes and their protein products are increased in
cortical neurons. We are using in vitro studies to define
the effects of the expression of these genes in neurons,
and we hypothesize that 2 possibly overlapping categories of responses will be seen: those that help protect the neurons in the milieu of viral/macrophage
inflammatory reactions and those that lead to dysfunction or death of neurons. Second, although we
and others have found chemotactic factors that induce
entry of macrophages into the brain, we are now investigating the factors that prevent macrophages from leaving the brain, favoring the accumulation of these cells.
One such molecule, osteopontin, is being investigated
in a number of model systems.
Currently, we are focusing on the chronic stage of
SIV infection, modeling the effects of patients who are
infected with HIV but have not yet progressed to AIDS.
At this stage, a chronic, low-level interaction between
virus and immune cells is occurring in the CNS. Although
this interaction is necessary for controlling the virus in
the brain, because of the unique functions of the CNS,
such a prolonged interaction can lead to damage and
dysfunction. In humans, the minor HIV cognitive/motor
complex, which includes cognitive, motor, and/or behavioral abnormalities, defines the spectrum of impairments
found in this stage, whereas in SIV-infected monkeys,
cognitive, motor, circadian, and neurophysiologic abnormalities occur. Neurostructural analysis has revealed a
distinct remodeling induced in populations of cortical
neurons in chronically infected monkeys, and transcriptional profiling has revealed the upregulation of select
immune mediators that also can directly affect neurons.
Studies on how the virus-host interaction leads to the
production of these mediators, and what their direct
effects on neurons are, will help define the pathogenesis of CNS HIV infection, as well as potentially other
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
299
chronic CNS disorders, and lead to preventative or therapeutic strategies.
PUBLICATIONS
Gaskill, P.J., Watry, D.D., Burdo, T.H., Fox, H.S. Development and characterization of positively selected brain-adapted SIV. Virol. J. 2:44, 2005.
Katner, S.N., Flynn, C.T., Von Huben, S.N., Kirsten, A.J., Davis, S.A., Lay, C.C.,
Cole, M., Roberts, A.J., Fox, H.S., Taffe, M.A. Controlled and behaviorally relevant
levels of oral ethanol intake in rhesus macaques using a flavorant-fade procedure.
Alcohol. Clin. Exp. Res. 28:873, 2004.
Madden, L.J., Flynn, C.T., Zandonatti, M.A., May, M., Parsons, L.H., Katner,
S.H., Henriksen, S.J., Fox, H.S. Modeling human methamphetamine exposure in
nonhuman primates: chronic dosing in the rhesus macaque leads to behavioral and
physiological abnormalities. Neuropsychopharmacology 30:350, 2005.
Marcondes, M.C.G., Furtado, G.C., Wensky, A., de Lafaille, M.A.C., Fox, H.S.,
LaFaille, J. Immune regulatory mechanisms influence early pathology in spinal cord
injury and in spontaneous autoimmune encephalomyelitis. Am. J. Pathol., in press.
Masliah, E., Roberts, E.S., Langford, D., Everall, I., Crews, L., Adame, A., Rockenstein, E., Fox, H.S. Patterns of gene dysregulation in the frontal cortex of
patients with HIV encephalitis. J. Neuroimmunol. 157:163, 2004.
Pardo, F.S, Lien, W.W., Fox, H.S., Efird, J.T., Aguilera, J.A., Burton, D.W., Deftos, L.J.
Parathyroid hormone-related protein expression is correlated with clinical course in
patients with glial tumors. Cancer 101:2622, 2004.
Roberts, E.S., Burudi, E.M.E., Flynn, C., Madden, L.J., Roinick, K.L., Watry,
D.D., Zandonatti, M.A., Taffe, M.A., Fox, H.S. Acute SIV infection of the brain
leads to upregulation of IL6 and interferon-regulated genes: expression patterns
throughout disease progression and impact on neuroAIDS. J. Neuroimmunol.
157:81, 2004.
Roberts, E.S., Masliah, E., Fox, H.S. CD163 identifies a unique population of
ramified microglia in HIV encephalitis (HIVE). J. Neuropathol. Exp. Neurol.
63:1255, 2004.
Tensing, E.K., Ma, J., Hukkanen, M., Fox, H.S., Li, T.F., Tornwall, J., Konttinen,
Y.T. Protein kinase C expression in salivary gland acinar epithelial cells in nonobese diabetic mice, an experimental model for Sjögren’s syndrome. Rheumatol.
Int. 25:28, 2005.
Wen, H., Watry, D.D., Marcondes, M.C.G., Fox, H.S. Selective decrease in paracellular conductance of tight junctions: role of the first extracellular domain of
claudin-5. Mol. Cell. Biol. 24:8408, 2004.
Interaction Between Lassa
Virus and Its Receptor and
Development of Antiviral
Drugs Against Lassa Fever
J.M. Rojek, D.L. Boger,* S. Kunz
* Department of Chemistry, Scripps Research
assa virus, a pathogenic arenavirus, causes more
than 500,000 infections per year, is the cause
of several thousand deaths, and is a severe
threat for human health. The identification of α-dystroglycan as the cellular receptor for Lassa virus has
enabled us to study binding of the virus to its recep-
L
300 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
tor, determine the biological effects of the binding, and
develop novel antiviral drugs.
A fundamental reason for the high mortality associated with Lassa virus infection in humans is a failure
of the host’s immune system to control viral replication,
a situation that leads to an unchecked viremia and
hemorrhagic disease. Because rapid dissemination of
Lassa virus critically depends on its attachment to receptor molecules on host cells, drugs that can block the
virus-receptor interaction will give the host’s immune
system more time to generate an efficient antiviral
immune response.
To identify specific inhibitors of the interaction
between Lassa virus and its receptor, we use combinatorial chemical libraries, generated in the laboratory of
D. Boger, Department of Chemistry. Because research
with Lassa virus requires maximum biosafety measures
and equipment, we use retroviral vectors that contain
Lassa virus glycoprotein in their envelope and a luciferase reporter gene to screen for compounds that block
attachment of Lassa virus to host cells. In a second
approach, we generate recombinant antibodies to the
receptors for Lassa virus in which the Fab part of an
IgG molecule is replaced by a virus-binding fragment
of α-dystroglycan. Because of the high binding affinity
of α-dystroglycan for Lassa virus, we expect such antibodies to efficiently neutralize free virus.
In fatal Lassa fever, death occurs in the absence
of extensive inflammation and tissue destruction, indicating that virus-induced changes in the function of
host cells rather than immunopathologic changes are
responsible. Studies of the mechanisms of the interactions between Lassa virus and host cells are important
for understanding viral pathogenesis and for developing
treatment for critically ill patients. Because of its relevance for normal cell function, the cellular receptor of
Lassa virus, α-dystroglycan, is of particular interest in
virus-induced pathologic changes in host cells. Because
the glycoprotein of Lassa virus binds α-dystroglycan with
high affinity and competes with ligands for α-dystroglycan in the extracellular matrix, virus binding most likely
interferes with the normal function of this receptor, contributing to virus-induced dysfunction in host cells.
Using a combination of biochemical and cell biological techniques, we are studying the impact of virus
binding on signal transduction mediated by α-dystroglycan and the function of α-dystroglycan in cell-matrix
adhesion. α-Dystroglycan–associated signaling pathways
and cellular proteins affected by virus binding most likely
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
are host factors involved in viral pathogenesis and may
be novel targets for drugs against Lassa virus.
PUBLICATIONS
Kanagawa, M., Saito, F., Kunz, S., Yoshida-Moriguchi, T., Barresi, R., Kobayashi,
Y.M., Muschler, J., Dumanski, J.P., Michele, D.E., Oldstone, M.B., Campbell,
K.P. Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell 117:953, 2004.
Kunz, S., Rojek, J.M., Perez, M., Spiropoulou, C.F., Oldstone, M.B. Characterization of the interaction of Lassa fever virus with its cellular receptor α-dystroglycan.
J. Virol. 79:5979, 2005.
Kunz, S., Sevilla, N., Rojek, J.M., Oldstone, M.B. Use of alternative receptors different than α-dystroglycan by selected isolates of lymphocytic choriomeningitis
virus. Virology 325:432, 2004.
Relieving the CNS of a
Persistent Viral Infection
D.B. McGavern, P. Truong, H. Lauterbach
variety of pathogens, such as HIV, herpesvirus,
measles virus, and human T-lymphotrophic virus
type I, can establish persistence within the CNS
after entry through a peripheral route. Because of its
importance in maintaining life, this vital tissue compartment must be equipped with a collection of protective
mechanisms that limit potential sources of damage.
Consequently, the CNS is fitted with an array of dampening mechanisms that limit the toxic effects (and in
some cases, the effectiveness) of a pathogen-specific
immune response. Because of this chink in the armor
of the immunologic defenses, a variety of pathogens
can use the CNS as a safe haven, a situation that can
result in severe neurologic disturbances.
The complexity of the CNS suggests that this compartment cannot be cleansed of a persistent pathogen.
However, a seminal series of studies with a model system with lymphocytic choriomeningitis virus (LCMV)
revealed that the body can be cleared of a pathogen
after the pathogen has established persistence. If mice
are infected at birth or in utero with LCMV (referred to
as LCMV carrier mice), the virus establishes persistence
in nearly every tissue compartment, including the CNS.
In addition, in these mice, neurons are the predominant
LCMV-infected cell population in the CNS parenchyma.
Except in extenuating circumstances (e.g., electrical
inactivation), neurons do not readily express MHC
class I or class II molecules, and thus T lymphocytes
are faced with a considerable challenge when attempting to eliminate a persistent infection from these cells.
A
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
However, the CNS (and peripheral tissues) of LCMV
carrier mice can be completely purged of virus by a
single injection of LCMV-specific memory T lymphocytes, a strategy referred to as immunocytotherapy,
and quite remarkably viral clearance within the CNS
appears to occur without any marked neuronal damage.
We are elucidating the mechanism by which memory
T lymphocytes cleanse the CNS of a persistent viral
infection without damaging neurons. We showed that
genetically tagged populations of memory T lymphocytes (both CD8+ and CD4+) specific for the LCMV glycoprotein migrate into the CNS of LCMV carrier mice
after immunocytotherapy and that this migration is associated with a massive recruitment of antigen-presenting cells, including dendritic cells and macrophages.
These antigen-presenting cells display LCMV peptides
and induce the memory T lymphocytes to produce antiviral cytokines (e.g., TNF-α) ex vivo.
On the basis of these observations, we hypothesize that interactions between LCMV-specific T lymphocytes and antigen-presenting cells recruited into the
brain parenchyma after immunocytotherapy contribute
to the noncytopathic clearance of persistently infected
neurons in an indirect, cytokine-dependent manner. If
this hypothesis is correct, therapeutic manipulation of
recruitment of antigen-presenting cells into the CNS may
be a practical strategy to facilitate (or accelerate) clearance of a persistent infection without harming neurons.
PUBLICATIONS
Brooks, D.G., Teyton, L., Oldstone, M.B.A., McGavern, D.B. Intrinsic functional
dysregulation of CD4 T cells occurs rapidly following persistent viral infection. J.
Virol., in press.
Christen, U., Edelmann, K.H., McGavern, D.B., Wolfe, T., Coon, B., Teague,
M.K., Miller, S.D., Oldstone, M.B.A., von Herrath, M.G. A viral epitope that mimics a self antigen can accelerate but not initiate autoimmune diabetes. J. Clin.
Invest. 114:1290, 2004.
McGavern, D.B. Immunotherapeutic relief from persistent infections and amyloid
disorders. Neurology, in press.
McGavern, D.B. The role of bystander T cells in the development of CNS pathology. Crit. Rev. Immunol., in press.
McGavern, D.B., Truong, P. Rebuilding an immune-mediated central nervous system disease: weighing the pathogenicity of antigen-specific versus bystander T cells.
J. Immunol. 173:4779, 2004.
Popkov, M., Jendreyko, N., McGavern, D.B., Rader, C., Barbas, C.F. III. Targeting
tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody. Cancer Res.
65:972, 2005.
Zuniga, E.I., McGavern, D.B., Pruneda-Paz, J.L., Teng, C., Oldstone, M.B.A.
Bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic
cells upon virus infection. Nat. Immunol. 5:1227, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
301
Viral-Immunobiology Laboratory
M.B.A. Oldstone, S. Kunz, D. McGavern, D. Brooks,
K. Edelmann, B. Hahm, L. Martin, M. Trifilo, E. Zuniga,
A. Tishon, H. Lewicki, P. Borrow,* E. Domingo,**
J.E. Gairin,*** R. Kiessling,**** N. Sevilla,**
C. Spiropoulou*****
* Edward Jenner Institute for Vaccine Research, Compton, England
** Universidad Autonoma de Madrid, Madrid, Spain
*** CNRS, Toulouse, France
**** Karolinska Institutet, Stockholm, Sweden
***** Centers for Disease Control and Prevention, Atlanta, Georgia
esearchers in the Viral-Immunobiology Laboratory study both viral diseases and transmissible
spongiform encephalopathies (e.g., prion diseases, scrapie). Included are basic analysis of the
mechanisms by which viruses persist, escape immune
recognition, and cause disease. Integral parts of our
program are understanding how viruses infect cells;
defining the cellular receptors used by viruses; and mapping the trafficking of viruses into cells and the subsequent viral uncoating, replication, assembly, exit, and
spread. Because the immune system has evolved to
recognize, attack, and remove these foreign substances,
we evaluate the immune response against viruses,
probe how viruses subvert this response to provide a
selective advantage for their survival, and study how
the host can correct this subversion to allow termination of viral persistence.
Other major interests are dissecting how viruses
alter differentiation processes of cells they persistently
infect, thereby disturbing homeostasis and causing
disease, and understanding the molecular basis by
which viruses induce autoimmune disease or induce
immunosuppression. Because different viruses have
different lifestyles, we focus our resources on 2 RNA
negative-stranded viruses: lymphocytic choriomeningitis virus and measles virus. We also investigate the
mechanisms by which agents cause transmissible
spongiform encephalopathies.
R
302 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Viruses Evade the Immune
System Through Type I InterferonMediated STAT2-Dependent, but
STAT1-Independent, Signaling
Reprinted from Immunity, Vol. 22, Hahm, B., Trifilo, M.J.,
Zuniga, E.I., Oldstone, M.B., pp. 247-257. Copyright 2005,
with permission from Elsevier.
nderstanding, treating, and preventing diseases
caused by immunosuppression and/or persistent
infections remain both a major challenge in biomedical research and an important health goal. For a
virus or any infectious agent to persist, it must utilize
strategies to suppress or evade the host’s immune
response. Here, we report that two dissimilar viruses
employ a common maneuver to cause a profound
immunosuppression. Measles virus (MV) and lymphocytic choriomeningitis virus (LCMV) interfere with dendritic cell (DC) development and expansion in vivo and
in vitro. The underlying mechanism for this is through
the generation of type I interferon (IFN) that acts via
a signal transducer and activator of a transcription
(STAT)2-dependent, but STAT1-independent, pathway.
Thus, viruses subvert the known antiviral effect of
type I IFN through STAT2-specific signaling to benefit
their survival. These observations have implications
for understanding and developing therapies to treat
diseases caused by immunosuppression and/or persistent infections.
U
Mechanism for the Association
Between Subacute Sclerosing
Panencephalitis and Persistent
Measles Virus Infection
L. Martin, A. Tishon, H. Lewicki, S. Dales,*
M.B.A. Oldstone
* Rockefeller University, New York, New York
e uncoded the precipitating events and biology that underlie the pathogenesis of subacute sclerosing panencephalitis (SSPE). Using
a transgenic mouse model humanized to express the
receptor for measles virus and allow measles virus replication, we found that virus persisted solely in mice in
which T and B lymphocytes were deleted. In mice
W
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
deleted of T and B lymphocytes, infection with measles
virus mimicked SSPE of humans in 4 ways: (1) a progressive fatal CNS disease occurred with replication of
measles virus in neurons; (2) virus in neurons was
defective; (3) cloning of measles virus from the CNS
revealed a predominance of uridine-to-cytosine– and
adenine-to-guanine–biased hypermutations in the matrix
gene, often encompassing 25%–50% of the entire matrix
genome, and (4) electron microscopy of virus in the
CNS revealed the pathognomonic structure of SSPE
measles virus.
Other experiments revealed that the triggering mechanism was an infection by a different virus or an environmental injury that both preceded measles virus
infection and transiently suppressed the host’s immune
system. The transient immunosuppression provided a
window of opportunity that allowed the virus to infect
neurons that became persistently infected during the
host’s lifetime. The recovery of the immune system
allowed the final 2 mimics of SSPE to occur: formation of high titers of antibodies to measles virus and
infiltration of T and B lymphocytes into the CNS.
Because neurons do not display MHC molecules on
their surface, they cannot present measles virus peptides for recognition by these immune T cells, but the
replication of measles virus provides a continuous
antigenic stimulation to expand the immune response
to the virus. Further, according to in vitro experiments,
through a mechanism of antibody-induced antigenic
(measles) modulation, the high titers of antibodies to
measles virus favor viral persistence because antibody
acts on the plasma membrane of infected cells, disordering viral transcription and assembly of viral proteins
inside the cells.
IL-7 Regulates Basal Homeostatic
Proliferation of Antiviral CD4+
T Cell Memory
Reprinted from with permission from Proc. Natl. Acad. Sci.
U. S. A., Vol. 101, Lenz, D.C., Kurz, S.K., Lemmens, E.,
Schoenberger, S.P., Sprent, J., Oldstone, M.B., Homann, D.,
pp. 9357-9362. Copyright 2004 National Academy of
Sciences, U.S.A.
eightened protection from infectious disease as
conferred by vaccination or pathogen exposure
relies on the effective generation and preservation of specific immunological memory. T cells are irre-
H
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
ducibly required for the control of most viral infections,
and maintenance of CD8+T cell memory is regulated
by at least two cytokines, IL-7 and IL-15, which support survival (IL-7, IL-15) and basal homeostatic proliferation (IL-15) of specific CD8+ memory T cells (TM).
In contrast, the factors governing the homeostasis of
pathogen-specific CD4+TM remain at present unknown.
Here, we used a physiologic in vivo model system for
viral infection to delineate homeostatic features and
mechanisms of antiviral CD4+TM preservation in direct
juxtaposition to CD8+T cell memory. Basal homeostatic proliferation is comparable between specific CD4+
and CD8+TM and independent of immunodominant
determinants and functional avidities but regulated in
a tissue-specific fashion. IL-7, identified as the dominant cytokine, and IL-15, an accessory cytokine, regulate basal homeostatic proliferation and survival of
antiviral CD4 +T M. Interestingly, a role for these cytokines in regulation of CD4+T cell memory is not readily
discernible in the generic “memory-phenotype” population, apparently a consequence of its heterogeneous
composition. We also describe a prominent, nonredundant role for IL-7 in supporting basal homeostatic proliferation of CD8 + T M . We propose that homeostatic
control of antiviral CD4+ and CD8+T cell memory is
fundamentally similar and characterized by quantitative, rather than qualitative, differences.
Anchorless Prion Protein Results
in Infectious Amyloid Disease
Without Clinical Scrapie
Reprinted with permission from Science, Vol. 308,
Chesebro, B., Trifilo, M., Race, R., Meade-White, K.,
Teng, C., LaCasse, R., Raymond, L., Favara, C., Baron, G.,
Priola, S., Caughey, B., Masliah, E., Oldstone, M., pp.
1435-1439. Copyright 2005 AAAS.
n prion and Alzheimer’s diseases, the roles played
by amyloid versus nonamyloid deposits in brain
damage remain unresolved. In scrapie-infected transgenic mice expressing prion protein (PrP) lacking the
glycosylphosphatidylinositol (GPI) membrane anchor,
abnormal protease-resistant PrPres was deposited as
amyloid plaques, rather than the usual nonamyloid
form of PrPres. Although PrPres amyloid plaques induced
brain damage reminiscent of Alzheimer’s disease, clinical manifestations were minimal. In contrast, combined
I
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
303
expression of anchorless and wild-type PrP produced
accelerated clinical scrapie. Thus, the PrP GPI anchor
may play a role in the pathogenesis of prion diseases.
Mice Devoid of Prion Protein
Have Cognitive Deficits That Are
Rescued by Reconstitution of
PrP in Neurons
Reprinted from Neurobiol. Dis., Vol. 19, Criado, J.R.,
Sánchez-Alavez, M., Conti, B., Giacchino, J.L., Wills, D.N.,
Henriksen, S.J., Race, R., Manson, J.C., Chesebro, B.,
Oldstone, M.B., pp. 255-265. Copyright 2005, with permission from Elsevier.
rion protein (PrPC) is a constituent of most normal mammalian cells and plays an essential
role in the pathogenesis of transmissible spongiform encephalopathies (TSE). However, the normal cellular function of PrPC remains unclear. Here, we document
that mice with a selective deletion of PrPC exhibited
deficits in hippocampal-dependent spatial learning, but
non-spatial learning remained intact. mPrP–/– mice also
showed reduction in paired-pulse facilitation and longterm potentiation in the dentate gyrus in vivo. These
deficits were rescued in transgenic mPrP–/– mice expressing PrPC in neurons under control of the neuron-specific
enolase (NSE) promoter indicating that they were due to
lack of PrPC function in neurons. The deficits were seen
in mPrP–/– mice with a homogeneous 129/Ola background and in mPrP–/– mice in the mixed (129/Ola x
C57BL/10) background indicating that these abnormalities were unlikely due to variability of background genes
or alteration of the nearby Prnd (doppel) gene.
P
PUBLICATIONS
Brooks, D.G., Teyton, L., Oldstone, M.B.A., McGavern, D.B. Intrinsic functional
dysregulation of CD4 T cells occurs rapidly following persistent viral infection. J.
Virol. 79:1051, 2005.
Christen, U., Edelmann, K.H., McGavern, D.B., Wolfe, T., Coon, B., Teague,
M.K., Miller, S.D., Oldstone, M.B.A., von Herrath, M.G. A viral epitope that mimics a self antigen can accelerate but not initiate autoimmune diabetes. J. Clin.
Invest. 114:1290, 2004.
Kanagawa, M., Saito, F., Kunz, S., Yoshida-Moriguchi, T., Barresi, R., Kobayashi,
Y.M., Muschler, J., Dumanski, J.P., Michele, D.E., Oldstone, M.B.A., Campbell,
K.P. Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell 117:953, 2004.
Kunz, S., Calder, L., Oldstone, M.B.A. Electron microscopy of an α-dystroglycan
fragment containing the receptor sites for lymphocytic choriomeningitis virus and
laminin, and use of the receptoid body as a reagent to neutralize virus. Virology
325:207, 2004.
304 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Kunz, S., Rojek, J.M., Perez, M., Spiropoulou, C.F., Oldstone, M.B.A. Characterization of the interaction of Lassa fever virus with its cellular receptor α-dystroglycan. J. Virol. 79:5979, 2005.
Kunz, S., Sevilla, N., Rojek, J., Oldstone, M.B.A. Use of alternative receptors different than α-dystroglycan by selected isolates of lymphocytic choriomeningitis
virus. Virology 325:432, 2004.
Naniche, D., Garenne, M., Rae, C., Manchester, M., Buchta, R., Brodine, S.K.,
Oldstone, M.B.A. Decrease in measles virus-specific CD4 T cell memory in vaccinated subjects. J. Infect. Dis. 190:1387, 2004.
Oldstone, M.B.A. Future trends in neurovirology: neuronal survival during virus
infection and analysis of virus-specific T cells in central nervous system tissues. J.
Neurovirol. 10:207, 2004.
Oldstone, M.B.A. Molecular and cellular mechanism, pathogenesis, and treatment
of insulin-dependent diabetes obtained through study of a transgenic model of
molecular mimicry. Curr. Top. Microbiol. Immunol., in press.
Oldstone, M.B.A. Molecular mimicry, microbial infection and autoimmune disease:
evolution of the concept. Curr. Top. Microbiol. Immunol., in press.
Zuniga, E.I., Edelmann, K.H., Oldstone, M.B.A. Viruses and dendritic cells: a
prominent mechanism for subverting the immune response. In: Microbial Subversion of the Host Immune Response. Lachmann, P., Oldstone, M.B.A. (Eds.). Horizon Scientific Press, London, in press.
Zuniga, E.I., Hahm, B., Edelmann, K.H., Oldstone, M.B.A. Immunosuppressive
viruses and dendritic cells: a multifront war. ASM News 71:285, 2005.
Zuniga, E.I., McGavern, D.B., Pruneda-Paz, J.L., Teng, C., Oldstone, M.B.A.
Bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic
cells upon virus infection. Nat. Immunol. 5:1227, 2004.
most recent data indicate that expression of receptors
for IFN-γ on CD8 + T cells is tightly regulated and that
cells lacking these receptors are at a selective disadvantage. Therefore, evolution appears to have used IFN-γ
to kill 2 birds with 1 stone; the cells that are best suited
to combat viral infection (i.e., the cells that most rapidly
elaborate IFN-γ) are the ones that are preferentially
expanded in the host. These studies of T-cell regulation are being extended to include CD4+ T cells.
In most studies of T-cell function, including ours,
synthetic peptides are used to stimulate T-cell responses
in vitro. We developed a novel method to identify T cells,
and other cell types, that are actively responding to
authentic antigen contact in vivo. Using this approach,
we showed that the in vivo response of CD8 + memory
T cells to viral infection is explosive. This method not
only will be useful for studies of immune responses to
infection but also may facilitate a better understanding
of autoimmune disease. In our analysis of antigen-specific activation in vivo, we also use in situ hybridization, and we have confirmed that the in vivo CD8 +
T-cell IFN-γ response to antigen contact is very rapid
and is regulated at the transcriptional level.
V I R A L PAT H O G E N E S I S
Viral Pathogenesis and
Antiviral Immunity
J.L. Whitton, N. Benning, C. Cornell, S. Crocker, R. Feuer,
R. Frausto, S. Harkins, I. Hunziker, F. Liu, A. Nussbaum,
R. Pagarigan, M.P. Rodriguez-Carreno, J. Whitmire
ANTIVIRAL T-CELL FUNCTION
D8 + T cells play a key role in combating most
viral infections, either by killing virus-infected
cells or by showering the cells with antiviral
cytokines such as IFN-γ. During microbial infection,
epitope-specific CD8 + T-cell responses usually exist
as a hierarchy; responses to some epitopes are much
stronger than are responses to others. The stronger
responses are termed dominant; the weaker, subdominant. The hierarchy is regulated by a poorly understood
phenomenon called immunodominance.
We have found that immunodominance depends
on the expression of IFN-γ. Our current hypothesis is
that the immunodominance hierarchy (i.e., the relative
abundances of the various epitope-specific T-cell populations) is defined by the rate at which the various
epitope-specific cells can initiate production of IFN-γ;
the fastest cells become the dominant population. Our
C
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Our ongoing studies of the molecular biology of
coxsackievirus B3 have suggested a new explanation
for the existence of highly conserved nucleotides flanking
the eukaryotic translation initiation codon. For the past
2 decades, researchers have assumed that these bases
are important only for efficient initiation of translation,
but we have shown that one of the constraints may
instead be a requirement for posttranslational modification
of the encoded protein. Coxsackievirus B3 is an important
human pathogen that causes a variety of clinical syndromes, including myocarditis and pancreatitis. Myocarditis is remarkably common (about 1 million cases per year
in the United States), currently is not treatable, and can
lead to 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 in the
related immunopathologic changes.
We are extending our studies of coxsackievirus B–
specific immune responses to ask why this virus does
not induce strong CD8+ T-cell responses, despite reaching
very high titers in various tissues. We are also investigating prophylactic measures by evaluating RNA immunization in the coxsackievirus B3 model by using variant
viral genomes with directed mutations that are intended
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
to retain immunogenicity while reducing virulence.
Recently, we found that blockade of the protein tissue inhibitor of metalloproteinase 1 can ameliorate
myocarditis and its consequences; this observation may
be of substantial clinical utility. Finally, our studies of
coxsackievirus B3 infection of the CNS in neonates
indicated that the virus may preferentially infect stem
cells and be carried into the brain parenchyma by these
cells as they migrate toward their final destinations.
305
Rodriguez-Carreno, M.P., Nelson, M.S., Botten, J., Smith-Nixon, K., Buchmeier, M.J.,
Whitton, J.L. Evaluating the immunogenicity and protective efficacy of a DNA vaccine encoding Lassa virus nucleoprotein. Virology 335:87, 2005.
Whitmire, J.K., Tan, J.T., Whitton, J.L. Interferon-γ acts directly on CD8+ T cells
to increase their abundance during virus infection. J. Exp. Med. 201:1053, 2005.
Whitton, J.L. Adaptive immune responses. J. Med. Microbiol., in press.
Whitton, J.L., Feuer, R. Myocarditis, microbes, and autoimmunity. Autoimmunity
37:375, 2004.
Whitton, J.L., Slifka, M.K., Liu, F., Nussbaum, A.K., Whitmire, J.K. The regulation and maturation of antiviral immune responses. Adv. Virus Res. 63:181, 2004.
AUTOIMMUNITY
Together with colleagues at the University of Utah
and the La Jolla Institute of Allergy and Immunology,
we are studying the molecular basis of autoimmunity
induced by viral infection. Some autoimmune diseases
(e.g., multiple sclerosis) appear to be triggered and/or
exacerbated by a wide variety of viral infections. Two
general mechanisms, molecular mimicry and activation of bystander cells, have been proposed to explain
this phenomenon. We have suggested an alternative
explanation that is based on changes in antigen presentation that occur during almost all viral infections.
Laboratory of Translational
Neurophysiology and the San
Diego Substance Abuse and
Minorities Project
C.L. Ehlers, C. Agneta, L. Corey, M. Dixon, P. Duro,
D.A. Gilder, A. Gross, J.W. Havstad, P. Lau, S.L. Lopez,
E. Phillips
D N A I M M U N I Z AT I O N
With our colleague M.J. Buchmeier, Molecular and
Integrative Neurosciences Department, we are evaluating DNA vaccines against the highly pathogenic arenavirus Lassa virus. We have developed a DNA vaccine
that encodes Lassa virus proteins, shown that this
vaccine induces Lassa virus–specific immune responses
in mice, and demonstrated that these mice are protected against challenge with a related, but less pathogenic, arenavirus.
PUBLICATIONS
Feuer, R., Pagarigan, R.R., Harkins, S., Liu, F., Hunziker, I.P., Whitton, J.L. Coxsackievirus targets proliferating neuronal progenitor cells in the neonatal CNS. J.
Neurosci. 25:2434, 2005.
Harkins, S., Cornell, C.T., Whitton, J.L. Analysis of translational initiation in coxsackievirus B3 suggests an alternative explanation for the high frequency of R+4 in
the eukaryotic consensus motif. J. Virol. 79:987, 2005.
Hunziker, I.P., Harkins, S., Feuer, R., Cornell, C.T., Whitton, J.L. Generation and
analysis of an RNA vaccine that protects against coxsackievirus B3 challenge.
Virology 330:196, 2004.
Leifert, J.A., Rodriguez-Carreno, M.P., Rodriguez, F., Whitton, J.L. Targeting plasmidencoded proteins to the antigen presentation pathways. Immunol. Rev. 199:40, 2004.
Liu, F., Whitton, J.L. Cutting edge: re-evaluating the in vivo cytokine responses of CD8+
T cells during primary and secondary viral infections. J. Immunol. 174:5936, 2005.
Liu, F., Whitton, J.L., Slifka, M.K. The rapidity with which virus-specific CD8+ T
cells initiate IFN-γ synthesis increases markedly over the course of infection and
correlates with immunodominance. J. Immunol. 173:456, 2004.
Nussbaum, A.K., Whitton, J.L. The contraction phase of virus-specific CD8+ T
cells is unaffected by a pan-caspase inhibitor. J. Immunol. 173:6611, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
ates of alcoholism and drug dependence within
a population are thought to reflect an almost
equal combination of sociocultural (environmental) and biological (genetically determined) factors.
Data from a large national survey suggest that demographic predictors of lifetime use of a drug differ from
predictors of the development of dependence (addiction). In general, environmental factors may be more
important in predicting the transition between initiation and use, whereas genetic factors may exert more
influence on transitions from substance use to substance dependence. Our goal is to identify candidate
genes that encode the neurophysiologic processes that
underlie drug dependence.
The prevalence of drug and alcohol dependence
among ethnic groups varies widely. These differences
provide an opportunity to investigate how genetic variation may influence substance abuse. One difference
between ethnic groups is a natural variation in the genes
that encode the structure of the enzymes that metabolize alcohol. We were the first to identify a role for
genetic variations in 2 genes, the gene for alcohol dehydrogenase (ADH1B3) and the gene for cytosolic aldehyde
dehydrogenase (ALDH1A1), in both African Americans
and Southwest California Indians. We also showed that
the response to alcohol in Asians is highly dependent
on variants of the gene for the enzyme mitochondrial
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306 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
aldehyde dehydrogenase (ALDH2*2). In Mexican-Americans, the presence of the gene for another form of alcohol dehydrogenase (ADH1B2) may also provide some
protection from heavy drinking and alcohol dependence.
Although variations in alcohol-metabolizing enzymes
clearly confer some protection from the development
of alcoholism, other genes need to be identified that
encode for risk for the disorder. In complex diseases
such as alcoholism, studies of genes, the environment,
and gene-environment interactions have advantages
when conducted in well-defined populations such as
Native American tribes. Compared with the general
population, such populations are often more environmentally and genetically homogeneous, are more geographically restricted, and more commonly have large
extended families. However, only a few studies have
been conducted in Native American populations in which
the investigators evaluated unique or genetic factors
associated with substance dependence.
We conducted a genomic screen in Southwest California Indian families for alcoholism and behaviors
related to substance use. We found that chromosomes
4 and 12 appear to have genes linked to the severity
of an individual’s drinking; chromosomes 6, 15, and
16 have genes linked to a severe form of alcoholism
with symptoms of withdrawal; and a locus on chromosome 5 is linked to “craving” for alcohol.
Additional genomic screens were conducted for use
of tobacco, dependence on marijuana, and dependence
on stimulants. We discovered that a unique locus on
chromosome 14 is linked to marijuana dependence; a
locus on chromosome 1, to stimulant dependence; and
a locus on chromosome 8, to smoking tobacco. In addition, several sites in the genome are linked not only to
multiple drugs of abuse but also to body mass.
One theoretical assumption concerning Native Americans is that the long history of dependence on foraging
and subsistence agriculture may have led to selective
enrichment of traits that improve genetic fitness, socalled thrifty or fat-sparing genes. In addition, such
genes might influence fat accumulation during food
availability, thus improving survival during times of
shortage. This same selective pressure may have
enriched for genetic variants that increase the risk for
consumption of energy-rich beverages such as alcohol
and perhaps of other drugs of abuse. Our data lend
support for this hypothesis and further suggest that
cigarette smoking, alcohol dependence, and other consumptive behaviors may share some common genetic
risk and/or protective factors.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
PUBLICATIONS
Cook, T.A.R., Luczak, S.E., Shea, S.H., Ehlers, C.L., Carr, L.G., Wall, T.L. Associations of ALDH2 and ADH1B genotypes with response to alcohol in Asian Americans. J. Stud. Alcohol, in press.
Ehlers, C.L., Gilder, D.A., Wall, T.L., Phillips, E., Feiler, H., Wilhelmsen, K.C.
Genomic screen for loci associated with alcohol dependence in Mission Indians.
Am. J. Med. Genet. B Neuropsychiatr. Genet. 129:110, 2004.
Ehlers, C.L., Phillips, E., Schuckit, M.A. EEG alpha variants and alpha power in
Hispanic American and white non-Hispanic American young adults with a family
history of alcohol dependence. Alcohol 33:99, 2004.
Ehlers, C.L., Phillips, E., Wall, T.L., Wilhelmsen, K., Schuckit, M.A. EEG alpha
and level of response to alcohol in Hispanic- and non-Hispanic-American young
adults with a family history of alcoholism. J. Stud. Alcohol 65:301, 2004.
Ehlers, C.L., Spence, J.P., Wall, T.L., Gilder, D.A., Carr, L.G. Association of
ALDH1 promoter polymorphisms with alcohol-related phenotypes in Southwest
California Indians. Alcohol. Clin. Exp. Res. 28:1481, 2004.
Ehlers, C.L., Wall, T.L., Betancourt, M., Gilder, D.A. The clinical course of alcoholism in 243 Mission Indians. Am. J. Psychiatry 161:1204, 2004.
Ehlers, C.L., Wilhelmsen, K.C. Genomic scan for alcohol craving in Mission Indians. Psychiatr. Genet. 15:71, 2005.
Gilder, D.A., Wall, T.L., Ehlers, C.L. Comorbidity of select anxiety and affective
disorders with alcohol dependence in Southwest California Indians. Alcohol. Clin.
Exp. Res. 12:1805, 2004.
Irwin, M., Rinetti, G., Redwine, L., Motivala, S., Dang, J., Ehlers, C. Nocturnal
proinflammatory cytokine-associated sleep disturbances in abstinent African American alcoholics. Brain Behav. Immun. 18:349, 2004.
Venner, K.L., Wall, T.L., Lau, P., Ehlers, C.L. Testing of an orthogonal measure of
cultural identification with adult Mission Indians. Cultur. Divers. Ethnic Minor. Psychol., in press.
Wilhelmsen, K., Ehlers, C.L. Heritability of substance abuse and alcohol symptoms in a Native American population. Psychiatr. Genet., in press.
Mouse Models for Unraveling
the Mystery of Schizophrenia
K.N. Fish, S. Morgan, A. Markou, A. Barr, T. Krucker
great need exists for animal models that mimic
the abnormalities in brain connectivity associated
with human psychiatric and neurologic disorders. Developmentally, altered brain connectivity can
result from the convergence of genetic predispositions
and environmental factors that affect neuronal positioning and/or function. Although the genes involved in the
etiology of connectivity disorders are mostly unknown,
the gene reelin is one that may be involved.
Reeler mice, which have a naturally occurring mutation in reelin, have been used to understand brain
development and identify crucial components of the
neuronal positioning machinery. The mutation in these
mice disrupts expression of the large extracellular matrix
protein Reelin. Reelin deficiency results in developmen-
A
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
tal deviations in neuronal positioning and circuitry formation in laminated brain regions that are reminiscent
of those hypothesized to occur in humans with connectivity disorders, such as schizophrenia and autism.
However, because the severe neuroarchitectural changes
associated with the mutation in reelin are so generalized, mice with the mutation are not a realistic and
useful model system for studies of how developmental
changes in neuroarchitecture alter brain connectivity.
In contrast to reeler mice, mice that lack the apolipoprotein E receptor 2 (ApoER2) have a subtler phenotype, making these mice a more useful model system
for studies of neuronal abnormalities in brain connectivity. ApoER2 is a receptor for Reelin, and the transmission of the Reelin signal via this receptor and/or
the very-low-density lipoprotein receptor (VLDLR) to
the cytoplasmic adaptor protein disabled-1 during
neuronal migration is required for the establishment of
normal brain architecture. Although brain development
in mice that lack the genes for both ApoER2 and
VLDLR is indistinguishable from development in reeler
mice, the distinct neuroanatomic phenotypes associated with deficiencies in the individual receptors suggest that the functions and/or brain expression patterns
of VLDLR and ApoER2 only partially overlap. In mice
lacking the gene for ApoEr2, mispositioned neurons are
found mostly in the cortex and hippocampus, whereas in
mice lacking the gene for VLDLR, the neuroarchitecture of the cerebellum is mostly altered.
We are analyzing behavioral deficits associated with
deletion of the genes for ApoER2 and VLDLR. In addition, we are using organotypic slice cultures and lowdensity isolated neuronal cultures to do a molecular
analysis of mice that lack these genes. A full characterization of these mice may allow us to generate new
mouse models of schizophrenia that faithfully reproduce
both pathologic processes and behavioral phenomena
associated with the disease. The validation of these new
mouse strains for their usefulness in improving existing
antipsychotic drugs and developing new ones is fundamental to achieving our long-term goal: improving the
well-being of patients who have schizophrenia, especially
those patients who are resistant to treatment with
presently available antipsychotic drugs.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
307
Cellular and Molecular
Mechanisms of Neuronal
Signaling in the CNS
D.L. Gruol, T.E. Nelson, J. Cho,* J. Sabeti, H. Bajova,
S. Phong, E. Vereyken,** P.N.E. de Graan**
* Dongguk University, Gyeong Buk, Korea
** University Medical Center Utrecht, Utrecht, the Netherlands
M O L E C U L A R TA R G E T S O F I L - 6 , A K E Y P L AY E R I N
CNS DISEASE
ytokines are thought to play an important role in
the neuroadaptive changes induced in the CNS
by a variety of conditions, including seizures,
hypoxia-ischemia, trauma, and bacterial or viral infections. In all of these conditions, elevations in cytokine
levels occur in the cerebrospinal fluid or CNS parenchyma, including elevation of the interleukin IL-6. Both
peripheral and CNS sources can contribute to the elevated levels of IL-6 in these conditions. CNS sources
include neurons, astrocytes, and microglia.
A specific receptor for IL-6 (IL-6R) mediates the
effects of the interleukin in the CNS. Recent studies
indicate that CNS neurons and glial cells express IL-6Rs
and that multiple signal transduction pathways are
associated with receptor activation, including the signal transducer and activator of transcription 3 and the
Ras–MAP kinase/extracellular signal–regulated kinase
pathways. These pathways ultimately lead to the regulation of gene expression.
Other studies indicate that short- or long-term
exposure to IL-6 can produce altered CNS function.
However, relatively little is known about the cellular
targets of IL-6 that mediate the altered CNS function.
To address this issue, we are examining neuroadaptive
changes produced by chronic exposure to IL-6 in CNS
neurons. We are using gene profiling to assess changes
at the mRNA level, Western blot analysis to assess
changes at the protein level, immunohistochemical
assays to identify cellular localization, and physiologic
analyses to detect functional changes.
Using cultured rodent hippocampal neurons, we
found that several proteins critical for normal neuronal
function are targets of IL-6 under conditions of IL-6
exposure that simulate a disease state. These targets
include proteins involved in synaptic transmission, neuron excitability, and intracellular calcium homeostasis.
Of particular interest is the downregulation at both the
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308 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
mRNA and protein levels of group II metabotropic glutamate receptors (mGluRs) induced by chronic IL-6
exposure. These G protein–coupled receptors control
release of neurotransmitters at CNS synapses and
thereby play a key role in the regulation of information
transfer through neuronal circuits. Consistent with the
decreased expression of group II mGluR protein, synaptic network activity in IL-6–treated hippocampal cultures was less sensitive to drugs that selectively act at
group II mGluRs than was synaptic network activity in
control cultures. Analysis of protein levels in hippocampi
from transgenic mice that express elevated levels of
IL-6 in the CNS and from control littermates also indicated reduced levels of group II mGluRs in the IL-6
mice. Taken together, our results implicate several
neuronal targets as important mediators of the altered
CNS function that occurs in disease states associated
with elevated levels of IL-6 in the CNS.
Nelson, T.E., Gruol, D.L. The chemokine CXCL10 modulates excitatory activity and
intracellular calcium signaling in cultured hippocampal neurons, J. Neuroimmunol.
156:74, 2004.
NEUROADAPTIVE CHANGES PRODUCED BY
S.J. Henriksen, R.-S. Lee, S. Huitrón-Reséndiz, R. Gallegos,
ALCOHOL ABUSE
M. Sánchez-Alavez, A. Ray, B.F. Cravatt,* B. Conti,
Alcohol abuse alters many aspects of CNS function
by inducing both short- and long-tern neuroadaptive
changes. Recent findings suggest that neuroadaptive
changes in response to alcohol in adolescents may differ from the changes in adults. However, our understanding of these age-dependent differences in the CNS
actions of alcohol is limited. To address this issue, we
are investigating in early adolescent and young adult rats
the effects of chronic alcohol exposure on hippocampal
long-term potentiation (LTP), a form of synaptic plasticity
thought to underlie memory and learning. We use an in
vitro model system, hippocampal slices obtained from
control rats and from rats exposed to alcohol.
We found an age-dependent difference in the effects
of alcohol on hippocampal LTP. In the adolescent rats,
chronic alcohol treatment significantly enhanced LTP,
whereas in the young adult rats, the same treatment
significantly reduced LTP. Such age-dependent differences in the effects of alcohol on hippocampal synaptic plasticity may be an important factor in increased
vulnerability of adolescents to alcohol dependence.
T. Bartfai, S. Steffensen,** O. Prospero-Garcia,***
PUBLICATIONS
Conroy, S.M., Nguyen, V., Quina, L.A., Blakely-Gonzales, P., Ur, C., Netzeband,
J.G., Prieto, A.L., Gruol, D.L. Interleukin-6 produces neuronal loss in developing
cerebellar granule neuron cultures. J. Neuroimmunol. 155:43, 2004.
Gruol, D.L., Nelson, T.E. Purkinje neuron physiology is altered by the inflammatory
factor interleukin-6. Cerebellum, in press.
Gruol, D.L., Netzeband, J.G., Quina, L.A., Blakely-Gonzalez, P.K. Contribution of
L-type channels to calcium regulation of neuronal properties in early developing
Purkinje neurons. Cerebellum, in press.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Nelson, T.E., Netzeband, J.G., Gruol, D.L. Chronic interleukin-6 exposure alters
metabotropic glutamate receptor-activated calcium signalling in cerebellar Purkinje
neurons. Eur. J. Neurosci. 20:2387, 2004.
Nelson, T.E., Ur, C.L., Gruol, D.L. Chronic intermittent ethanol exposure enhances
NMDA receptor-mediated synaptic responses and NMDA receptor expression in
hippocampal CA1 region. Brain Res., in press.
Rempel, J.D., Quina, L.A., Blakely-Gonzales, P.K., Buchmeier, M.J., Gruol, D.L.
Viral induction of central nervous system innate immune responses. J. Virol.
79:4369, 2005.
van Gassen, K.L.I., Netzeband, J.G., de Graan P.N.E., Gruol, D.L. The chemokine
CCL2 modulates calcium dynamics and electrophysiological properties of cultured
cerebellar Purkinje neurons. Eur. J. Neurosci., in press.
Laboratory of In Vivo
Neurophysiology
T. Phillips,**** G. Berg, C. Pun, V. Zhukov, D. Wills
* Department of Cell Biology, Scripps Research
** Brigham Young University, Provo, Utah
*** University Nacional Autónoma de México, México City, México
**** Western University of Health Sciences, Pomona, California
e investigate the functional organization of
neural circuits that have intrinsic capacities
for both normal and abnormal brain functions.
To understand how the brain initiates, promotes, and
alters behavior, we must understand the hierarchy that
neuronal circuits and their chemical messengers have
in producing appropriate responses to external and internal sensory events.
Our approach requires studies in whole animals and
is best described as a “systems” analysis of behavior.
We use anatomic, neuropharmacologic, electrophysiologic, molecular, and behavioral tools to reveal the
hierarchies of the cellular components of brain circuits
that underlie behavioral integration and functional
abnormalities. We study animals in which the CNS is
either intact or has been compromised by viral infection, genetic manipulations, or molecular engineering.
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MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
309
Role of γ-Aminobutyric Acid
Neurons in the Reticular Core in
Cortical Activation and Arousal
R. Gallegos, R.-S. Lee, A. Ray, C. Pun, E. Crawford,
S. Steffensen,* S.J. Henriksen
* Brigham Young University, Provo, Utah
esolimbic dopamine transmission has been
implicated in mediating drug-induced reward
as well as behavioral and electrocortical
arousal. However, dopamine neurons in the ventral
tegmental area (VTA) and the substantia nigra pars
compacta are not markedly modulated by anesthetics
or by the sleep-wake cycle. On the other hand, nondopamine neurons, that is, neurons containing γ-aminobutyric acid (GABA), have increased firing rates during
active wakefulness and REM sleep, suggesting that
ongoing discharge rates of these neurons may either
reflect or, in part, mediate states of neocortical activation and arousal.
Recently, precise, anatomic investigations have
definitively described monosynaptic projections from
the VTA directly to the neocortex and, importantly, to
basal forebrain cholinergic and GABAergic neurons,
which are the primary mediators of low-voltage, highfrequency cortical activity. The connectivity of neurons
in this primarily nonthalamic arousal process involves
first a cascade of hypothalamic, central autonomic,
associational cortical and brain stem tegmental input
onto these GABAergic projecting neurons in the VTA.
These projection neurons then, we hypothesize, integrate
this information by preparing the organism for motive
behavior, including drug seeking, by using specific, highly
regulated disinhibition at the level of the basal forebrain and the cortex itself, inducing cortical activation.
In support of this hypothesis, in a series of electrophysiologic and anatomic studies, we elucidated
both proximal and distal components of this proposed
circuit (Fig. 1). The results of these ongoing studies
suggest that VTA nondopamine neurons are GABA neurons and part of a column of GABA-containing neurons
made up, in part, of long projecting components that
extend from the nucleus pontis oralis of the pontine
reticular formation to regions of the posterior part of
the hypothalamus. The findings also suggest that this
neuronal cellular area has a primary role in initiating
neocortical activation by eliciting disinhibition of both
M
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . VTA neurons filled with the anterograde tracer biotinylated
dextran project to the diagonal bands of Broca in the basal forebrain.
A, Confocal microscopic image of the vertical limb of the diagonal
bands of Broca filtered to show solely fibers filled with biotinylated
dextran. B, The same field filtered to show solely the diffuse and
punctate labels representing GABAergic cells and terminals, respectively. C, Merged fields of A and B. The arrowhead points to a fiber
positive for biotinylated dextran. The arrow points to a GABAergic
projection from the VTA to the forebrain. The asterisk represents an
apposition (possible synaptic contact) of that fiber on a putative
GABAergic cell body.
basal forebrain and thalamocortical neuronal circuits
in response to convergent information from sensory,
cortical, limbic, and other brain stem areas responsive
to internal and external sensory events.
Differential Modulation by
Urethane of Subtypes of
Nondopaminergic Neurons in
the Ventral Tegmental Area
R. Gallegos, R.-S. Lee, S. Huitrón-Reséndiz,
M. Sánchez-Alavez, A. Ray, S.J. Henriksen
resumed GABAergic cells in the ventral tegmental area (VTA) have been linked to cortical and
behavioral arousal; in freely moving animals, the
discharge rates of these cells increase during periods
of cortical arousal. Anatomic evidence indicates that
the cells have projections to the basal forebrain area.
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310 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Urethane, which is widely used in studies because of
its relatively minimal effects on autonomic function, is
thought to enhance the function of γ-aminobutyric acid.
We recorded the activity of VTA neurons while simultaneously recording cortical electroencephalograms (EEGs)
in rats anesthetized with both halothane and urethane.
Fast-firing neurons in the VTA with action potentials of short duration were identified, and firing rates
were recorded. The cortical EEG findings were also
digitized and recorded. Urethane was injected after
stable baseline neuronal recordings were obtained
with halothane anesthesia. Halothane was then discontinued, and EEG findings and neuronal discharge
were recorded continuously.
We found that nondopaminergic (GABAergic) neurons had at least 3 distinct types of responses to urethane. A stabilization of irregular firing occurred in one
group of cells, whereas another group of cells had a
dramatic reduction in firing rate. The third major
response, often observed in slower firing cells, was
characterized by an apparent entrainment of neuronal
discharge to the phasic, spiking cortical EEG wave
pattern produced by urethane. This relationship did
not occur with halothane.
We conclude that urethane significantly affects firing patterns and firing rates of subgroups of VTA nondopaminergic neurons. In particular, a subtype of VTA
neurons begins firing in synchrony with the EEG changes
in rats anesthetized with urethane. Experiments are under
way to determine how urethane exerts these effects.
Sleep Patterns in Mice That
Lack the Gene for Fatty Acid
Amide Hydrolase
S. Huitrón-Reséndiz, R. Gallegos, D. Wills, V. Zhukov,
B.F. Cravatt,* S.J. Henriksen
*Department of Cell Biology, Scripps Research
atty acid amides have been implicated in the
regulatory mechanisms of sleep. However, because
of their prompt catabolism by fatty acid amide
hydrolase (FAAH), the behavioral effects of these amides
in vivo are transient. We characterized the sleep-wake
patterns in mice lacking the gene for FAAH before and
after sleep deprivation.
Instruments for recording electroencephalograms
(EEGs) were implanted in transgenic mice lacking the
F
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
gene for FAAH and in nontransgenic control mice. Two
weeks later, all the mice were habituated to EEG recording conditions for 48 hours, and 24-hour baseline
recordings were obtained. The mice were then deprived
of sleep for 8 hours, and EEGs were recorded during a
recovery phase of 16 hours. EEGs were recorded for 3
stages: wakefulness, slow-wave sleep, and REM sleep.
Compared with control mice, homozygous transgenic mice spent more time in slow wave sleep during
the light period. In addition, rebound in the amount of
slow wave sleep was reduced in homozygous transgenic mice after sleep deprivation, and a long-lasting
increase in the levels of slow wave EEG frequencies
occurred. Our results support the role of FAAH as an
important regulator of the hypnogenic processes associated with fatty acid amides in vivo. In the absence
of FAAH, the hypnogenic amides induced an increase
in the duration of sleep.
PUBLICATIONS
Criado, J.R., Sánchez-Alavez, M., Conti, B., Giacchino, J.L., Wills, D.N., Henriksen, S.J., Race, R., Manson, J.C., Chesebro, B., Oldstone, M.B. Mice devoid of
prion protein have cognitive deficits that are rescued by reconstitution of PrP in
neurons. Neurobiol. Dis. 19:255, 2005.
de Rozieres, S., Swan, C.H., Sheeter, D.A., Clingerman, K.J., Lin, Y.C., HuitrónReséndiz, S., Henriksen, S., Torbett, B.E., Elder, J.H. Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3.
Retrovirology 1:38, 2004.
Huitrón-Reséndiz, S., Sánchez-Alavez, M., Wills, D.N., Cravatt, B.F., Henriksen,
S.J. Characterization of the sleep-wake patterns in mice lacking fatty acid amide
hydrolase. Sleep 27:857, 2004.
Madden, L.J., Flynn, C.T., Zandonatti, M.A., May, M., Parsons, L.H., Katner,
S.N., Henriksen, S.J., Fox, H.S. Modeling human methamphetamine exposure in
nonhuman primates: chronic dosing in the rhesus macaque leads to behavioral and
physiological abnormalities. Neuropsychopharmacology 30:350, 2005.
Xu, Y.L., Reinscheid, R.K., Huitrón-Reséndiz, S., Clark, S.D., Wang, Z., Lin, S.H.,
Brucher, F.A., Zeng, J., Ly, N.K., Henriksen, S.J., de Lecea, L., Civelli, O. Neuropeptide
S: a neuropeptide promoting arousal and anxiolytic-like effects. Neuron 43:487, 2004.
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Neurobiology of Addiction
and Stress
G.F. Koob, F. Weiss, A. Markou, L.H. Parsons, A.J. Roberts,
M.A. Taffe, E.P. Zorrilla, H. Richardson, M. Le Moal,*
E. Riley,** L. Stinus,* M. Cador,* L. Pulvirenti,***
R. Purdy,**** K. Inoue,***** A. Tabarin,† S. Ahmed,*
B. Boutrel, L. O’Dell, S. Chen, C. Reiter-Funk, E. Fekete,
T. Greenwell, B. Walker, S. Ghozland, R. Lintz, R. Schroeder,
T. Kimber, M. Cole, M. Arends, M. Brennan, R. Smith,
M. Mattock, S. Specio, Y. Grant
* Institut National de la Santé et de la Recherche Médicale, Unité 259, and
Université Victor Ségalen Bordeaux 2, Bordeaux, France
** San Diego State University, San Diego, California
*** Claude Bernard Neuroscience Institute, Pozzilli, Italy
**** University of California, San Diego, California
***** Osaka City University Medical School, Osaka, Japan
†
Université Victor Ségalen Bordeaux 2, Hopital du Haut-Lévêque, Pessac, France
ADDICTION
n studies on the neurobiology of addiction, we continue to explore the role of neurochemical systems
in the extended amygdala in the neuroadaptations
associated with the transition from drug taking to drug
dependence that is an integral part of the development
of addiction. We are developing animal models for
excessive drug intake and are charting the changes in
motivated behavior associated with such intake.
Previously, we established that prolonged access
to cocaine can produce progressive increases in drug
intake that are paralleled by decreases in reward function. This escalation is paralleled by a loss of sensitization to the locomotor-activating effects of cocaine.
These findings are consistent with the idea that the
opposing processes of homeostasis (i.e., maintenance
of a relatively stable psychologic condition, or "stability
through constancy") and allostasis (i.e., the ability of a
system to dynamically adopt varying states to accommodate changing demands or to vary the internal milieu
to match environmental demands, or "stability through
change") are involved in addiction. Escalation in drug
intake is accompanied by increased sensitivity to the
blockade of cocaine self-administration by a mixed dopamine D1-D2 receptor antagonist and increased sensitivity
to a corticotropin-releasing factor 1 (CRF1) antagonist.
Studies in animal models of heroin dependence have
revealed similar neuropharmacologic adaptations to
chronic administration of opioids. We found that chronic
opioid exposure increased heroin self-administration in
I
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The Scripps Research Institute. All rights reserved.
311
rats, and this increase could be blocked selectively by a
cannabinoid B1 receptor antagonist. In other studies, a
CRF1 antagonist effectively blocked the development of
place aversion to precipitated opioid withdrawal. These
results suggest that the reward dysregulation associated
with extended access to drugs of abuse that leads to
addiction may depend on neuroadaptive changes in both
dopamine (a reward transmitter) and CRF (a brain stress
neurotransmitter) systems.
We continue to develop animal models for excessive drinking of alcohol that will be useful for identifying compounds that have potential as medications for
treatment of drug addiction. The excessive drinking
associated with alcohol dependence can be exacerbated
by intermittent repeated withdrawal from chronic alcohol exposure. Research with highly selective CRF 2
agonists has shown that the CRF2 system may have
potential in blocking excessive drinking associated with
dependence and the anxiogenic-like effects of alcohol
withdrawal. Because exposure to stressors is a major
stimulus for relapse in humans with alcoholism, these
data suggest a possibly novel role for the CRF2 system
in the prevention of relapse.
We are refining our conceptual framework that the
neurochemical changes in reward and stress neurotransmitter systems lead to an allostatic change in
motivated behavior. Consistent with a role for self-medication of emotional states in humans with drug addiction, in the allostatic view, individuals, through genetic
vulnerability or environmental events, may use drugs
in an attempt to return to a state of motivational homeostasis or stability. Because of the time lag between cause
and effect in the neuroadaptational capabilities of the
brain motivational systems, however, such individuals
defend an allostatic state (an altered reward set point)
rather than return to a homeostatic state. Further
refinement of this hypothesis is the possibility that
taking the drugs themselves can either produce the
allostatic state or exaggerate preexisting genetic or environmental vulnerability.
NEUROPEPTIDES AND STRESS
We are examining the functional significance of
members of the CRF brain stress neurotransmitter system. We found that human urocortin 2, a selective
agonist for the CRF2 receptor, decreased feeding and
drinking in rats but did not alter locomotor activity or
produce malaise. In contrast, a CRF1 agonist reduced
food intake but produced locomotor activation, anxiogenic-like effects, and malaise. Restricted feeding in a
312 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
rodent model produced prolonged anxiolytic-like effects
that persisted beyond normalization of food intake. These
results broaden the functional selectivity of the actions
of the brain CRF-urocortin systems and provide a rich
area for future studies of the pathophysiology of stress
and appetite disorders.
PUBLICATIONS
Ahmed, S.H., Koob, G.F. Changes in response to a dopamine antagonist in rats
with escalating cocaine intake. Psychopharmacology (Berl.) 172:450, 2004.
Azar, M.R., Ahmed, S.H., Lintz, R., Stinus, L., Koob, G.F. A non-invasive gating
device for continuous drug delivery that allows control over the timing and duration
of spontaneous opiate withdrawal. J. Neurosci. Methods 135:129, 2004.
Ben-Shahar, O., Ahmed, S.H., Koob, G.F., Ettenberg, A. The transition from controlled to compulsive drug use is associated with a loss of sensitization. Brain Res.
995:46, 2004.
O’Dell, L.E., Bruijnzeel, A.W., Ghozland, S., Markou, A., Koob, G.F. Nicotine
withdrawal in adolescent and adult rats. Ann. N. Y. Acad. Sci. 1021:167, 2004.
O’Dell, L.E., Roberts, A.J., Smith, R.T., Koob, G.F. Enhanced alcohol self-administration after intermittent versus continuous alcohol vapor exposure. Alcohol. Clin.
Exp. Res. 28:1676, 2004.
Solbrig, M.V., Koob, G.F. Epilepsy, CNS viral injury and dynorphin. Trends Pharmacol. Sci. 25:98, 2004.
Stinus, L., Cador, M., Zorrilla, E.P., Koob, G.F. Buprenorphine and a CRF1 antagonist block the acquisition of opiate withdrawal-induced conditioned place aversion
in rats. Neuropsychopharmacology 30:90, 2004.
Valdez, G.R., Koob, G.F. Allostasis and dysregulation of corticotropin-releasing factor and neuropeptide Y systems: implications for the development of alcoholism.
Pharmacol. Biochem. Behav. 79:671, 2004.
Valdez, G.R., Sabino, V., Koob, G.F. Increased anxiety-like behavior and ethanol
self-administration in dependent rats: reversal via corticotropin-releasing factor-2
receptor activation. Alcohol. Clin. Exp. Res. 28:865, 2004.
Boutrel, B., Koob, G.F. What keeps us awake: the neuropharmacology of stimulants and wakefulness-promoting medications. Sleep 27:1181, 2004.
Weed, M.R., Gold, L.H., Polis, I., Koob, G.F., Fox, H.S., Taffe, M.A. Impaired performance on a rhesus monkey neuropsychological testing battery following simian
immunodeficiency virus infection. AIDS Res. Hum. Retroviruses 20:77, 2004.
Breese, G.R., Chu, K., Dayas, C.V., Funk, D., Knapp, D.J., Koob, G.F., Le, D.A.,
O’Dell, L.E., Overstreet, D.H., Roberts, A.J., Sinha, R., Valdez, G.R., Weiss, F.
Stress enhancement of craving during sobriety: a risk for relapse. Alcohol. Clin.
Exp. Res. 29:185, 2005.
Zorrilla, E.P., Inoue, K., Valdez, G.R., Tabarin, A., Koob, G.F. Leptin and postprandial satiety: acute central leptin more potently reduces meal frequency than
meal size in the rat. Psychopharmacology (Berl.) 177:324, 2004.
Carrera, M.R.A., Ashley, J.A., Hoffman, T.Z., Isomura, S., Wirsching, P., Koob,
G.F., Janda, K.D. Investigations using immunization to attenuate the psychoactive
effects of nicotine. Bioorg. Med. Chem. 12:563, 2004.
Carrera, M.R.A., Kaufmann, G.F., Mee, J.M., Meijler, M.M., Koob, G.F., Janda,
K.D. Treating cocaine addiction with viruses. Proc. Natl. Acad. Sci. U. S. A.
101:10416, 2004.
Finn, D.A., Purdy, R.H., Koob, G.F. Animal models of anxiety and stress-induced
behavior: effects of neuroactive steroids. In: Neurosteroid Effects in the Central
Nervous System: The Role of the GABAA Receptor. Smith, S.S. (Ed.). CRC Press,
Boca Raton, FL, 2004, p. 317.
Heinrichs, S.C., Koob, G.F. Corticotropin-releasing factor in brain: a role in activation, arousal, and affect regulation. J. Pharmacol. Exp. Ther. 311:427, 2004.
Inoue, K., Zorrilla, E.P., Tabarin, A., Valdez, G.R., Iwasaki, S., Kiriike, N., Koob,
G.F. Reduction of anxiety after restricted feeding in the rat: implication for eating
disorders. Biol. Psychiatry 55:1075, 2004.
Zorrilla, E.P., Koob, G.F. The therapeutic potential of CRF1 antagonists for anxiety.
Expert Opin. Investig. Drugs 13:799, 2004.
Zorrilla, E.P., Reinhardt, L.E., Valdez, G.R., Inoue, K., Rivier, J.E., Vale, W.W.,
Koob, G.F. Human urocortin 2, a corticotropin-releasing factor (CRF)2 agonist, and
ovine CRF, a CRF1 agonist, differentially alter feeding and motor activity. J. Pharmacol. Exp. Ther. 310:1027, 2004.
Physiology of
Neurodegenerative Diseases
T. Krucker, R. Vlkolinsky, S. Morgan, B. Chi, A. Mosley,
G.R. Siggins, S.J. Henriksen, J. Wood,* M. Staufenbiel,*
N. Baumann,* E.P. Meyer,** A. Schuler,** S. Heinzer,**
Koob, G.F. Allostatic view of motivation: implications for psychopathology. Nebr.
Symp. Motiv. 50:1, 2004.
R. Müller,*** P. Schneider,*** M. Stampanoni,****
Koob, G.F. A role for GABA mechanisms in the motivational effects of alcohol. Biochem.
Pharmacol. 68:1515, 2004.
M. Pecaut,***** J. Fike†
R. Abela,**** G. Nelson,***** A. Obenaus,*****
* Novartis Pharma AG, Basel, Switzerland
Koob, G.F., Ahmed, S.H., Boutrel, B., Chen, S.A., Kenny, P.J., Markou, A., O’Dell,
L.E., Parsons, L.H., Sanna, P.P. Neurobiological mechanisms in the transition from
drug use to drug dependence. Neurosci. Biobehav. Rev. 27:739, 2004.
Koob, G.F., Le Moal, M. Drug addiction and allostasis. In: Allostasis, Homeostasis,
and the Costs of Physiological Adaptation. Schulkin, J. (Ed.). Cambridge University
Press, New York, 2004, p. 150.
Martin, G., Guadano-Ferraz, A., Morte, B., Ahmed, S., Koob, G.F., de Lecea, L., Siggins, G.R. Chronic morphine treatment alters N-methyl-D-aspartate receptors in freshly
isolated neurons from nucleus accumbens. J. Pharmacol. Exp. Ther. 311:265, 2004.
Navarro, M., Carrera, M.R.A., del Arco, I., Trigo, J.M., Koob, G.F., Rodriguez de
Fonseca, F. Cannabinoid receptor antagonist reduces heroin self-administration
only in dependent rats. Eur. J. Pharmacol. 501:235, 2004.
O’Dell, L.E., Alomary, A.A., Vallee, M., Koob, G.F., Fitzgerald, R.L., Purdy, R.H.
Ethanol-induced increases in neuroactive steroids in the rat brain and plasma are absent
in adrenalectomized and gonadectomized rats. Eur. J. Pharmacol. 484:241, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
** University of Zürich, Zürich, Switzerland
*** ETH and University of Zürich, Zürich, Switzerland
**** Paul Scherrer Institute, Villigen, Switzerland
***** Loma Linda University, Loma Linda, California
†
University of California, San Francisco, California
ur objective is to elucidate common physiologic
processes that underlie neuronal degeneration in
diseases such as Alzheimer’s disease, senile
dementia, and AIDS dementia by using transgenic mice
as models. We primarily use electrophysiologic techniques that allow direct measurement of neuronal function to assess brain plasticity, which is fundamental not
O
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
only to brain development but also to learning and memory formation. These studies enable us to characterize
the time-dependent consequences of neurophysiologic
and neuropharmacologic alterations and to test pharmacologic interventions.
Recently, we began using techniques that enable us
to study the architecture and morphology of the brain
vasculature, including microvessels and capillaries.
Altered blood flow is one of the most consistent physiologic deficits in neurodegenerative disease, and it may
be an important factor in mental illness. In collaboration with M. Staufenbiel and N. Baumann at Novartis;
R. Müller from the ETH and the University of Zürich;
M. Stampanoni and R. Abela at the Paul Scherrer Institute; and E.P. Meyer, S. Heinzer, and A. Schuler at the
University of Zürich, we are studying transgenic mice
in which pathologic features reminiscent of Alzheimer’s
disease develop (Fig. 1). In these models, we found
that significant physiologic changes precede pathologic
changes such as plaque load, a hallmark of Alzheimer’s
disease. Our goal is to determine if cerebrovascular
insufficiencies such as reduced blood supply to the
brain or disrupted microvascular integrity are a response
to neuronal damage or a factor that initiates the neuropathologic changes characteristic of the disease.
Angiogenesis plays a fundamental role in many
normal physiologic processes and in a number of
313
pathologic conditions, including tumor growth, rheumatoid arthritis, ischemic retinopathies, macular degenerations, and chronic asthma. With J. Wood at Novartis,
we are further developing the technology that allows
us to examine the vascular pattern in normal and pathologic tissue and to assess the 3-dimensional architecture in tissues and organs.
As part of an effort initiated and funded by the
National Aeronautics and Space Administration to identify the most significant health risks to astronauts who
are engaging in space flight activities, we are establishing the level of risk to the CNS in terms of possible
decrements in performance that could endanger ongoing
missions or lead to decreased latency to late-onset CNS
dysfunction syndromes such as Alzheimer’s disease or
stroke. Together with research teams headed by G. Nelson, A. Obenaus, and M. Pecaut at Loma Linda University and J. Fike at the University of California, San
Francisco, we are using animal models to quantify the
function of the CNS after exposure to space radiation.
With S.J. Henriksen, Molecular and Integrative
Neurosciences Department, we continued our studies
on the neuronal effects of cytokines and the HIV envelope protein gp120. Using a transgenic mouse model
of neuroAIDS, we are assessing the functional consequences of the neurotoxic effects of methamphetamine
and of viral or host-derived factors. In these models,
we specifically focus on the signal transduction pathways involving the chemokine CXCL 10.
PUBLICATIONS
Krucker, T., Schuler, A., Meyer, E.P., Staufenbiel, M., Beckmann, N. Magnetic
resonance angiography and vascular corrosion casting as tools in biomedical
research: application to transgenic mice modeling Alzheimer’s disease. Neurol.
Res. 26:507, 2004.
Vlkolinsky, R., Siggins, G.R., Campbell, I.L., Krucker, T. Acute exposure to CXC
chemokine ligand 10, but not its chronic astroglial production, alters synaptic plasticity in mouse hippocampal slices. J. Neuroimmunol. 150:37, 2004.
Williams, B., Watanabe, C.M.H., Schultz, P.G., Rimbach, G., Krucker, T. Agerelated effects of Ginkgo biloba extract on synaptic plasticity and excitability. Neurobiol. Aging 25:955, 2004.
1 . Reconstruction of blood vessels in a transgenic mouse
model for Alzheimer’s disease. A, Scanning electron micrograph of
a vascular corrosion cast of the whole brain. B, Detailed view of the
dense network of capillaries in the cortex. C, Sequential slices from
a 3-dimensional reconstruction made by using synchrotron-based
micro–computed tomography of the cortex of a 12-month-old
mouse. The missing microvasculature is forming holes in the dense
vascular network. In a surface view (left), such holes cannot be
detected. It is only by making use of the 3-dimensional nature of
the micro–computed tomographic images that an appropriate field
of view can be digitally selected to “zoom in” on the holes.
Fig
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Role of the Neuregulins in the
Nervous System
C. Lai, J.L. Weber, T. Fischer, I. Cheung, A. Dowell
he focus of our research is understanding the signaling mechanisms that underlie the establishment and maintenance of mature neuronal and
glial cell phenotypes. We are studying the roles played
T
314 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
by a subfamily of receptor protein-tyrosine kinases, the
ErbBs (EGFR, ErbB2, ErbB3, and ErbB4), and their
ligands, the neuregulins (NRG-1–NRG-4). NRG-1 was
first identified as the Schwann cell mitogen glial growth
factor. NRG-1 was also termed ARIA (for acetylcholine
receptor inducing activity), which was thought to regulate expression of acetylcholine receptors at the developing neuromuscular junction. These distinct functions
are now thought to be served by discrete types of NRG-1
(I, II, and III) that arise by alternative splicing. A primary goal of our research program is to understand
the specific roles of each of these types of NRG-1 in
the nervous system.
NRG-1 supports the survival of Schwann cells and
is thought to regulate the number of premyelinating
Schwann cells. The results of genetic studies suggested
that the type III isoform serves in this capacity, and we
helped determine that this isoform also plays a key role
in regulating the thickness of the myelin sheath. Currently, we are investigating whether the type I or the
type II isoform functions in the role of ARIA. The emerging picture is that different NRG-1 isoforms act as signaling molecules from neuron to glial cell and from
neuron to muscle cell to carry out distinct biological
activities. We are also pursuing the roles of these NRG-1
isoforms in the brain, which became an area of considerable interest after NRG-1 was identified as a susceptibility gene for schizophrenia.
We have 4 areas of primary interest. The first is
the roles of the 3 types of NRG-1 in the developing and
mature nervous systems. We developed transgenic mice
that permit the tetracycline-regulated expression of specific NRG-1 isoforms. With these mice, we can assess
the distinct biological functions served by each isoform.
The second area is neurogenesis and migration. We
found that the neuregulin receptor ErbB4 is expressed
by multiple tangentially migrating populations of neuronal cells in the developing and mature nervous systems. ErbB4 is expressed at high levels in the mature
subventricular zone and rostral migratory stream, one
of the few regions in the brain in rats where neurogenesis occurs in adults. We are searching for the endogenous ligands and are testing the effects of the NRGs
on cells derived from the subventricular zone. Our data
suggest that ErbB4 influences both the proliferation of
neural progenitor cells and the migration of neuroblasts
in the rostral migratory stream.
The third area of interest is the effects of the loss
of ErbB4 function in the mature brain. We are analyzPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
ing the phenotype of mice that lack the gene for ErbB4
in the nervous system. Last, we are developing transgenic tools that allow regulated gene expression in specific subsets of neurons. We developed lines of mice
that permit regulated gene expression in cholinergic
neurons, and we are evaluating similar lines that permit regulated expression in either dopaminergic neurons or in the medium spiny neurons of the striatum.
These animal models may be useful for investigating
addiction and neurodegenerative disorders such as
Alzheimer’s, Parkinson’s, and Huntington’s diseases.
PUBLICATIONS
Anton, E.S., Ghashghaei, H.T., Weber, J.L., McCann, C., Fischer, T.M., Cheung,
I.D., Gassmann, M., Messing, A., Klein, R., Schwab, M.H., Lloyd, K.C.K., Lai, C.
Receptor tyrosine kinase ErbB4 modulates neuroblast migration and placement in
the adult forebrain. Nat. Neurosci. 7:1319, 2004.
Flames, N., Long, J.E., Garratt, A.N., Fischer, T.M., Gassmann, M., Birchmeier, C.,
Lai, C., Rubenstein, J.L.R., Marin, O. Short- and long-range attraction of cortical
GABAergic interneurons by neuregulin-1. Neuron 44:251, 2004.
Neurobiology of Reward,
Motivation, and Emotion in
Psychiatric Disorders
A. Markou, K.N. Fish, S.G. Semenova, N.E. Paterson,
P.J. Kenny, A. Bruijnzeel, B. Boutrel, B. Henry, N. Amitai,
S. Jonkman, J. Benedict, G. Finnerman, C. Onifer,
J. Cryan,* W. Froestl,* F. Gasparini.* D. Hoyer,*
D. Slattery*
* Novartis Pharma AG, Basel, Switzerland
he focus of our research is the neurobiology of
reward, motivation, and emotion in 3 psychiatric disorders: drug abuse, depression, and
schizophrenia.
T
NICOTINE DEPENDENCE
A factor that contributes to drug dependence, including habitual use of tobacco, is the depression-like symptoms that occur when a person quits smoking. These
symptoms motivate the person to reinitiate tobacco
use to alleviate this negative affective state. Because
nicotine is the main ingredient in tobacco that leads to
addiction, the focus of preclinical studies is the neurobiology of nicotine dependence.
We are examining the neuroadaptations that occur
in specific brain sites during the development of nicotine dependence that mediate the negative affective
states that characterize nicotine withdrawal. We use a
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
brain stimulation reward procedure that provides a
quantitative measure of brain reward function (i.e., brain
reward thresholds) to operationally assess the anhedonic
depression-like state in rats. Previously, we found that
withdrawal from chronic nicotine administration induced
elevations in reward thresholds indicative of a depressionlike state. Therefore, we examined whether administration of various pharmacologic agents would precipitate
withdrawal-like signs in nicotine-dependent rats but
not in control rats, revealing adaptations that occur with
the development of nicotine dependence.
We assessed the effects of chronic nicotine administration on the activity of nicotinic acetylcholine receptors in the ventral tegmental area (VTA) and the nucleus
accumbens shell and on the activity of dopamine D1like and D 2 -like receptors in the VTA. Injections of
dihydro-β-erythroidine, an antagonist of nicotinic
acetylcholine receptors, into, but not outside, the VTA
resulted in elevations in thresholds in nicotine-dependent rats but not in control rats. In contrast, injections
of dihydro-β-erythroidine into the nucleus accumbens
shell had no effect on thresholds in nicotine-treated
rats or control rats. Further, injections of the D1-like
receptor antagonist SCH 23390 into the VTA differentially elevated thresholds; nicotine-dependent rats
were less sensitive than control rats to D1-like antagonism. By contrast, injection of the D2-like receptor
antagonist eticlopride into the VTA slightly elevated
thresholds in both control rats and nicotine-treated
rats, with no differential effects.
In conclusion, decreased sensitivity of nicotinic
acetylcholine receptors and increased sensitivity of
dopamine D 1 -like receptors in the VTA most likely
mediate, at least partly, the affective depression-like
signs of nicotine withdrawal. Our previous work also
indicated increased activity of metabotropic glutamate
2/3 receptors in the VTA, decreased activity of ionotropic
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/
kainate glutamate receptors but no changes in γ-aminobutyric acid B receptors in the VTA in nicotine dependence. These findings contribute to our growing
understanding of the neurobiological changes leading
to the manifestation of the depression-like aspects of
nicotine withdrawal.
DEPRESSION
In another line of research, we are investigating the
similarities in the neurobiology of drug-induced and
non–drug-induced depression. On the basis of the phenomenologic similarity between drug withdrawal and
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
315
non–drug-induced depression, we hypothesized that
treatment with clinical antidepressants would alleviate
the depression-like aspects of amphetamine or nicotine
withdrawal. Previously, we found that increasing serotonin neurotransmission by coadministering the selective serotonin reuptake inhibitor fluoxetine and the
serotonin1A receptor antagonist p-MPPI reversed reward
deficits observed during nicotine or amphetamine withdrawal. Most recently, we found that coadministration
of paroxetine, a selective serotonin reuptake inhibitor
that increases both serotonergic and noradrenergic neurotransmission, with p-MPPI alleviated reward deficits
associated with amphetamine withdrawal. Finally, chronic
treatment with the tricyclic antidepressant desipramine,
which primarily increases noradrenergic transmission,
abolished nicotine withdrawal.
Thus, the affective symptoms of amphetamine and
nicotine withdrawal, similar to those of non–drug-induced
depressions, may be partly mediated by reduced serotonergic and noradrenergic neurotransmission. Accordingly,
these data support the hypothesis that the neurobiology of drug-induced and non–drug-induced depression
is similar.
In other ongoing work, we are developing and using
rat and murine models of the cognitive deficits of schizophrenia in humans, and we are extending our nicotine
dependence investigations to mice, the most commonly
used species in genetic studies.
PUBLICATIONS
Barr, M.A., Markou, A. Psychostimulant withdrawal as an inducing condition in
animal models of depression. Neurosci. Biobehav. Rev. 29:675, 2005.
Bruijnzeel, A.W., Markou, A. Adaptations in cholinergic transmission in the ventral
tegmental area associated with the affective signs of nicotine withdrawal in rats.
Neuropharmacology 47:572, 2004.
Jonkman, S., Henry, B., Semenova, S., Markou, A. Mild anxiogenic effects of
nicotine withdrawal in mice. Eur. J. Pharmacol. 516:40, 2005.
Kenny, P.J., Boutrel, B., Gasparini, F., Koob, G.F., Markou, A. Metabotropic glutamate 5 receptor blockade may attenuate cocaine self-administration by decreasing
brain reward function in rats. Psychopharmacology (Berl.) 179:247, 2005.
Markou, A. Nicotine dependence perpetuating tobacco smoking may be treatable
by drugs acting at glutamate receptors. Discov. Med. 4:246, 2004.
Markou, A., Harrison, A.A., Chevrette, J., Hoyer, D. Paroxetine combined with a
5-HT1A receptor antagonist reversed reward deficits observed during amphetamine
withdrawal in rats. Psychopharmacology (Berl.) 178:133, 2005.
Markou, A., Paterson, N.E., Semenova, S. Role of γ-aminobutyric acid (GABA)
and metabotropic glutamate receptors in nicotine reinforcement: potential pharmacotherapies for smoking cessation. Ann. N. Y. Acad. Sci. 1025:491, 2004.
Paterson, N., Froestl, W., Markou A. Repeated administration of the GABAB receptor
agonist CGP44532 decreased nicotine self-administration, and acute administration
decreased cue-induced reinstatement of nicotine-seeking in rats. Neuropsychopharmacology 30:119, 2005.
316 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Paterson, N.E., Bruijnzeel, A.W., Kenny, P.J., Wright, C.D., Froestl, W., Markou, A.
Prolonged nicotine exposure does not alter GABAB receptor-mediated regulation of
brain reward function. Neuropharmacology, in press.
Paterson, N.E., Markou, A. The metabotropic glutamate receptor 5 antagonist
MPEP decreased break points for nicotine, cocaine and food in rats. Psychopharmacology (Berl.) 179:255, 2005.
Slattery, D.A., Markou, A., Froestl, W., Cryan, J.F. The GABAB receptor-positive
modulator GS39783 and the GABAB receptor agonist baclofen attenuate the
reward-facilitating effects of cocaine: intracranial self-stimulation studies in the rat.
Neuropsychopharmacology, in press.
Medication Development in
Alcohol Dependence
B.J. Mason, K. Buffkins, R. Corrias, K. Coveney, T. Escher,
C. Libby, S. Payton, J. Diamant,* F. Shadan,* S. Rao,*
M. Kyle,* M. Adusumalli,* P. Campbell,* L.J. Quinton,*
B. Turner*
* Scripps Green Hospital, La Jolla, California
CLINICAL RESEARCH
e recently completed a 1-year maintenance
study of naltrexone for the long-term treatment of alcohol dependence. Results of shortterm trials were variable but generally supported the
effectiveness of naltrexone in reducing the effects of
binge drinking and relapse to heavy drinking. However,
in the only long-term trial (i.e., 1 year), no advantage
was found for naltrexone when patients were treated with
placebo for 3 months or 1 year. We thought it unlikely
that a physician would treat a patient for 1 year with
a medication to which the patient had not initially
responded. Therefore, we treated patients with the
standard 3-month, fixed daily dose of naltrexone of
50 mg, and only the patients who responded to the
drug were then randomized to either continue treatment
with naltrexone or treatment with a placebo.
Our data indicated that initial responders who continued treatment with naltrexone had significantly
fewer relapses to heavy drinking during the year of
treatment than did those treated with placebo. These
results provided the first evidence of the long-term
(1 year) efficacy of naltrexone for patients who respond
to the standard 3-month course of therapy. Because
the first year of recovery is associated with a heightened vulnerability to relapse, these findings have
important implications for optimizing recovery in alcohol dependence.
In a double-blind, placebo-controlled multicenter
trial in patients with alcoholism, treatment with the
W
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antirelapse medication acamprosate increased the
number of alcohol-free days, especially in patients
motivated to be abstinent from alcohol. In July 2004,
the Food and Drug Administration approved acamprosate calcium (Campral) for the maintenance of
abstinence from alcohol in patients with alcohol
dependence who are abstinent at the time treatment
is started. This approval brings an important new
medicine to the aid of patients committed to beating
alcohol dependence. This new medication is the first
one for the treatment of alcohol dependence in almost
a decade.
Also, the results of a recent study of the pharmacokinetic and pharmacodynamic interactions between
acamprosate and naltrexone supported the safety of
coadministration of these drugs. An additional finding
was a significant increase in the rate and extent of
acamprosate absorption, which may be associated with
improved clinical outcome. Currently, we are analyzing
data from trial completed in 2005 to evaluate the clinical effectiveness of acamprosate and naltrexone, alone
or in combination, for treatment of alcohol dependence.
Previously, using our human laboratory model for
the risk of relapse in protracted abstinence from alcohol, we found that treatment with gabapentin could
prevent relapse. Now we are doing a 12-week, double-blind, placebo-controlled study to further explore
gabapentin as a treatment for symptoms, such as anxiety and insomnia, that may occur after alcohol withdrawal and that can lead to a relapse to drinking.
D E V E L O P M E N T O F A H U M A N L A B O R AT O R Y M O D E L
During the past year, using reactivity to alcohol
and affective cues (i.e., emotional conditions) as
analogs of high-risk situations, we refined our human
laboratory model of the risk for relapse in protracted
abstinence from alcohol. Our goal is to facilitate the
identification of medications that can be tested in clinical trials. We showed that our model evoked greater
responsivity to alcohol than to water and that this
responsivity was heightened in the presence of negative emotional conditions.
We then used the model to determine if treatment
with gabapentin could be used to prevent relapse. Preliminary results indicated a beneficial effect of gabapentin
on measures of protracted abstinence commonly associated with relapse. We are using the human laboratory
model to further explore gabapentin as a possible treatment for patients who have both cannabis and alcohol
dependence. It is thought that gabapentin may help
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
with symptoms, such as anxiety and insomnia, that
may occur when patients stopping using alcohol and
cannabis. Such a drug may help reduce the risk of
relapse to both alcohol and cannabis use.
We are using this model to explore the role of various candidate drugs that affect a range of brain systems that may have a role in the potential treatment
of alcohol dependence, including the corticotropin-releasing factor, dopamine, opioid peptide, and γ-aminobutyric acid systems.
PUBLICATIONS
Mason, B.J. Acamprosate in the treatment of alcohol dependence. Expert Opin.
Pharmacother., in press.
Mason, B.J. Rationale for combining acamprosate and naltrexone for treating alcohol dependence. J. Stud. Alcohol Suppl. 15:148, 2005.
O’Malley, S.S., Martin, D.J., Hosking, J., Mason, B.J. How pilot studies improve clinical trials: lessons from the COMBINE study. J. Stud. Alcohol Suppl. 15:66, 2005.
Chemokine Effects on
Neuronal Physiology
T.E. Nelson, D.L. Gruol, H. Bajova, J. Cho*
* Dongguk University, Gyeong Buk, South Korea
hemokines are members of the cytokine family
of immunoregulatory proteins whose primary
role is the activation and trafficking of leukocytes to sites of infection or injury. Expression of chemokines in the CNS is upregulated in a number of
neurologic diseases and disorders, including HIV-associated dementia, multiple sclerosis, Alzheimer ’s disease, brain tumors, CNS trauma, and stroke. In the
CNS, chemokines are expressed predominantly by glial
cells (astrocytes and microglia), whereas chemokine
receptors are expressed by both neurons and glial cells,
indicating that the latter 2 cell types are potential targets of chemokine actions in the CNS. In addition to
their role in neuroinflammation, chemokines are also
involved in regulating normal brain functions, including neuronal migration, modulation of synaptic activity
and plasticity, and neuronal survival.
Currently, we are using primary organotypic cultures
of rat hippocampus to investigate the effects of acute
and chronic chemokine exposure on neuronal physiology. We have focused on the chemokine CXCL10 (previously known as IFN-γ–inducible protein-10 or IP-10).
Elevated levels of CXCL10 are highly prevalent in the
cerebrospinal fluid in patients with HIV type 1 infec-
C
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317
tion and correlate strongly with the severity of the
neurologic disorders associated with the infection.
Using fluorescence-based calcium imaging and
intracellular electrophysiologic recording, we found
that acute exposure to CXCL10 enhanced ongoing
electrical activity (i.e., generation of action potentials)
of cultured hippocampal neurons. In addition, exposure to CXCL10 resulted in elevations of intracellular
calcium that coincided with the increased neuronal
excitability and were abolished when neuronal activity
was blocked, although activity-independent calcium
signaling remained in a small percentage (<10%) of
cells. Moreover, acute exposure to CXCL10 enhanced
spontaneous synaptic activity and synchronous calcium oscillations occurring in networks of cultured
hippocampal neurons.
Using immunoblotting and immunohistochemistry,
we investigated CXCL10-dependent signal transduction
and the effects of chronic CXCL1O exposure on the
expression of receptors for the neurotransmitters glutamate and γ-aminobutyric acid in hippocampal cultures.
We found that acute exposure (5–30 min) to CXCL10
activated the p44/p42 (extracellular signal–regulated
kinase 1/2) MAP kinases in hippocampal neurons. In
addition, chronic exposure (4–7 days) resulted in persistent, elevated activation of the kinases in culture.
Moreover, protein levels of the NR1 and NR2B subunits
of the N-methyl-D-aspartate subtype of glutamate receptors were increased, and the R1 subunit of the γ-aminobutyric acid B receptor was decreased after chronic
exposure to CXCL10.
In future studies, we will address the neurophysiologic implications of changes in signal transduction in
the expression of neurotransmitters induced by chronic
exposure to CXCL1O. Our results indicate that chemokines have a role in modulating CNS function under normal physiologic conditions as well as during periods of
immune challenge, neurotrauma, or neurologic disease.
PUBLICATIONS
Gruol, D.L., Nelson, T.E. Purkinje neuron physiology is altered by the inflammatory
factor interleukin-6. Cerebellum, in press.
Nelson, T.E., Gruol, D.L. The chemokine CXCL10 modulates excitatory activity and
intracellular calcium signaling in cultured hippocampal neurons. J. Neuroimmunol.
156:74, 2004.
Nelson, T.E., Netzeband, J.G., Gruol, D.L. Chronic interleukin-6 exposure alters
metabotropic glutamate receptor-activated calcium signalling in cerebellar Purkinje
neurons. Eur. J. Neurosci. 20:2387, 2004.
Nelson, T.E., Ur, C.L., Gruol, D.L. Chronic intermittent ethanol exposure enhances
NMDA receptor-mediated synaptic transmission and NMDA receptor expression in
hippocampal CA1 region. Brain Res., in press.
318 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Neurochemistry of Addiction
L.H. Parsons, S. Caillé, I. Polis, D. Stouffer
e are elucidating neurochemical mechanisms
that participate in the development and maintenance of drug dependence. During the past
year, we concentrated on evaluating the influence of
endogenous cannabinoids on the rewarding and neurochemical effects produced by opiates, ethanol, and psychostimulants.
As described previously, we found that blockade of
cannabinoid-1 (CB 1 ) receptors by the selective CB 1
antagonist SR141716A (rimonabant) reduced operant
heroin and ethanol self-administration by Wistar rats.
In contrast, the antagonist did not alter self-administration of either cocaine or amphetamine, suggesting
that the endogenous cannabinoid system does not play a
role in modulating the reinforcing properties of psychostimulant drugs. We extended these behavioral findings
by investigating possible neurochemical mechanisms
through which the central cannabinoid system may
modulate drug reward.
We found that blockade of CB1 receptors attenuated
ethanol-induced increases in the levels of dopamine in
the nucleus accumbens, both after noncontingently
administered ethanol and during ongoing ethanol selfadministration. In contrast, blockade did not alter the
stimulatory effects of the self-administration of either
morphine or heroin on the levels of dopamine in the
nucleus accumbens but rather attenuated the suppressant effects of opiates on the release of γ-aminobutyric
acid in the ventromedial ventral pallidum.
These findings provide preliminary evidence that
CB 1 receptors modulate ethanol and opiate reward by
altering the effects of these substances on neurotransmission in the “reward circuit” that includes the nucleus
accumbens and ventral pallidum. Psychostimulant drugs
also increase the levels of dopamine in the nucleus
accumbens and decrease the levels of γ-aminobutyric
acid in the ventral pallidum, effects implicated in the
mediation of psychostimulant reward. However, the
blockade of CB 1 receptors had no effect on either
cocaine-induced increases in dopamine in the nucleus
accumbens or cocaine-induced decreases in γ-aminobutyric acid in the ventral pallidum, consistent with
the lack of effect of SR141716A on the self-administration of psychostimulants.
We are beginning to evaluate the neural circuits
through which the endogenous cannabinoid system
W
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alters opiate and ethanol reward. We found that selfadministration of heroin was reduced by infusions of
SR141716A into the nucleus accumbens but not by
infusions into the ventral pallidum. On the basis of
this evidence of a role for nucleus accumbens CB 1
receptors in the modulation of opiate (and perhaps
ethanol) reward, we are evaluating the effects of opiate
and ethanol administration on the formation of endocannabinoids in the nucleus accumbens. We have
optimized a microdialysis procedure for sampling fatty
acid amides from the brain interstitial environment
and have developed a liquid chromatography-mass
spectrometry assay to measure these substances in
brain microdialysates.
Using these techniques, we detected low nanomolar
concentrations of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in nucleus
accumbens dialysates. Importantly, we found that the
levels of anandamide were significantly increased by
either intraperitoneal administration of morphine or
intravenous self-administration of heroin. Interestingly,
neither morphine nor heroin had any effects on the levels of 2-AG. Conversely, both intraperitoneal administration of ethanol by us and oral self-administration by the
experimental animals significantly increased the levels of
2-AG without altering the levels of anandamide.
These observations provide important evidence that
endogenous cannabinoids are released into the extracellular environment of the brain and that their levels
are increased by opiates and ethanol. We are evaluating the significance of the differential effects of opiates
and ethanol on the formation of anandamide and 2-AG.
Preliminary data indicate that self-administration of
ethanol is selectively altered by pharmacologic manipulations of clearance mechanisms for 2-AG but not
anandamide. Additional experiments are under way to
evaluate potential alterations in endocannabinoid signaling induced by chronic exposure to opiates or ethanol
and the usefulness of chronic administration of CB 1
antagonists in reducing both ethanol and opiate consumption and the propensity to relapse after a period
of drug abstinence.
PUBLICATIONS
Caillé, S., Parsons, L.H. Cannabinoid modulation of opiate reinforcement through
the ventral striatopallidal pathway. Neuropsychopharmacology, in press.
Caillé, S., Parsons, L.H. Intravenous heroin self-administration decreases GABA
efflux in the ventral pallidum: an in vivo microdialysis study in rats. Eur. J. Neurosci. 20:593, 2004.
Madden, L.J., Flynn, C.T., Zandonatti, M., May, M., Parsons, L.H., Katner, S.,
Henriksen, S.J., Fox, H.S. Modeling human methamphetamine exposure in nonhuman primates: chronic dosing of the rhesus macaque leads to behavioral and physiological abnormalities. Neuropsychopharmacology 30:350, 2005.
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
319
O’Dell, L.E., Parsons, L.H. Serotonin1B receptors in the ventral tegmental area
modulate cocaine-induced increases in nucleus accumbens dopamine levels. J.
Pharmacol. Exp. Ther. 311:711, 2004.
Purdy, R.H., Fitzgerald, R.L., Alomary, A.A., Parsons, L.H. The analysis of neuroactive steroids by mass spectrometry. In: Practical Neurochemistry (Methods):
Handbook of Neurochemistry and Molecular Biology, 3rd ed. Baker, G., Dunn, S.,
Holt, A. (Eds.). Kluwer Academic/Plenum Publishers, New York, in press.
Roberto, M., Madamba, S.G., Stouffer, D.G., Parsons L.H., Siggins, G.R. Increased GABA
release in the central amygdala of ethanol-dependent rats. J. Neurosci. 24:10159, 2004.
Imaging of Alzheimer’s Disease
Via Magnetoencephalography
C. Amo,* J.R. Criado,* S.M. Otis,* J. Polich
* Scripps Clinic, La Jolla, California
A L Z H E I M E R ’ S D I S E A S E A N D B R A I N WAV E S
euroelectric measures can provide direct imaging of CNS function. Indeed, electroencephalography (EEG) and sensory evoked potentials are
routinely used to assess brain systems. Cognitive eventrelated potential methods also are used, because considerable research has indicated systematic differences
between healthy volunteers and patients. In particular,
Alzheimer’s disease has been studied by using EEG
analysis and cognitive event-related potentials that
typically require an active discrimination task, a testing
situation that patients with even moderate Alzheimer’s
disease find difficult. What is needed is a means to
easily identify changes in neuronal cells that precede
the development of clinically frank Alzheimer’s disease.
N
MAGNETIC BRAIN SIGNALS
Magnetoencephalography (MEG) is a noninvasive
neuroimaging technique that records the magnetic
counterpart of EEG signals via many sensors that surround the head. More important, unlike electrical measures, MEG recordings allow the localization of dipole
sources because the magnetic signal is transmitted
through the brain, dura, and skull without alteration
by the biological material. This technology therefore
can facilitate the neural identification of potential markers in patients in whom MEG generation is compromised
by neurologic disease such as stroke, epilepsy, and
migraine. This approach recently has been used to
assess patients with Alzheimer ’s disease and elderly
healthy volunteers (control subjects) in a collaborative
study with Scripps Clinic.
Figure 1 illustrates MEG signals from a control subject and a patient with Alzheimer’s disease. The MEG
recording for the control subject is similar to EEG
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . A, MEG recording (left hemisphere channels) with a coro-
nal magnetic resonance image imposed on the waveforms shows
normal baseline activity in a control subject. B, Similar MEG recording (left hemisphere channels) with a coronal magnetic resonance
image imposed on the waveforms shows pronounced slow wave
activity (5–6 Hz) in a patient with Alzheimer’s disease. White dots
indicate dipole localization of increased slow wave activity in the
left temporal lobe. The dipole localization is consistent with the
neuronal destruction patterns associated with Alzheimer’s disease.
recordings from a healthy elderly subject; the MEG
recording for the patient shows pronounced slow wave
activity (5–6 Hz) across the recording channels. The
magnetic strength of this slow wave activation can be
used to localize the generating neurons that produce a
dipole field pattern. The patient has a regular cluster
of slow wave dipole sources in the left temporal lobe.
This activation pattern is consistent with pathologic
changes associated with Alzheimer’s disease.
These preliminary findings suggest that MEG measures may be helpful in assessing patients who have
Alzheimer’s disease and could provide a noninvasive,
relatively inexpensive tool for assessing therapeutic
treatments. Future studies will focus on developing a
normative database, assessing patients in the very
early stages of Alzheimer’s disease, and tracking deterioration in the patients’ conditions over time.
320 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
PUBLICATIONS
Cahn, B.R., Polich, J. Medication states and traits: EEG, ERP, and neuroimaging
studies. Psychol. Bull., in press.
Hagen G.F., Gatherwright, J.R., Lopez, B.A., Polich, J. P3a from visual stimuli:
primary task difficulty effects. Int. J. Psychophysiol., in press.
Polich, J., Corey-Bloom, J. Alzheimer’s disease and P300: review and evaluation
of task and modality. Curr. Alzheimer Res., in press.
Polich, J., Criado, J.R. Neuropsychology and neuropharmacology of P3a and P3b.
Int. J. Psychophysiol., in press.
Wetter, S., Polich, J., Murphy, C. Olfactory, auditory, and visual ERPs from single
trials: no evidence for habituation. Int. J. Psychophysiol. 54:263, 2004.
Mouse Models for Studies of the
Neural Bases of Behavior
A.J. Roberts, C.L. Levy, K. Chu, C. Paneda
ur overall goal is to investigate the neural bases
of behavior by using mouse models. In particular, we are interested in motivated behaviors
such as drug and alcohol self-administration, exploratory
drive, and feeding. In addition, we have developed
batteries of tests that model behavioral correlates of
several neuropsychiatric disorders, including anxiety
disorders, depression, compulsive and impulsive disorders, and disorders of learning and memory. We are
particularly interested in exploring and developing
behavioral tests as a means to meet the translational
needs of investigators at Scripps Research, for example, to make transitions from laboratory findings to
clinical application. Behavioral testing fits into the
translational, bench-to-bedside approach because it
enables the modeling of human diseases and the preliminary development of medications.
We are involved in a multisite integrated neuroscience initiative on alcoholism sponsored by the
National Institute on Alcohol Abuse and Alcoholism in
which the overall goal is to examine the neural basis
of excessive alcohol drinking. We developed a model
of excessive alcohol drinking after a period of abstinence in alcohol-dependent mice. The corticotropinreleasing factor 1 receptor is an important player in
this phenomenon, and we are examining the roles of
the opioid receptors and the intracellular factors,
δ-FosB and adenylate cyclase 7. In addition, we are
investigating the role of the extended amygdala circuitry, implicated in addictive behaviors, in excessive
alcohol drinking after abstinence by examining the
effects of several receptor antagonists administered
directly into specific brain sites.
O
Published by TSRI Press®. © Copyright 2005,
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Another focus of our group is studies of self-administration of intravenous cocaine, morphine, and methamphetamine in mice. For example, we have examined
self-administration in several genetically manipulated
mouse strains to determine potential neuropharmacologic mechanisms of drug-seeking behavior. In addition,
we developed a mouse model of stress-induced relapse
to cocaine self-administration. Mice are trained to press
a lever for cocaine, and then this behavior is extinguished by removing the cocaine. Finally, mice are
subjected to various stressors and how they respond
to the lever previously associated with cocaine is
recorded. This effect of stress on relapse is of critical
importance in human addicts; therefore, understanding the underlying neural mechanisms can enhance
the ability to treat and prevent addictive disorders.
Finally, we continue to use our behavioral test batteries to characterize mice generated in other investigators’ laboratories. Currently, we are testing mice in
collaboration with C. Lai, Molecular and Integrative
Neurosciences Department; L. de Lecea, E.A. Thomas,
and J.G. Sutcliffe, Department of Molecular Biology;
and M. Riewald, Department of Immunology.
PUBLICATIONS
Breese, G.R., Chu, K., Dayas, C.V., Funk, D., Knapp, D.J., Koob, G.F., Le, D.A.,
O’Dell, L.E., Overstreet, D.H., Roberts, A.J., Sinha, R., Valdez, G.R., Weiss, F.
Stress enhancement of craving during sobriety: a risk for relapse. Alcohol. Clin.
Exp. Res. 29:185, 2005.
Ghozland, S., Chu, K., Kieffer, B.L., Roberts, A.J. Lack of stimulant and anxiolytic-like effects of ethanol and accelerated development of ethanol dependence in
µ-opioid receptor knockout mice. Neuropharmacology, in press.
Katner, S.N., Flynn, C.T., Von Huben, S.N., Kirsten, A.J., Davis, S.A., Lay, C.C,
Cole, M., Roberts, A.J., Fox, H.S., Taffe, M.A. Controlled and behaviorally relevant
levels of oral ethanol intake in rhesus macaques using a flavorant-fade procedure.
Alcohol. Clin. Exp. Res. 28:873, 2004.
O’Dell, L.E., Roberts, A.J., Smith, R.T., Koob, G.F. Enhanced alcohol self-administration after intermittent versus continuous alcohol vapor exposure. Alcohol. Clin.
Exp. Res. 28:1676, 2004.
Risbrough, V.B., Hauger, R.L., Roberts, A.J., Vale, W.W., Geyer, M.A. Corticotropin-releasing factor receptors CRF1 and CRF2 exert both additive and opposing
influences on startle defensive behavior. J. Neurosci. 24:6545, 2004.
Winsky-Sommerer, R., Yamanaka, A., Diano, S., Borok, E., Roberts, A.J., Sakurai, T., Kilduff, T.S., Horvath, T.L., de Lecea, L. Interaction between the corticotropin-releasing factor system and hypocretins (orexins): a novel circuit mediating
stress response. J. Neurosci. 24:11439, 2004.
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
321
Molecular Mechanisms of
Adaptive and Maladaptive
Neuronal Plasticity
P.P. Sanna, T. Behnisch,* F. Berton, M. Cammalleri,**
Z. Chen, D. Lekic, M. Mendez-Diaz, V. Mendoza-Fernandez,
M. Morales,*** V. Repunte-Canonigo,* V. Romano-Spica,****
K. Slanina, D. Thurbon, L. van der Stap, W. Francesconi
* Leibniz Institute for Neurobiology, Magdeburg, Germany
** Università degli Studi di Pisa, Pisa, Italy
*** National Institute on Drug Abuse, Baltimore, Maryland
**** Istituto Universitario di Scienze Motorie, Rome, Italy
ong-lasting changes in neuronal function, also
known as neural plasticity, involve structural and
functional modifications of synaptic connections
and changes in gene expression. We are interested in
the molecular bases of forms of neuronal plasticity associated with physiologic and pathologic events such as
learning and the development of substance dependence.
The cystine prodrug N-acetylcysteine can prevent
relapse to cocaine-seeking behavior in rats with a history of cocaine self-administration. For this reason, we
are investigating the effects of cystine, the active principle of N-acetylcysteine, on synaptic transmission at
Schaffer collateral/commissural fiber–CA1 synapses in
rat hippocampal slices. In addition to being a valuable
system for studies of synaptic plasticity, the hippocampus plays a crucial role in gating cortical throughput
in the nucleus accumbens, and electrical stimulation
of the hippocampus elicits relapse to cocaine-seeking
behavior in rats with a history of cocaine self-administration. We found that cystine induced a long-term
depression at Schaffer collateral/commissural fiber–CA1
synapses that depended on glutamate release via the
cystine-glutamate antiporter for its induction (Fig. 1).
Drugs of abuse can affect synaptic plasticity in multiple brain regions involved in drug-induced adaptations.
Our results indicate that induction of synaptic plasticity is also a prominent effect of the activation of the
cystine-glutamate antiporter.
In other studies, we discovered a novel form of regulation of the excitability of the juxtacapsular subdivision
of the bed nucleus of the stria terminalis (BNST). The
BNST has been implicated in stress responses and in
the motivational dysregulation associated with drug
dependence. We observed a long-term potentiation of
the intrinsic excitability in the juxtacapsular BNST
L
Published by TSRI Press®. © Copyright 2005,
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F i g . 1 . A, A 20-minute application of cystine, the active principle
of the putative anticraving drug N-acetylcysteine, induced a protracted
decrease of field excitatory postsynaptic potentials (fEPSPs) at Schaffer
collateral/commissural fiber–CA1 synapses in rat hippocampal slices.
Representative fEPSP traces recorded at the times marked (a, b) are
shown in the inset. Cystine-induced depression of fEPSPs was
reversed by delivery of theta-burst stimulation (TBS). B, Long-term
depression dependent on metabotropic glutamate receptors was
induced to saturation by delivery of 3 trains of low-frequency stimulation (LFS) in the presence of a competitive antagonist (D-AP5)
of N-methyl-D -aspartate receptors. Addition of cystine 25 minutes
after the last train of low-frequency stimulation did not induce any
further reduction of fEPSP slopes. C, Application of L-homocysteate,
an inhibitor of the cystine-glutamate antiporter, before and during
cystine application prevented cystine-induced depression of fEPSP
slopes. D, Application of L-homocysteate after cystine washout did
not reverse cystine-induced depression of fEPSP slopes.
expressed as a decrease in the firing threshold of juxtacapsular BNST neurons mediated by changes in
D-type potassium current. This form of plasticity was
impaired during protracted withdrawal from various
322 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
drugs of abuse, including alcohol, cocaine, and heroin.
The impairment was graded and was more pronounced
in rats that self-administered greater amounts of the
drugs and that also had increased expression of the
Kv1.2 channel, the main contributor to the D-type
potassium current. These results indicate that multiple
drugs of abuse induce a common change of the integration properties of juxtacapsular BNST neurons and
suggest that adaptations of Kv1.2-containing potassium
channels may be involved in the development of compulsive drug intake.
To understand the molecular mechanisms responsible for the development of compulsive drug use and
vulnerability to relapse, we are using microarray-based
strategies to identify changes in gene expression associated with self-administration of drugs of abuse. We
found that the lateral part of the hypothalamus was the
most transcriptionally responsive brain region. Genes
involved in synaptic plasticity were mostly affected,
suggesting that intrinsic circuitry in the lateral part of
the hypothalamus could undergo a structural remodeling during the transition to drug addiction.
Last, to profile gene expression in brain subregions
that are below the level of anatomic resolution of manual dissecting techniques, we are using a laser microdissection approach and 2 rounds of in vitro transcription
to generate target RNAs for microarray analyses. Because
biologically meaningful changes in RNA and protein in
the brain are often of limited magnitude, increased anatomic accuracy in the dissections greatly increases the
capability of genomics and proteomics approaches for
detecting differences.
PUBLICATIONS
Ahmed, S.H., Lutjens, R., van der Stap, L.D., Lekic, D., Romano-Spica, V.,
Morales, M., Koob, G.F., Repunte-Canonigo, V., Sanna, P.P. Gene expression evidence for remodeling of lateral hypothalamic circuitry in cocaine addiction. Proc.
Natl. Acad. Sci. U. S. A., in press.
Conti, B., Sugama, S., Lucero, J., Winsky-Sommerer, R., Wirz, S.A., Maher, P.,
Andrews, Z., Barr, A.M., Morale, M.C., Paneda, C., Pemberton, J., Gaidarova, S.,
Behrens, M.M., Beal, F., Sanna, P.P., Horvath, T., Bartfai, T. Uncoupling protein 2
protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity. J. Neurochem. 93:493, 2005.
Francesconi, W., Cammalleri, M., Berton, F., Sanna, P.P. The metabotropic glutamate receptor 5 is necessary for late-phase long-term potentiation in the hippocampal CA1 region. Brain Res. 1022:12, 2004.
Lu, X., Barr, A.M., Kinney, J.W., Sanna, P., Conti, B., Behrens, M.M., Bartfai, T.
A role for galanin in antidepressant actions with a focus on the dorsal raphe nucleus.
Proc. Natl. Acad. Sci. U. S. A. 102:874, 2005.
Sanna, P.P., King, A.R., van der Stap, L., Repunte-Canonigo, V. Gene profiling of
laser microdissection of brain regions and subregions. Brain Res. Brain Res. Protoc., in press
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Cellular Physiology of Brain
Cannabinoids and Peptides
P. Schweitzer, M. Roberto, K. Slanina, G.R. Siggins,
B. Lambolez,* D. Piomelli**
* Ecole Superiéure de Physique et de Chimie Industrielles, Paris, France
** University of California, Irvine, California
N E U R O B I O L O G Y O F C A N N A B I N O I D S U B S TA N C E S
annabinoid substances contained in marijuana
have powerful psychoactive properties and alter
cognitive processes via activation of cannabinoid-1 (CB 1) receptors, but in order to function properly, the brain produces its own cannabinoid ligands.
Our objectives are to uncover the cellular mechanisms
that underlie the central effects of cannabinoid ligands
and to determine the role played by endogenously
formed cannabinoids.
Using a physiologic approach, we are investigating
the modulation of synaptic transmission and plasticity.
In this approach, we record from neurons in brain tissue from the hippocampus and neocortex, 2 structures
that have high levels of CB1 receptors and are involved
in learning and memory processes. In collaboration with
D. Piomelli, University of California, Irvine, we are using
various pharmacologic tools to study the routes of degradation of endogenous cannabinoids. In our collaboration with B. Lambolez, Ecole Superiéure de Physique
et de Chimie Industrielles, we are using single-cell
reverse transcriptase–polymerase chain reaction after
whole-cell recording to characterize the neuronal populations that express transcripts for CB1 receptors.
The role played by endogenous cannabinoids in hippocampal excitatory synaptic transmission and plasticity
remains controversial. We found that endogenous cannabinoids acting at CB1 receptors selectively decrease
excitatory transmission and restrict synaptic plasticity.
Consistent with such a role for endogenous cannabinoids, our research in collaboration with Dr. Lambolez
indicates that more than half of pyramidal neurons
express CB1 receptors in the neocortex. We also discovered that cyclooxygenase-2 has a predominant role in
controlling the tonic level of endogenous cannabinoids
that modulate synaptic activity and plasticity.
Thus, endogenous cannabinoids acting at CB1 receptors decrease excitatory synaptic transmission and plasticity in the hippocampus. In addition, the endogenous
cannabinoid system interacts with the cyclooxygenase-2
C
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
pathway, and inhibitors of this enzyme may increase
levels of endogenous cannabinoids.
CELLULAR MECHANISMS OF ACTION OF
BRAIN PEPTIDES
Neuropeptides are found throughout the brain and
strongly influence neuronal activity. We are studying
the postsynaptic mechanisms of action of cortistatin
and somatostatin, 2 closely related peptides that have
different effects on sleep activity. We found that cortistatin, but not somatostatin, augments the H-current.
This conductance modulates synaptic integration and
oscillatory activity, and the selective effect of cortistatin
on the H-current may therefore be a cellular target for
regulation of sleep behavior by cortistatin.
Peptides such as corticotropin-releasing factor (CRF)
and nociceptin interact with ethanol. In collaboration
with G.R. Siggins, Molecular and Integrative Neurosciences Department, we are examining the actions of
ethanol in the central amygdala, a brain region prominently involved in alcohol dependence and reinforcement. Our results indicate that CRF1 receptors mediate
the ethanol enhancement of inhibitory transmission,
providing a cellular mechanism for the involvement of
CRF in the effects of ethanol and supporting a role for
the peptide in the motivational effects of ethanol. CRF1
receptors could be an important therapeutic target for
the treatment of stress-induced alcohol drinking.
In other studies, we investigated the cellular effects
of γ-hydroxybutyrate (GHB), a compound used to induce
absence seizures in animals. GHB has become a recreational drug of abuse and is also known as “acquaintance rape” drug. We found that GHB postsynaptically
activated γ-aminobutyric acid B (GABAB) receptors to
augment potassium conductances and decrease the
H-current in the hippocampus, the brain region that
has the highest endogenous levels of GHB. Because
the H-current participates in bursting activity and neuronal oscillations, this cellular target may be involved
in the ability of GHB to induce absence seizures. In
addition, high doses of GHB act at GABAB receptors,
and treatment of GHB intoxication or abuse should take
into account the GABAB system.
PUBLICATIONS
Schweitzer, P., Roberto, M., Madamba, S.G., Siggins, G.R. γ-Hydroxybutyrate
increases a potassium current and decreases the H-current in hippocampal neurons
via GABAB receptors. J. Pharmacol. Exp. Ther. 311:172, 2004.
Slanina, K.A., Roberto, M., Schweitzer, P. Endocannabinoids restrict hippocampal
long-term potentiation via CB1. Neuropharmacology, in press.
323
Neurobiology of Developmental
Drug Exposure and Alcohol
Reinforcement
C.J. Slawecki, J. Roth, A. Thorsell, C.L. Ehlers
CONSEQUENCES OF EXPOSURE TO DRUGS
DURING ADOLESCENCE
se of drugs during adolescence is associated
with increases in risks for substance abuse and
cognitive deficits that persist into adulthood.
Because alcohol and nicotine are highly abused by
adolescents, we have sought to develop models that
allow us to assess the long-term consequences of exposure to alcohol or nicotine during adolescence in rats.
In these models, periadolescent rats (30–45 days old)
are exposed to alcohol or nicotine, and then brain function and behavior are examined.
Our results continue to indicate that adolescents
and adults respond differently to chronic exposure to
alcohol. After 2–3 weeks of abstinence, rats exposed
to alcohol during adolescence seem to have depressivelike behavior. Ongoing studies also support our hypothesis that exposure to alcohol during adolescence or
adulthood differentially alters cognitive function. Exposure during adulthood impairs acquisition of a sustained
attention task, whereas exposure during adolescence
impairs baseline sustained attention performance. These
behavioral deficits accompany impaired function in brain
regions that mediate these cognitive processes (i.e., the
cortex and hippocampus) and appear to mimic changes
seen in human adolescents who abuse alcohol.
We also found that exposure of rats to nicotine
during adolescence caused persistent changes in
behavior and brain function. After protracted abstinence
from nicotine, increases in anxiety-like and decreases
in depressive-like behaviors occurred. In the brain, corticotropin-releasing factor and neuropeptide Y levels were
enhanced by exposure to nicotine. Because these peptides play an important role in anxiety and depression,
alterations in their brain levels could provide the basis
for the behavioral changes that occur in rats exposed
to nicotine during adolescence. These protracted changes
could also contribute to continued use and/or relapse
to use of nicotine.
U
N E U R O B I O L O G I C A L M E D I AT O R S O F
ALCOHOL CONSUMPTION
Slanina, K.A., Schweitzer, P. Inhibition of cyclooxygenase-2 elicits a CB1-mediated
decrease of excitatory transmission in rat CA1 hippocampus. Neuropharmacology,
in press.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Alcoholism can be associated with intense “cravings” and excessive drinking, despite known adverse
324 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
consequences. We continue to examine neurobiological
mediators of excessive alcohol drinking in novel rat models. In a newly developed model, a prolonged period of
alcohol exposure sufficient to induce physical dependence produced a 20% increase in alcohol drinking that
persisted for more than 9 weeks. Importantly, the elevated alcohol intake was reduced after administration
of neuropeptide Y, a brain peptide that plays a critical
role in feeding and anxiety. Alcohol-seeking behavior,
as indexed by pressing of a lever, was not altered by
neuropeptide Y in this model. These data further indicate that neuropeptide Y plays an important role in
mediating alcohol drinking but not alcohol seeking in
rats with a history of alcohol dependence.
PUBLICATIONS
Slawecki, C.J., Jimenez-Vasquez, P., Mathe, A.A., Ehlers, C.L. Effect of ethanol on
brain neuropeptides in adolescent and adult rats. J. Stud. Alcohol 66:46, 2005.
Slawecki, C.J., Purdy, R.H., Ehlers, C.L. Electrophysiological effects of allopregnanolone
in rats with a history of ethanol exposure. Alcohol. Clin. Exp. Res. 29:66, 2005.
Slawecki, C.J., Roth, J. Assessment of sustained attention in ad libitum fed Wistar
rats: effects of MK-801. Physiol. Behav., in press.
Slawecki, C.J., Thomas J.D., Riley, E.P., Ehlers, C.L. Neurophysiologic consequences of neonatal ethanol exposure in the rat. Alcohol 34:187, 2004.
Slawecki, C.J., Thorsell, A., Ehlers, C.L. Long-term neurophysiological effects of
alcohol or nicotine exposure in adolescent animal models. Ann. N. Y. Acad. Sci.
1021:448, 2004.
Thorsell, A., Slawecki, C.J., Ehlers, C.L. Effects of neuropeptide Y and corticotropin-releasing factor on ethanol intake in Wistar rats: interaction with chronic
ethanol exposure. Behav. Brain Res. 161:133, 2005.
Thorsell, A., Slawecki, C.J., Ehlers, C.L. Effects of neuropeptide Y on appetitive
and consummatory behaviors associated with alcohol drinking in Wistar rats with a
history of ethanol exposure. Alcohol. Clin. Exp. Res. 29:584, 2005.
Neuronal Communication,
Neuropeptides, Abused Drugs,
and Neuropathology
G.R. Siggins. P. Schweitzer, S. Madamba, Z. Nie,
M. Roberto, T. Krucker, R. Vlkolinsky, M. Bajo, A. Mosley,
K. Lindsley, I.L. Campbell, L.H. Parsons, L. de Lecea,*
S. Moore,** G. Martin***
* Department of Molecular Biology, Scripps Research
** Duke University, Raleigh-Durham, North Carolina
*** University of Massachusetts Medical School, Worcester, Massachusetts
e assess the electrophysiologic and molecular mechanisms of neuronal and synaptic
function and the effects of neuropeptides,
abused drugs, and neuropathologic insults on these
W
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
functions. We use extracellular, intracellular, and patch
recording of brain neurons in vitro, and we administer
transmitters, drugs, cytokines, and neurotoxins by
micropipettes and by bath superfusion. We activate
synaptic transmission via stimulating electrodes. We
also use molecular biological methods to assess druginduced alterations of transmitter receptors.
We study synaptic mechanisms and drug effects in
the hippocampus, nucleus accumbens, and central
amygdala, brain regions prominently involved in memory, learning, stress, and drug abuse. As described
previously, we found inhibitory roles in the hippocampus for the neuropeptides somatostatin, cortistatin, and
the opioid-like peptide nociceptin. All 3 peptides
depressed epileptiform events in the hippocampus,
suggesting that they could dampen seizures. Our new
patch-clamp studies of neurons in the central amygdala
indicate that nociceptin also decreases presynaptic
release of the inhibitory transmitter γ-aminobutyric acid
(GABA) and reverses the effect of ethanol in enhancing
GABA release. We also found that a δ opioid receptor
agonist appears to reduce transmitter release in neurons in the central amygdala, with little effect on postsynaptic membrane properties.
Our previous findings suggested that receptors for
N-methyl-D-aspartate (NMDA) play a role in opiate and
ethanol dependence; thus, glutamatergic synapses
could be involved in opiate and alcohol abuse. Using
patch-clamp recording and pharmacologic studies, we
found that chronic morphine treatment altered several
pharmacologic and biophysical properties of NMDA
receptor–mediated excitatory postsynaptic potentials
(EPSPs) in slices and freshly isolated neurons from the
nucleus accumbens in ways that suggested changes in
the composition or function of the subunits of NMDA
receptors. Using quantitative real-time polymerase chain
reaction and Western blots of NMDA receptor subunits
in tissue from the nucleus accumbens, we found that
the RNA for the 3 major subunits (NR1, NR2A, and
NR2B) does not change in morphine-dependent rats,
but the protein levels of NR1 and NR2B increase significantly, suggesting a posttranscriptional effect of
chronic morphine. In contrast, in the central amygdala,
chronic morphine significantly increased RNA levels for
the NR1 subunit but had no effect on the protein levels
of any of the 3 subunits.
Our single-cell reverse transcriptase–polymerase
chain reaction profiling data suggested that chronic morphine treatment reduced the number of neurons in the
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
nucleus accumbens with multiple NR2 (heteromultimeric) subunit types. Our combined data now suggest
that morphine dependence leads to regionally specific
and subunit-specific changes in NMDA receptors.
In slices of the central amygdala from rats never
exposed to ethanol and rats chronically exposed to
ethanol, acute exposure to ethanol consistently increased
the amplitude of GABAergic inhibitory postsynaptic
potentials (IPSPs) and decreased glutamatergic EPSPs,
indicating reciprocal alteration of GABAergic and glutamatergic systems in the central amygdala; no tolerance to these effects were seen after chronic ethanol
treatment. The results of quantal synaptic analysis
and microdialysis studies, done in collaboration with
L.H. Parsons, Molecular and Integrative Neurosciences
Department, indicate that ethanol’s effect on IPSPs is
largely presynaptic, enhancing vesicular GABA release.
Corticotropin-releasing factor (CRF), reportedly
involved in stress-related alcohol dependence, also
presynaptically enhanced IPSPs in the central amygdala
of mice and rats, and CRF 1 antagonists or a mutation that deleted the gene for the CRF1 receptor abolished the effects of both CRF and ethanol, suggesting
mediation of ethanol’s effects by activation of endogenous CRF1 receptors (Fig. 1). Interestingly, sensitivity
to the enhancing effect of CRF on IPSPs increased
after chronic ethanol treatment. Our recent studies
with S. Moore, Duke University, indicate that ethanol
increases GABA release in neurons in the central amygdala from mice with null mutations in either δ or µ
opioid receptors significantly more than in neurons
from control mice, suggesting that endogenous opioid
peptides act opposite to CRF, reducing the effects of
ethanol on the GABAergic system.
As we reported previously, acute ethanol also
reduced glutamatergic transmission, including NMDAEPSPs, in the central amygdala. However, the depressant effect of acute ethanol on NMDA-EPSPs was
enhanced, and glutamate release increased (as determined by quantal analysis and microdialysis in vivo),
after chronic ethanol treatment and withdrawal, suggesting possible presynaptic and postsynaptic mechanisms underlying behavioral sensitization to ethanol.
The postsynaptic effect of chronic ethanol appears to
involve recomposition of NMDA receptors to a preponderance of NR2B subunits in the central amygdala, as
suggested by changes in responses to subunit-selective
NMDA receptor antagonists and in RNA and protein
levels. In sum, these neuropeptide interactions with
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
325
F i g . 1 . Schematic of hypothetical action of ethanol (EtOH) on
GABAergic synapses in the central amygdala. Top synapse, Ethanol
could enhance the release of GABA (light gray spheroids) from another GABAergic interneuron, either via (1) the release from the same
terminal of CRF (dark gray triangles) that then acts on CRF-R1 on
the terminal to elicit (black arrow) release of more GABA or (2) direct
activation of CRF-R1 to elicit the release of more GABA. Bottom
synapse, A terminal (or dendrite) could feed back onto the same
neuron to act on GABAergic autoreceptors. Ethanol then may enhance
GABA release onto these GABA receptors via CRF release or via
direct activation of CRF-1R receptors, as in the top synapse. Thus,
ethanol may augment the autoinhibition of this inhibitory interneuron,
leading to excitation of downstream neurons by disinhibition. Reprinted
from Siggins, G.R., Roberto, M., Nie, Z. The tipsy terminal: presynaptic effects of ethanol. Pharmacol. Ther. 107:80, 2005, with
permission from Elsevier. Copyright 2005.
synaptic effects of ethanol may represent novel cellular
neuroadaptations underlying ethanol dependence.
We also use mouse models to study dementias
due to viral infections, cytokines, and chemokines, as
described in the report of T. Krucker, Molecular and
Integrative Neurosciences Department. For example,
we found that exogenous CXC chemokine ligand 10
(CXCL10), but not its chronic production in transgenic
mice, reduced long-term potentiation by acting on a
CXCR3 receptor, suggesting that chemokines (and cytokines) can disrupt brain function and therefore perhaps
memory and learning. Other studies indicate that the
psychostimulant methamphetamine may exacerbate the
increased glutamate release and further reduce the
diminished long-term potentiation that occurs in transgenic mice expressing the HIV coat protein gp120.
PUBLICATIONS
Martin, G., Guandano-Ferraz, A., Morte, B., Ahmed, S., Koob, G.F., de Lecea, L.,
Siggins, G.R. Chronic morphine treatment alters N-methyl-D-aspartate receptors in
freshly isolated neurons from nucleus accumbens. J. Pharmacol. Exp. Ther.
311:265, 2004.
326 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Roberto, M., Madamba, S.G., Stouffer, D.G., Parsons, L.H., Siggins, G.R. Increased
GABA release in the central amygdala of ethanol-dependent rats. J. Neurosci.
24:10159, 2004.
Schweitzer, P., Roberto, M., Madamba, S.G., Siggins, G.R. γ-Hydroxybutyrate
increases a potassium current and decreases the H-current in hippocampal neurons
via GABAB receptors. J. Pharmacol. Exp. Ther. 311:172, 2004.
Siggins, G.R., Roberto, M., Nie, Z. The tipsy terminal: presynaptic effects of ethanol.
Pharmacol. Ther. 107:80, 2005.
Primate Neurobehavioral
Laboratory
M.A. Taffe, S.N. Katner, R.D. Crean, S.A. Davis, C.C. Lay,
S.N. Von Huben
he drug 3,4-methylenedioxymethamphetamine
(MDMA), known as “Ecstasy,” continues to be
used recreationally, with about 7% of U.S. high
school seniors and 13% of U.S. college students reporting use. Persons who have used substantial amounts
of Ecstasy have a range of cognitive, mood, and sleep
disturbances even after prolonged abstinence from the
drug. Because such persons usually have high rates of
exposure to many other illicit drugs, determining the
precise role of MDMA itself in the reported signs and
symptoms is difficult.
Laboratory studies during past decades have indicated that MDMA can produce a widespread reduction
in serotonergic function in the brain in many species,
including nonhuman primates. Although much has been
accomplished in describing the mechanisms of the neurotoxic effects of MDMA, whether such brain changes
produce the cognitive or mood disruptions associated
with use of Ecstasy in humans remains unclear. We are
determining how MDMA-induced brain changes may
impair cognition, mood, circadian patterns of temperature
and activity, or brain electrophysiologic characteristics.
Recently, we have focused on disruptions of body
temperature caused by MDMA. An elevation in temperature is a critical factor in the neurotoxic effects of
MDMA in rodents, but the thermoregulatory response
differs in larger bodied primates. We found that the
temperature of rhesus monkeys is elevated by MDMA
under conditions in which body temperature in rats is
decreased. Body temperature in monkeys is also elevated by the R(–) stereoisomer of MDMA, which has a
minimal effect in rodents; humans consume the racemic
mixture of both stereoisomers. In monkeys, but not in
rodents, MDMA produces temperature elevations independently of marked increases in locomotor activity.
T
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Thus, our data suggest that the thermoregulatory
responses to MDMA in primates may be more sensitive than would be predicted on the basis of studies in
rodents. Our ongoing studies will determine if such
sensitivity is also associated with increased risk for the
neurotoxic effects of MDMA.
PUBLICATIONS
Katner, S.N., Davis, S.A., Kirsten, A.J. Taffe, M.A. Effects of nicotine and mecamylamine on cognition in rhesus monkeys. Psychopharmacology (Berl.) 175:225, 2004.
Madden, L.J., Flynn, C.T., Zandonatti, M.A., May, M., Parsons, L.H., Katner,
S.N., Henriksen, S.J., Fox, H.F. Modeling human methamphetamine exposure in
nonhuman primates: chronic dosing in the rhesus macaque leads to behavioral and
physiological abnormalities. Neuropsychopharmacology 30:350, 2005.
Roberts, E.S., Burudi, E.M.E., Flynn, C.T., Madden, L.J., Roinick, K.L., Watry,
D.D., Zandonatti, M.A., Taffe, M.A., Fox, H.S. Acute SIV infection of the brain
leads to upregulation of IL6 and interferon-regulated genes: expression patterns
throughout disease progression and impact on neuroAIDS. J. Neuroimmunol.
157:81, 2004.
Neurobiology of Addiction
F. Weiss, L.H. Parsons, R. Ciccocioppo,* M. Massi,*
P.P. Sanna, R. Martin-Fardon, E.P. Zorrilla, M.A. Baptista,
C.V. Dayas, Y. Zhao, H. Aujila, T.M. Kerr, N.D. Stuempfig,
K.M. Santerre
* University of Camerino, Camerino, Italy
e study signaling mechanisms and neurocircuitries that mediate the addictive actions of
drugs of abuse. One area of particular interest
is mechanisms responsible for the development of lasting vulnerability to relapse, a defining feature of substance dependence and a central problem for successful
treatment. We recently identified a set of novel regulatory systems and potential treatment targets for protracted withdrawal symptoms, hyperresponsiveness to
stress, and drug “craving,” conditions that are major risk
factors for relapse. The set includes group II metabotropic
glutamate receptors (mGluRs), the nociceptin/orphanin
FQ opioid peptide system, and nitric oxide signaling.
Pharmacologic manipulation of these systems was
highly effective in preventing resumption of drug seeking in animal models of relapse and did not interfere
with motor, cognitive, or normal motivational functions.
This past year we began to investigate the neurocircuitry through which mGluR-modulated glutamate
transmission and nitric oxide–dependent signaling participate in regulating drug-seeking behavior. In addition,
we extended our search for novel treatment targets,
focusing on the σ1 intracellular receptor.
W
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Group II mGluRs are Gi/cAMP-coupled receptors
located at the presynaptic (mGluR 2) and postsynaptic
(mGluR 3) level, where they provide negative feedback
by decreasing the release of glutamate and reducing
excitability by glutamate, respectively. We reported
earlier that LY379268, a potent and selective mGluR
2/3 agonist, blocks the initiation of cocaine seeking in
rats in an experimental model of drug craving. We have
now found that these protective effects of LY379268
extend to the initiation of drug seeking associated with
another risk factor for relapse, stress, as modeled by
brief exposure to electric footshock.
Using a c-fos neural mapping approach, we discovered that the CA1, CA3, and dendate gyrus regions
of the hippocampus are putative sites of action for the
anticraving actions of LY379268, consistent with the
established role of the hippocampus in associative
learning of contingencies between contextual stimuli
and reward. Additionally, our findings implicate the
lateral and medial divisions of the central nucleus of
the amygdala in the attenuation of stress-induced drug
seeking by LY379268. In these sites, LY379268
increased expression of c-fos in a benzodiazepine-like
manner, presumably reflecting activation of inhibitory
GABAergic output from the central amygdala resulting
in anxiolytic effects known to be associated with activation of group II mGluRs.
Nitric oxide, a gaseous intercellular messenger
present throughout the CNS, participates in regulating
numerous neurobiological functions, including synaptic
plasticity. Our previous data that a nonselective inhibitor of the synthesis of nitric oxide (L-NAME) attenuates
the motivating effects of alcohol-related contextual
stimuli in rats suggested a role for nitric oxide signaling in drug-directed behavior. In support of this hypothesis, we have replicated this finding by using a neuronal
nitric oxide–selective inhibitor (N-propyl- L -arginine)
and have traced these effects to the hypothalamic paraventricular nucleus. Inhibition of nitric oxide synthesis
in the paraventricular nucleus interferes with activation
of the hypothalamic-pituitary-adrenal axis, and thus
this interference is a potential mechanism for the effects
of the inhibition of nitric oxide synthesis on alcoholseeking behavior. This hypothesis is consistent with
emerging evidence that craving and relapse associated
with exposure to drug cues depend on the functional
integrity of the hypothalamic-pituitary-adrenal axis, but
direct confirmation is required.
The σ1 receptor is an intracellular protein present
on the endoplasmic reticulum as well as on mitochonPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
327
drial, nuclear, and plasma membranes. Physiologically,
activation of this receptor modifies the composition of
the microdomains of membrane lipid rafts. Functionally, activation of the receptor has been implicated in
learning and memory, anxiety, responses to stress, and,
recently, the appetitive effects of cocaine. We examined
whether pharmacologic blockade of σ1 receptors via a
potent and selective antagonist (BD1047) alters the
direct reinforcing actions of cocaine or the motivating
effects of cocaine-related contextual stimuli. The σ1
receptor antagonist dose-dependently reduced the initiation of drug seeking normally associated with exposure
to cocaine cues, without altering behavior maintained
either by cocaine itself or by a highly palatable nutrient.
The results indicate that the σ 1 receptor is another
novel potential target for preventing craving and relapse
associated with cocaine addiction.
PUBLICATIONS
Breese, G.R., Chu, K., Dayas, C.V., Funk, D., Knapp, D.J., Koob, G.F., Le, D.A.,
O’Dell, L.E., Overstreet, D.H., Roberts, A.J., Sinha, R., Valdez, G.R., Weiss, F.
Stress enhancement of craving during sobriety and the risk for relapse. Alcohol.
Clin. Exp. Res. 29:185, 2005.
Little, H.J., Stephens, D.N., Ripley, T.L., Borlikova, G., Duka, T., Schubert, M.,
Albrecht, D., Becker, H.C., Lopez, M.F., Weiss, F., Drummond, C., Peoples, M.,
Cunningham, C. Alcohol withdrawal and conditioning. Alcohol. Clin. Exp. Res.
29:453, 2005.
Liu, X., Weiss, F. Nitric oxide synthesis inhibition attenuates conditioned reinstatement of ethanol-seeking, but not the primary reinforcing effects of ethanol. Alcohol.
Clin. Exp. Res. 8:1194, 2004.
Weiss, F. Agents interacting with dopamine neurotransmission: effects on ethanolseeking in animal models of relapse. In: Milestones in Drug Therapy: Drugs for
Relapse Prevention of Alcoholism. Spanagel, R., Mann, K. (Eds.). Birkhäuser,
Basel, Switzerland, 2005, p. 135.
Weiss, F. Neurobiology of craving, conditioned reward and relapse. Curr. Opin.
Pharmacol. 5:9, 2005.
Neurobiology of Feeding
and Stress
E.P. Zorrilla, G.F. Koob, A.J. Roberts, K. Inoue,*
A. Tabarin,** É. Fekete, Y. Zhao, V. Sabino, P. Cottone,
L. O’Dell, S. Chen, G.R. Valdez, R. Lintz, M. Arends,
M. Brennan, M. Mattock, M. Hoefer, J. Becker, J. Stampe
* Osaka City University Medical School, Osaka, Japan
** Université de Bordeaux II, USN du Haut-Leveque, Pessac, France
e study the regulation of motivated behavior,
such as appetite. In a study fundamental for
understanding ingestion, we showed that
drinking is an integrated component of meal taking in
rats, a finding that redefined what a meal is. Using
W
328 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
the meal model, we discovered that leptin and fenfluramine suppress appetite through different behavioral
mechanisms, as had been suggested by clinical experience but not reproduced in animal models. The model
opens the way for differentiating appetite suppressants
according to modes of action and perhaps for identifying those that act as long-term, and not only short-term,
signals of energy balance.
One goal of our group is to understand the influence
of stress and stress-related peptides, such as corticotropin-releasing factor (CRF) and the urocortins, on
motivated behavior. We found that CNS infusion of
urocortin 2, a selective CRF2 receptor agonist, reduced
intake of a palatable cafeteria diet, which otherwise
promotes overeating, without malaise. Urocortin 2 was
more potent than ovine CRF, a preferential CRF1 receptor agonist, and did not have the anxiogenic-like or
stimulant properties of the CRF1 agonist. Urocortin 2
suppressed appetite via a different mode of action than
ovine CRF did, because the peptides had qualitatively
different effects on diet self-selection. CNS infusion of
urocortin 3, another CRF 2 agonist, also suppressed
intake of palatable food without malaise, and hypothalamic CRF2 receptors were the site of action.
To understand further the role of signaling by members of the CRF/urocortin family in energy balance, we
expanded meal-pattern analysis to mice and examined
the effects of CRF1, CRF2, and urocortin 2 deficiencies on the microstructure of ingestion. The findings
support our hypothesis that CRF1 and CRF2 pathways
participate differentially in the regulation of feeding
and support the potential efficacy of CRF2 agonists as
appetite suppressants.
We also studied the relation of scheduled dieting
to changes in feeding and stress-related behavior. A
history of scheduled, restricted feeding (i.e., eating
less than what would be typically eaten in an entire
day and predominantly within a single large meal)
reduced anxiety-like behavior not only during food
restriction but also after the resumption of normal daily
intake. The results indicate that scheduled dieting
interacts with stress-related neurocircuitry in a manner
with implications for the treatment of eating disorders.
Finally, we found that small-molecule, selective
CRF1 receptor antagonists had antistress effects, consistent with the finding that CNS infusion of a novel, selective CRF1 agonist, stressin-1, had anxiogenic-like effects
in animal models. The findings support the therapeutic
potential of CRF1 antagonists for treatment of anxiety.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
PUBLICATIONS
Stinus, L., Cador, M., Zorrilla, E.P., Koob, G.F. Buprenorphine and a CRF1 antagonist block the acquisition of opiate withdrawal-induced conditioned place aversion
in rats. Neuropsychopharmacology 30:90, 2005.
Zorrilla, E.P., Inoue, K., Fekete, E.M., Tabarin, A., Valdez, G.R., Koob, G.F. Measuring meals: structure of prandial food and water intake of rats. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 288:R1450, 2005.
Zorrilla, E.P., Inoue, K., Valdez, G.R., Tabarin, A., Koob, G.F. Leptin and postprandial satiety: acute central leptin more potently reduces meal frequency than
meal size in the rat. Psychopharmacology (Berl.) 177:324, 2005.
Zorrilla, E.P., Koob, G.F. The therapeutic potential of CRF1 antagonists for anxiety.
Expert Opin. Investig. Drugs 13:799, 2004.
Zorrilla, E.P., Reinhardt, L.E., Valdez, G.R., Inoue, K., Rivier, J., Vale, W.W.,
Koob, G.F. Human urocortin 2, a corticotropin-releasing factor (CRF) agonist, and
ovine CRF, a CRF1 agonist, differentially alter feeding and motor activity. J. Pharmacol. Exp. Ther. 310:1027, 2004.
Harold L. Dorris Neurological
Research Institute
Tamas Bartfai, Ph.D., Director
esearchers at the Harold L. Dorris Neurological
Research Institute focus on molecular and cellular studies of schizophrenia, major depression,
and cognitive decline related to aging and neurodegenerative disease. The center has 3 full-time faculty members with complementary research backgrounds: Tamas
Bartfai in neuropharmacology and biochemistry; M. Margarita Behrens in neuronal signaling, neuronal cultures,
and excitotoxicity; and Bruno Conti in molecular neurobiology and neuroimmunology. The center also funds
the Helen Dorris Fellow in Schizophrenia, a named fellowship position for a postdoctoral researcher to study
aspects of schizophrenia and depression from the neurobiological perspectives. The current fellow is Lisa
Sharkey. The visiting professors at the center in 2004
were the noted electrophysiologist Henri Korn from the
Pasteur Institute in Paris, France, and the famous neuroanatomist Tomas Hökfelt from the Karolinska Institutet in Stockholm, Sweden.
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MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Galanin, a Neuropeptide
Involved in Depression
329
Lu, X., Lundström, L., Bartfai, T. Galanin (2-11) binds to GalR3 in transfected cell
lines: limitations for pharmacological definition of receptor subtypes. Neuropeptides
39:165, 2005.
Lu, X., Lundström, L., Langel, Ü., Bartfai, T. Galanin receptor ligands. Neuropeptides 39:143, 2005.
T. Bartfai, X. Lu, H. Badie-Mahdavi, A. Barr, J. Kinney
alanin is a neuropeptide involved in regulation
of cognition, mood, seizure, and pain threshold.
Antagonists of galanin receptors enhance release
of acetylcholine, noradrenaline, and serotonin and thus
may be useful in the treatment of cognitive decline and
major depression.
After treating rats with selective serotonin reuptake inhibitors such as fluoxetine, electroconvulsive
shock, and sleep deprivation, measures used to treat
depression, we used transcriptional profiling to examine different areas of the brain. We found that galanin
mRNA was upregulated in cells of the dorsal raphe
nucleus and locus coeruleus that are the major monoaminergic nuclei.
We also found that levels of the type 2 galanin
receptor were elevated in the dorsal raphe nucleus,
suggesting that this depolarizing galanin receptor subtype may promote release of serotonin and contribute
to antidepressant actions. In line with these findings,
galnon, a galanin receptor agonist, produced antidepressant-like effects similar to those of the antidepressants fluoxetine and imipramine. Many antidepressants
also promote neurogenesis, and we found that the type
2 galanin receptor mediates effects that promote neuroprotection or neurogenesis in the hippocampus. On the
basis of these data, we have made several important
steps toward defining the galanin receptor 2 as a putative drug target for a new class of antidepressant drugs.
G
PUBLICATIONS
Badie-Mahdavi, H., Behrens, M.M., Rebek, J., Bartfai, T. Effect of galnon on
induction of long-term potentiation in dentate gyrus of C57BL/6 mice. Neuropeptides 39:249, 2005.
Badie-Mahdavi, H., Lu, X., Behrens, M.M., Bartfai, T. Role of galanin receptor 1
and galanin receptor 2 activation in synaptic plasticity associated with 3′,5′-cyclic
AMP response element-binding protein phosphorylation in the dentate gyrus: studies with a galanin receptor 2 agonist and galanin receptor 1 knockout mice. Neuroscience 133:591, 2005.
Holmberg, K., Kuteeva, E., Brumovsky, P., Kahl, U., Karlström, H., Lucas, G.A.,
Rodriguez, J., Westerblad, H., Hilke, S., Theodorsson, E., Berge, O.G., Lendahl, U.,
Bartfai, T., Hökfelt, T. Generation and phyenotypic characterization of a galanin
overexpressing mouse. Neuroscience 133:59, 2005.
Hua, X.Y., Salgado, K.F., Gu, G., Fitzsimmons, B., Kondo, I., Bartfai, T., Yaksh,
T.L. Mechanisms of antinociception of spinal galanin: how does galanin inhibit
spinal sensitization? Neuropeptides 39:211, 2005.
Lu, X., Barr, A.M., Kinney, J.W., Sanna, P., Conti, B., Behrens, M.M., Bartfai, T.
A role for galanin in antidepressant actions with a focus on the dorsal raphe
nucleus. Proc. Natl. Acad. Sci. U. S. A. 102:874, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Lu, X., Mazarati, A., Sanna, P., Shinmei, S., Bartfai, T. Distribution and differential regulation of galanin receptor subtypes in rat brain: effects of seizure activity.
Neuropeptides 39:147, 2005.
Lundström, L., Lu, X., Langel, Ü., Bartfai, T. Important pharmacophores for binding to galanin receptor 2. Neuropeptides 39:169, 2005.
Malkmus, S., Lu, X., Bartfai, T., Yaksh, T.L., Hua, X.Y. Increased hyperalgesia
after tissue injury and faster recovery of allodynia after nerve injury in the GalR1
knockout mice. Neuropeptides 39:217, 2005.
Pirondi, S., Kuteeva, E., Giardino, L., Ferraro, L., Antonelli, T., Bartfai, T., Ögren,
S.O., Hökfelt, T., Calzà, L. Behavioral and neurochemical studies on brain aging in
galanin overexpressing mice. Neuropeptides 39:303, 2005.
Sergeyev, V., Fetissov, S., Mathe, A.A., Jimenez, P.A., Bartfai, T., Mortas, P.,
Gaudet, L., Moreau, J.-L., Hökfelt, T. Neuropeptide expression in rats exposed to
chronic mild stresses. Psychopharmacology (Berl.) 178:115, 2005.
Sollenberg, U., Bartfai, T., Langel, Ü. Galnon: a low-molecular weight ligand of
the galanin receptors. Neuropeptides 39:161, 2005.
Zheng, K., Kuteeva, E., Xia, S., Bartfai, T., Hökfelt, T., Xu, Z.Q. Age-related
impairments of synaptic plasticity in the lateral perforant path input to the dentate
gyrus of galanin overexpressing mice. Neuropeptides 39:257, 2005.
Search for Fast-Acting
Antidepressants
T. Bartfai, B. Conti, J. Lucero, A. Barr, S. Wirz,
M.C., Morale
urrent antidepressants such as the selective
serotonin reuptake inhibitors fluoxetine (Prozac)
and paroxetine (Paxil) must be taken for 14–21
days before clinically significant improvement occurs.
This delay is a large problem, particularly in the treatment of patients with depression who are at risk for
suicide. In collaboration with scientists at Novartis
Pharma, Basel, Switzerland, we are trying to develop
a fast-acting antidepressant.
We are using 2 techniques that produce rapid,
albeit short-lived, antidepressant effects in rats: sleep
deprivation and electroconvulsive therapy. We are comparing the transcriptional changes that occur in different brain areas in response to these treatments with
the changes produced by 14 days of treatment with
fluoxetine. We have identified some putative new drug
targets that if validated in behavioral experiments such
as learned helplessness may form the basis of efforts
to generate a fast-acting antidepressant.
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330 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Mechanisms of
Thermoregulation:
Thermosensitivity in the Brain
T. Bartfai, I. Tabarean, B. Conti, M.M. Behrens, H. Korn
he thermosensitivity of sensory neurons that
underlies the ability to sense cold and heat as
painful stimuli is just now being clarified through
the discovery of temperature-sensitive ion channels in
peripheral neurons. We are investigating neurons in the
anterior part of the hypothalamus that sense temperature and regulate core body temperature.
We showed that individual neurons without the
presence of a neuronal network can sense cold and warm
temperatures and can change firing rate in response
to these changes in temperature. These neurons express
receptors for several pyrogenic agents, such as prostaglandin E2, IL-1, and calcitonin gene–regulated peptide,
that are involved in mediating fever in response to
inflammation and infection and in hot flashes. The molecular and cellular understanding of the central temperature set point will be helpful in the treatment of feeding
and sleep disorders because these phenomena are closely
coordinated with and depend mutually on changes in
the temperature set point.
T
PUBLICATIONS
Conti, B., Sugama, S., Lucero, J., Winsky-Sommerer, R., Wirz, S.A., Maher, P.,
Andrews, Z., Barr, A.M., Morale, M.C., Paneda, C., Pemberton, J., Gaidarova, S.,
Behrens, M.M., Beal, F., Sanna, P.P., Horvath, T., Bartfai, T. Uncoupling protein 2
protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity. J. Neurochem. 93:493, 2005.
Tabarean, I.V., Conti, B., Behrens, M.M., Korn, H., Bartfai, T. Electrophysiological
properties and thermosensitivity of mouse preoptic and anterior hypothalamic neurons in culture. Neuroscience, in press.
Role of N-Methyl-D-Aspartate
Receptor Activity in the
GABAergic Function of
Parvalbumin-Positive
Interneurons in Schizophrenia
J. Kinney, C. Davis, B. Conti, T. Bartfai, M.M. Behrens
S
chizophrenia is a common psychiatric disorder
with a genetic basis but a complex mode of
inheritance. The onset is generally during late
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
adolescence or early adulthood, and the lifetime risk
for the disorder is approximately 1%–2% in the general population. Accumulating evidence suggests that
a hypoglutamatergic condition may cause the interneuronal derangements found in the prefrontal cortex in
autopsy material from patients with schizophrenia; for
example, the levels of several GABAergic markers, such
as the 67-kD form of glutamic acid decarboxylase
(GAD67) and the calcium-binding protein parvalbumin,
are decreased.
We analyzed whether cortical interneurons positive
for parvalbumin are the specific targets of antagonists
of the receptors of N-methyl-D-aspartate (NMDA). Using
primary cultures of cortical neurons, which allow us to
study the neurons in the absence of inputs from outside
the cortex, we characterized the development of parvalbumin-positive interneurons and analyzed the subunit
composition of NMDA receptors and NMDA signaling.
We found that expression of parvalbumin had a
developmentally regulated pattern; 50% of the GABAergic population of interneurons were parvalbumin positive after 3 weeks in culture. Real-time polymerase
chain reaction with mRNA isolated from single neurons indicated that parvalbumin-positive interneurons
had a 4:1 higher ratio of NR2A to NR2B (subunits of
the NMDA receptor) than pyramidal neurons did. Brief
exposure to NMDA induced the activation of the extracellular signal–regulated kinase and the 3′,5′-cyclic
AMP response element–binding protein signaling pathways, indicating the functionality of NMDA receptors
in these interneurons.
When cultures were exposed to sublethal concentrations of ketamine, a time- and dose-dependent
decrease in parvalbumin and GAD67 immunoreactivity
was induced only in parvalbumin-positive interneurons.
These effects were reproduced by the NR2A-selective
antagonist NVP-AAM077; the NR2B-selective antagonist Ro-25-6981 only reduced GAD67 expression.
Increasing intracellular calcium by coexposure to the
calcium channel opener BayK or the group I metabotropic glutamate receptor agonist DHPG attenuated
the decrease in GAD67 and parvalbumin. These results
suggest that the activity of NMDA receptors plays a
pivotal role in the maintenance of the GABAergic function of parvalbumin-positive interneurons.
PUBLICATIONS
Badie-Mahdavi, H., Lu, X., Behrens, M.M., Bartfai, T. Role of galanin receptor 1
and galanin receptor 2 activation in synaptic plasticity associated with 3′,5′-cyclic
AMP response element-binding protein phosphorylation in the dentate gyrus: studies with a galanin receptor 2 agonist and galanin receptor 1 knockout mice. Neuroscience 133:591, 2005.
MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
Conti, B., Sugama, S., Lucero, J., Winsky-Sommerer, R., Wirz, S.A., Maher, P.,
Andrews, Z., Barr, A.M., Morale, M.C., Paneda, C., Pemberton, J., Gaidarova, S.,
Behrens, M.M., Beal, F., Sanna, P.P., Horvath, T., Bartfai, T. Uncoupling protein 2
protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity. J. Neurochem. 93:493, 2005.
Dube, C., Vezzani, A., Behrens, M.M., Bartfai, T., Baram, T.Z. Interleukin-1 contributes to the generation of experimental febrile seizures [published correction
appears in Ann. Neurol. 57:609, 2005]. Ann. Neurol. 57:152, 2005.
331
Uncoupling Protein 2 and
Neuroprotection in a Mouse
Model of Parkinson’s Disease
B. Conti, T. Bartfai
Lu, X., Barr, A.M., Kinney, J.W., Sanna, P., Conti, B., Behrens, M.M., Bartfai, T.
A role for galanin in antidepressant actions with a focus on the dorsal raphe
nucleus. Proc. Natl. Acad. Sci. U. S. A. 102:874, 2005.
Hypothalamic-Pituitary-Adrenal
Axis Dysregulation in Mice
Devoid of Prion Protein
M. Sánchez-Alavez, J.R. Criado, G. Moroncini,
S.J. Henriksen, M.B.A. Oldstone, T. Bartfai, B. Conti
ransmissible spongiform encephalopathies are
lethal neurodegenerative disorders characterized
by posttranslational modification of the cellular
prion protein PrPC, leading to the accumulation of the
abnormal protease-resistant isoform PrP Sc . Although
PrP C is required for PrP Sc -mediated neuropathologic
changes, the normal physiologic role of PrPC remains
largely unknown. Disturbances of hypothalamic and
pituitary functions have long been recognized as clinical indications of naturally occurring scrapie. Ewes
naturally affected with scrapie, as well as experimentally infected hamsters and humans affected with the
prion disease fatal insomnia, have elevated levels of
circulating corticosteroids often associated with enlargement of the adrenal glands.
Specifically, scrapie hypercorticism is characterized
by an impaired central glucocorticoid negative feedback
inhibition of the hypothalamic-pituitary-adrenal axis.
We observed a similar phenotype in mice devoid of
PrPC; they have constitutive elevation of plasma corticosterone levels. When subjected to acute restraint
stress, mice devoid of PrP C had a normal activation of
the hypothalamic-pituitary-adrenal axis, but did not
downregulate secretion of corticotropin when the concentrations of glucocorticoids reached inhibitory levels.
Finally, pretreatment with a high dose of corticosterone
did not block synthesis and secretion of corticotropin
in mice devoid of PrP C. These data suggest that PrPC
plays a specific physiologic role in the glucocorticoidmediated negative feedback modulation of the hypothalamic-pituitary-adrenal axis.
T
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
xidative stress is implicated in the death of
dopaminergic neurons in some forms of Parkinson’s disease. Because oxidative stress can be
modulated endogenously by uncoupling proteins (UCPs),
we hypothesized that specific neuronal expression of
UCP2, a member of the UCP family that is rapidly
induced in the CNS after injury, could confer neuroprotection in a mouse model of Parkinson’s disease.
We generated transgenic mice that overexpressed
UCP2 in catecholaminergic neurons under the control
of the tyrosine hydroxylase promoter. Compared with
wild-type mice, the transgenic mice had 2-fold higher
levels of UCP2 expression in the dopaminergic neurons
of the substantia nigra, elevated uncoupling of their
mitochondria, and marked reduction in indicators of
oxidative stress, an effect also observed in the striatum.
Upon acute exposure to 1,2,3,6-methyl-phenyl-tetrahydropyridine, the transgenic mice had indications of
neuroprotection and retention of locomotor functions.
Our data suggest that UCP2 may be a drug target for
slowing the progression of Parkinson’s disease.
O
PUBLICATIONS
Conti, B., Sugama, S., Lucero, J., Winsky-Sommerer, R., Wirz, S.A., Maher, P.,
Andrews, Z., Barr, A.M., Morale, M.C., Paneda, C., Pemberton, J., Gaidarova, S.,
Behrens, M.M., Beal, F., Sanna, P.P., Horvath, T., Bartfai, T. Uncoupling protein 2
protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity. J. Neurochem. 93:493, 2005.
Effects of Core Body
Temperature on Aging
B. Conti, M. Sánchez-Alavez, R. Winsky-Sommerer,
M.C. Morale, J. Lucero, J. . Pemberton, V. Fabre,
L. de Lecea, S.J. Henriksen, T. Bartfai
eduction of core body temperature has antiaging effects and prolongs life in poikilotherms.
In homeotherms, lowered core body temperature is often achieved by restricting energy intake (also
referred to as calorie restriction), a controlled dietary
regimen that prolongs life span in rodents and monkeys and delays the effects of a variety of diseases,
including neoplastic and neurodegenerative diseases.
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332 MOLECUL AR AND INTEGRATIVE NEUROSCIENCES 2005
To investigate the effects of reduced core body temperature on aging and longevity, we generated transgenic mice that had reduced core body temperature.
The mice were obtained by inducing local heat production in the hypothalamus, in proximity to the neurons
that make up the “core body temperature thermostat,” by overexpressing uncoupling protein 2 in the
hypocretin neurons.
The transgenic mice had an elevated hypothalamic
temperature, indicating that the central thermostat had
been reset, resulting in lowered core body temperature
during the wake period of the day. Fed ad libitum, these
mice developed normally to adulthood, with a growth
curve similar to that of wild-type mice. However, the
transgenic mice had an increased median life span,
suggesting that local heat production in the hypothalamus is sufficient to produce a sustained decrease in
core body temperature resulting in prolonged median
life span independent of altered diet or restriction of
energy intake.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.