1985 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biochemistry of Gaba Receptor Mechanisms @ University of California Los Angeles
The objectives of this project are to determine the molecular mechanism of action of the mammalian brain inhibitory neurotransmitter substance gamma-aminobutyric acid (GABA). The approach is a biochemical study of the structure and function of the GABA receptor protein, its associated chloride ion channel, and its interaction with the modulatory drugs, benzodiazepines and barbiturates and picrotoxin-like convulsants. The receptor will be further defined and characterized in the brain homogenate membrane-bound state, solubilized in mild detergent, characterized and purified, and used for production of antibodies. The functional consequences of GABA and drug binding to these receptors will be analyzed by quantitative radioactive ligand binding, kinetics and equilibrium, and by radioactive chloride flux measurements in brain slices, cultured nerve cells, and in cell-free membrane vesicles. We will attempt to reconstitute functional membrane-bound proteins from defined purified soluble components. Binding and ion flux data will be compared in order to develop a model for the action of GABA and drugs. Protein purification will involve classical methods including lectin and affinity chromatography and affinity labels. Partially purified receptors now available will be used in attempts to make monoclonal antibodies, which will be screened for ability to bind to receptor proteins, inhibit binding activity in membranes or to precipitate soluble binding activity following secondary reaction with antibodies against mouse immunoglobulins and insoluble protein A. Monoclonal or conventional antibodies will be employed for (a.) collaborations in immunocytochemistry, (b.) studies on the turnover, development, and plastic changes in brain, (c.) structural probes of the receptors, (d.) investigation of the functional role of the various GABA and drug binding sites known to exist, (e.) total purification of the receptors if not already achieved. This basic research is relevant to clinical problems of muscle and nerve, especially epilepsy, Huntington's chorea, anxiety, and sleep disorders.
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1 |
1985 — 1995 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba/Benzodiazepine/Barbiturate Receptors in Epilepsy @ University of California Los Angeles |
1 |
1988 — 1995 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba Receptor Complex in Alcohol Dependence @ University of California Los Angeles
The objectives of this study are to determine if a kindling- like phenomenon, mediated via the GABA receptor complex, contributes significantly to the development of physical dependence on ethanol. The numerous episodes of ethanol induced depression of the CNS and the following rebound hyperexcitability are postulated to exert a kindling like effect during the periods of hyperexcitability. Studies are proposed in which ethanol will be administered to rats under a regimen designed to promote kindling effects. The development of ethanol dependence will be assessed during the course of treatment by measurements of seizure threshold with electroshock and two agents with convulsant properties, pentylenetetrazole (PTZ) and a beta-carboline, DMCM, which are known to interact with the GABA receptor complex. Concurrent studies will be directed towards determining whether ethanol dependence and changes of seizure threshold can be correlated with alterations in the neurochemical properties of the GABA receptor complex. The neurochemical studies will include assay of receptor density and allosteric modulation of radioligand binding, quantitative receptor autoradiography and chloride flux assays to assess changes in GABA receptor function. A major goal of this research is to determine if a long-lasting decrease of seizure threshold, similar to that seen with PTZ kindling, can be induced with the proposed method of chronic ethanol administration. Results from this study may be of value for establishing pharmacological and biochemical correlates of ethanol dependence in humans, which develops after years of excessive drinking and results in a long- lasting susceptibility to readdiction.
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1 |
1990 — 1993 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba-a Receptor Structure &Function @ University of California Los Angeles
This project will study the structure and function of the receptor protein for the major brain inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The GABA-A receptor complex is a ligand-gated chloride channel which is also the site of action of many important anticonvulsant, anxiolytic, and anesthetic drugs. The GABA-A receptor is actually a family of receptors, differing in gene sequence, subunit composition, pharmacological properties, and brain regional distribution. We can purify the receptor protein in large quantities by affinity chromatography. We will produce subunit-, subtype-, and functional domain-specific antibodies against synthetic peptides in the subunit sequences derived from cloning. Different subunits detected by gel electrophoresis of the purified receptor will be matched with different cDNA clones using clone-specific antibodies in Western blots and comparing regional location. The pharmacological specificity of ligand binding to GABA, benzodiazepine, barbiturate, and convulsant sites on the isolated subunits detected by photoaffinity labeling will be matched with brain regional ligand binding detected by autoradiography. This will allow us to determine the subunit composition, pharmacological properties, and cellular location of the receptor subtypes. The importance of receptor subtypes is that they vary in their function, susceptibility to disease processes, and sensitivity to pharmacological intervention. Domain-specific antibodies and active site sequencing of photolabeled protein will define functional domains within the protein structure. The purified protein will be reconstituted into membrane vesicles to study functional regulation, including phosphorylation by protein kinase C, which we have demonstrated in vitro on a specific A subunit. The information gained will be valuable in the understanding of normal and diseased brain function. Human disorders of particular relevance include epilepsy, anxiety, alcohol dependence, and sleep disorders.
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1 |
1992 — 1993 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba/Benzodiazepine/Barbiturate Preceptors in Epilepsy @ University of California Los Angeles
This project investigates the possible involvement of the GABA/benzodiazepine/barbiturate receptor complex (now termed the GABAA receptor) in the pathophysiology of epilepsy. We propose that epileptogenesis may in some cases involve an aberration of normal plasticity mechanisms in which GABA receptors are altered at the level of subunit gene expression or at the level of protein functional regulation in response to some environmental stress. GABAA receptors and also excitatory amino acid receptors are to be compared in human focal epilepsy patients and normal and in an animal model of epilepsy in which rats are 'kindled' with GABA antagonist convulsant drugs. We have found that GABA receptors are lower than normal in midbrain of gerbils and mice with genetic susceptibility to generalized seizures. We have found a loss of GABA receptors in hippocampus and a structural reorganization of GABA receptors in dentate gyrus of human patients undergoing surgery for focal temporal lobe epilepsy. We have found in another project that GABA receptor properties and subunits are altered following chronic intermittent administration of ethanol to rats, producing repeated withdrawals, followed by a long-lasting hypersensitivity to seizures elicited by GABA antagonists. We propose that repeated over-activity of GABA synapses may produce an adaptive plastic response that could increase seizure susceptibility, i.e., a unified theory of epileptogenesis. Receptor density, distribution, and pharmacological properties will be studied by Northern blot analysis and radioligand binding autoradiography in sections from cortical and hippocampal brain tissue surgically removed from patients with partial complex seizures, and from chemically kindled rats compared to normal. Receptor function including pharmacological subtype characterization and second messenger/phosphorylation will be assessed by brain slice and membrane homogenate 36C1 flux assays, and by electrophysiological studies on GABA and EAA-elicited currents in Xenopus oocytes following injection of mRNA from brain tissue of human epilepsy patients and normal and chemically kindled rats. The proposed work in animals and human brain should further understanding of basic mechanisms and possible new therapies for the epilepsies.
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1 |
1994 — 2003 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba-a Receptor Structure and Function @ University of California Los Angeles
This project will study the structure and function of the receptor protein for the major brain inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The GABA-A receptor complex is a ligand-gated chloride channel which is also the site of action of many important anticonvulsant, anxiolytic, and anesthetic drugs. The GABA-A receptor is actually a family of receptors, differing in gene sequence, subunit composition, pharmacological properties, and brain regional distribution. We can purify the receptor protein in large quantities by affinity chromatography. We will produce subunit-, subtype-, and functional domain-specific antibodies against synthetic peptides in the subunit sequences derived from cloning. Different subunits detected by gel electrophoresis of the purified receptor will be matched with different cDNA clones using clone-specific antibodies in Western blots and comparing regional location. The pharmacological specificity of ligand binding to GABA, benzodiazepine, barbiturate, and convulsant sites on the isolated subunits detected by photoaffinity labeling will be matched with brain regional ligand binding detected by autoradiography. This will allow us to determine the subunit composition, pharmacological properties, and cellular location of the receptor subtypes. The importance of receptor subtypes is that they vary in their function, susceptibility to disease processes, and sensitivity to pharmacological intervention. Domain-specific antibodies and active site sequencing of photolabeled protein will define functional domains within the protein structure. The purified protein will be reconstituted into membrane vesicles to study functional regulation, including phosphorylation by protein kinase C, which we have demonstrated in vitro on a specific A subunit. The information gained will be valuable in the understanding of normal and diseased brain function. Human disorders of particular relevance include epilepsy, anxiety, alcohol dependence, and sleep disorders.
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1 |
1995 — 2000 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba a Receptor Structure and Function @ University of California Los Angeles
The GABAA receptors are ligand-gated chloride ion channels formed as heteropentamers from 17 subunit possibilities. Of the thousands of potential combinations, only a few, 15-20 isoforms of such complexes occur in nature. The relationship between the subunit composition/ stoichiometry and functional characteristics is poorly defined. Our work seeks a) to analyze the structural basis of receptor heterogeneity; b) to determine what controls assembly of subunits into oligomers; c) to identify subunit specificity of biological regulatory mechanisms involving zinc, phosphorylation, and neurosteroids; d) to define functional domains within the subunits; and e) to test models of structure, using expression of recombinant receptor cDNAs in heterologous host cells. We employ traditional systems such as frog oocytes, and a new technology using Baculovirus shuttle vectors to test several subunit combinations and site-directed mutagenesis in insect cell line Sf9 expression. We also use mouse mutants to prove pharmacological biological roles of individual subunits. We will test the hypothesis that isoforms of GABAA receptors with different subunit composition have different biological functions/ regulatory mechanisms, and coincidentally pharmacological specificity. Using electrophysiology and binding assays, we seek to identify recombinant receptor subunit combinations that reconstitute functional subtypes observed in nature using binding, in vitro functional assays, an subunit-specific antibodies to isolate protein isoforms. For example, isoforms differ in their sensitivity to GABA, to modulatory drugs, to endogenous regulatory mechanisms including neurosteroids, zinc, and phosphorylation. We will also test the hypothesis that there is a subunit specificity and functional domain sequence specificity that controls assembly of subunits into functional membrane channel oligomers. We will test the hypotheses that a portion of the extracellular domain contains parts of the GABA and benzodiazepine binding sites on two beta- strands that surround an alpha-helix, a secondary structural element involved in channel gating; and that the binding sites for GABA, benzodiazepines, and anesthetics (barbiturates, steroids, and volatile agents) are all allosterically coupled to each other and to the structural element of the extracellular domain of the protein that mechanically moves the ion channel into open and shut conformations. This will involve some carefully chosen site-directed mutagenesis to identify amino acids involved in functional domains and test structural models deduced from our previous results. In addition, structural biochemistry on the receptor protein in Sf9 cell membranes is made possible by the overexpression of the genetically engineered system. Since the GABA A receptors are brain proteins that mediate the bulk of inhibitory communication in the brain, understanding of the molecular heterogeneity of GABA A receptors will be useful to knowledge of normal brain function as well as important disease processes.
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1 |
1997 — 2006 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Gaba Receptor Complex in Alcohol Depencence @ University of California Los Angeles
DESCRIPTION: (Adapted from Applicant's Abstract) The objectives of this study are to determine whether persistent alterations in the GABAA receptor complex (GABAR) can provide a molecular explanation for the development of physical dependence on ethanol in an animal model of alcoholism. Chronic intermittent ethanol (CIE) administration to rats has many features resembling human alcohol abuse behavior, including long-lasting susceptibility to readdiction. The numerous episodes of ethanol-induced depression of the nervous system and the following rebound hyperexcitability (withdrawal) have been shown to exert a kindling-like effect, i.e., a persistent increased severity of the hyperexcitable withdrawal symptoms. Rats treated in this manner become ethanol dependent, one measure being a decreased seizure threshold to the convulsant drug pentylenetetrazol (PTZ), a blocker of the GABAR. The hyperexcitability to PTZ (kindling) lasts at least 40 days after cessation of ethanol. Neurochemical and electrophysiological studies have been focused on whether this ethanol withdrawal kindling can be associated with alterations in the molecular properties of the GABAR, and have demonstrated a significant reduction in GABAR function. In addition, several pharmacological properties of GABAR are altered in hippocampus, although tolerance to eth enhancement of GABAR by ethanol was not found. We showed that GABAR are positively regulated by protein kinase C and neurosteroids Measurements of receptor subunit mRNA by reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization techniques reveal significant changes in isoform composition, with elevated levels of alpha4 and gamma2short subunits. In combination with protein biochemistry studies, we can compare naive and CIE rats in order to define the subunit isoforms present. Then we express the different hippocampal GABAR recombinant subunit isoforms in Xenopus oocytes and study what physiological consequences might arise from changing the subunit composition. We are testing initial suggestions that hypoinhibition may result from reduced positive modulation of GABAR by PKC and neurosteroids. We suggest that reduced GABAR function in the hippocampus of ethanol-dependent individuals has profound effects on various emotional and intellectual aspects of brain activity. Finding the molecular mechanisms responsible may help in treatment of withdrawal symptoms and hopefully in reduction of ethanol dependence.
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1 |
1998 — 2002 |
Olsen, Richard W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Benzodiazepine-Induced Gabaa Receptor Plasticity @ University of California Los Angeles
The GABA inhibitory synaptic system plays a major role in the central nervous system and is implicated in human neurological and psychiatric disorders such as epilepsy, stress, anxiety and panic disorders, sleep disorders, and drug dependence, especially to benzodiazepines and ethanol. The major postsynaptic GABA receptors involved in rapid inhibitory neurotransmission are the GABA/A receptors (GABA). GABAR proteins are subject to regulation at the level of transcription, translation, assembly, cell targeting, and the functional level. Endogenous regulation includes modulation by phosphorylation, zinc ions, and neuroactive steroids. GABAR are the known target of numerous clinically relevant drugs, including anti-epileptic anti-anxiety, and sedative/hypnotic/aesthetic agents. These include the widely used benzodiazepines, barbiturates, and possibly alcohol. GABAR are widely accepted as the major candidate molecular target of general anesthetic action. Their predominant role in the brain makes GABA likely players in the normal plasticity mechanisms that accompany ordinary and extraordinary experiences. By subjecting rats, or in some cases, cells, to somewhat extraordinary experiences that are considered to involve GABAR, we will investigate whether plastic changes in GABAR occur and the molecular and cellular mechanisms of the long-term modifications. In particular, chronic exposure of rats to benzodiazepines, but probably an elevation of GABAR function, leads to tolerance, especially to the anti-epileptic actions of these drugs. Tolerance is accompanied by a reduced GABAR function, reduced enhancement of GABAR function by benzodiazepines, and uncoupling of GABA- benzodiazepine binding measured in vitro. Tolerance to benzodiazepines can be mimicked in cells expressing recombinant GABAR that lack normal transcriptional control, and can be reversed rapidly by exposure in rats and in cells by exposure to the benzodiazepine antagonist flumazenil. This strongly suggests that the tolerance and reversal result from a physicochemical modification of the GABAR protein itself. This project will attempt to unearth this molecular mechanisms of plasticity. Ultimately therapeutic strategies could be based on our studies, aimed rationally at preventing the unwanted or pathological alterations in GABA/A receptors characteristic of several neurological and psychiatric disorders.
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1 |
1998 — 2002 |
Olsen, Richard W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Plasticity of Gaba Receptors @ University of California Los Angeles
In this program project application, a group of three independent faculty at the same institution, UCLA, have combined expertise in electrophysiology, neuroanatomy, biochemistry, and molecular biology to approach important questions in basic neurobiology which would be difficult for any one individual. The them chosen for collaborative research is "Plasticity of GABA Receptors". Program Director Richard Olsen has assembled, like the structure of GABA/A receptors themselves, a "heterooligomer" of scientists. Olsen's component project is 'Benzodiazepine-induced GABA/A receptor plasticity'. Carolyn Houser's project is 'Plasticity of GABA/A receptor subunit localization', and involves the kindling model of chronic epilepsy in rats. All projects focus on GABA/A receptors, the major postsynaptic receptors involved in rapid inhibitory neurotransmission. These receptors, and 'plastic' alterations in them that occur in response to a variety of extraordinary experiences, are implicated in many neurological and psychiatric disorders. The wide variety of employed techniques promises a level of investigation aimed at understanding the assembly, functioning, and plasticity of GABA/A receptors in the mammalian nervous system. It is both hoped and expected that the proposed studies will serve as a leading inquisitive collaboration to unveil the short and long-term control of inhibition in the mammalian brain. The proposed studies deal with the nature of the alterations in the molecular structure and function of GABA/A receptors that contribute to chronic changes in excitability of neurons or to the mechanism of tolerance and withdrawal from chronic drug exposure. Ultimately, therapeutic strategies could be based on our studied, aimed rationally at preventing the unwanted or pathological alterations in GABA/A receptors characteristic of several neurological and psychiatric disorders.
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1 |
1998 — 2000 |
Olsen, Richard W |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Research Training in Pharmacological Sciences @ University of California Los Angeles |
1 |
1999 — 2002 |
Olsen, Richard W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Sites of Anesthetic Action On Gaba a Receptors @ Massachusetts General Hospital
This program project seeks to identify the sites of action of general anesthetics on protein targets in the brain, concentrating on the ligand- gated ion channel super-family of neurotransmitter receptors, this project deals with the inhibitory GABA/A receptor-chloride ion channel (GABAR), the major candidate molecular target for the general anesthetics. As with all members of the super-family, GABAR are heteropentamers, comprised of three different sorts of subunits chosen from a repertoire of 18. This results in about two dozen different isoforms which differ in pharmacological properties. Different sensitivity to anesthetics of GABAR of varying subunit composition allow the use of the genetically engineered site-directed chimerae and mutagenesis to identify amino acid residues involved in anesthetic modulation. Others recently have identified several residues in the membrane-spanning domains; we will carry out a series of site-directed mutagenesis studies on the N-terminal extracellular domain of GABAR subunits of varying sensitivity to modulation by anesthetics in order to identify which residues are responsible. More direct identification of anesthetic binding sites can be obtained by photoaffinity labeling with radioactive anesthetics and determination of the amino acid residues that are covalently bound by microsequencing. This study will employ recombinant GABAR expressed in the insect Sf9 cell line using Baculovirus. In particular we will develop a new radioactive anesthetic steroid photoaffinity label for the GABAR work. The importance of labeled residues will be verified by mutagenesis and analysis of anesthetic sensitivity using binding and electrophysiology. The combination of these approaches, and comparison with sites of interaction in other members of the receptor super-family especially nicotinic acetylcholine receptors, will lead to a greater understanding of the mechanism of action of these clinically important drugs.
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0.909 |
2005 — 2009 |
Olsen, Richard W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Plasticity of Gaba-Mediated Inhibition @ University of California Los Angeles
DESCRIPTION (provided by applicant) In this renewal application of the program project (revised), two additional faculty at the same institution, UCLA, have combined efforts with the ongoing three investigators. Continuing are Drs. Richard Olsen, Carolyn Houser, and Istvan Mody. New are Drs. Michael Fanselow and Tom Otis. The Program Project is named "Plasticity of GABA-mediated Inhibition". The research brings together expertise in biochemistry, behavior, electrophysiology, neuroanatomy, and molecular and cell biology. Program members will combine to approach important questions in basic neurobiology which would be difficult for any one individual. Several important topics in which plasticity of GABAergic inhibition plays a crucial role will be addressed. Each set of studies will involve interactions among several investigators as indicated: 1) Effects of neurosteroids on GABARmediated inhibition;2) Effects of ethanol on specific types of GABAR-mediated inhibition;3) Neuronal plasticity following withdrawal of GABAR modulators;4) Post-translational modulation and trafficking of GABAR;5) Homeostatic neuronal plasticity; 6) GABAR-mediated inhibition in learning;7) Involvement of extrasynaptic alpha4/6-beta-delta subunit-containing GABAR-mediated tonic inhibition in the action of neurosteroids, ethanol, and anesthetics, and in various sorts of plasticity. Components in the program also will examine hippocampal inhibition, with a focus on alpha4delta-containing GABAR, and aspects of inhibition in hippocampus and cerebellum in the GABAR delta subunit knockout mice and other genetically engineered animals. One project will consider the balance of excitation and inhibition during and after plasticity-inducing activities with various time frames. Its predominant role in the brain makes GABA a major player in the normal plasticity mechanisms that accompany experiences. By subjecting rodents, or in some cases, cells and slices, to somewhat extraordinary experiences that are considered to involve GABA, we will investigate the molecular and cellular mechanisms of the long-term modifications resulting. Better understanding of plasticity phenomena involving GABA has high relevance to normal brain function and to disease processes and may suggest treatments. Most relevant are epilepsy, stress, anxiety, and panic disorders, sleep disorders, and drug dependence (alcohol and benzodiazepines). The complexity of the proposed experimental procedures requires the expertise of a team of investigators, each with a wide spectrum of analytical tools. Together, these tools yield a powerful combination of experimental approaches vested in the group rather than in any single team member. All members of the Program Project are committed to working together to successfully bridge the gap between behavior and molecular events in the CNS.
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1 |
2005 — 2009 |
Olsen, Richard W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Scientific @ University of California Los Angeles |
1 |
2005 — 2009 |
Olsen, Richard W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Mechanisms of Ligand-Induced Gaba Receptor Plasticity @ University of California Los Angeles
The objectives of this component are to analyze the molecular mechanisms of chronic GABAergic ligand-induced plasticity in the central nervous system (CNS). Various plasticity-inducing treatments produce changes in levels of the GABAA receptor (GABAR) subunit alpha4 and its associated partners, gamma2 or 8, accompanied by altered sensitivity to endogenous neurosteroids. In this program, we have determined that alpha4beta3delta subunit-containing GABAR are extrasynaptically localized, involved in tonic inhibition, and the major target of modulation of CNS excitability by neurosteroids and anesthetics including ethanol, while being insensitive to benzodiazepines. In particular we showed by several lines of evidence that the alpha6beta3delta subtype of GABAR is very likely the target of action of ethanol in the cerebellum. This important discovery needs substantiation and forms the rationale for Aim I. We suggest that the alpha4/6beta3delta GABAR are both susceptible to plasticity and positioned to regulate excitability via neurosteroid-modulated tonic inhibition. In this project Aim II we will compare and contrast changes in GABAR following chronic steroid (extrasynaptic delta subunit-containing target) and chronic BZ (synaptic gamma2 subunit-containing target). In drug-treated mice, we will measure biochemically the levels of GABAR subunits in hippocampus and cerebellum and partnering of subunits using antibodies, as well as binding assays for GABAR levels and allosteric modulation indicative of subunit switches. In drug-treated primary cultured hippocampal neurons, we will measure the content of cell surface GABAR subunits and subunit partners using biotinylation. Plastic changes following chronic GABAergic drugs are relevant not only to the development of tolerance to clinically useful agents like benzodiazepines (BZ) as well as withdrawal and dependence, but also to the control of excitability by the GABA system. This in turn is critically important to normal CNS information processing and changes that occur in response to usual or unusual experiences (plasticity), including epileptogenic phenomena.
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1 |
2008 — 2012 |
Olsen, Richard W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gaba(a) Receptor Complex in Alcohol Dependence @ University of California Los Angeles
[unreadable] DESCRIPTION (provided by applicant): The objectives of this study are to determine whether persistent alterations in the GABAA receptor complex (GABAR) can provide a molecular explanation for the development of physical dependence on ethanol in an animal model of alcoholism. Chronic intermittent ethanol (CIE) administration to rats has many features resembling human alcohol abuse behavior, including long-lasting susceptibility to readdiction. The numerous episodes of ethanol (EtOH)-induced depression of the nervous system and the following rebound hyperexcitability (withdrawal) have been shown to exert a kindling-like effect, i.e., a persistent increased severity of the hyperexcitable withdrawal symptoms. Rats treated in this manner become EtOH-dependent, one measure being a decreased seizure threshold to the convulsant drug pentylenetetrazol (PTZ), a blocker of the GABAR. The hyperexcitability to PTZ (kindling) lasts at least 40 days after cessation of EtOH. The CIE rats exhibit elevated anxiety, show tolerance to the sedative action of EtOH and cross-tolerance to other sedatives, and impaired hippocampal memory. Neurochemical and electrophysiological studies have been focused on whether this ethanol withdrawal syndrome can be associated with alterations in GABAR, and have demonstrated a significant reduction, specifically in the hippocampal formation, in GABAR function, as well as multiple alterations in the molecular properties of GABAR. We showed a restructuring of GABAR subunit composition consistent with changes in electropharmacology of GABAR-mediated synaptic and extrasynaptic tonic currents. These biochemical and physiological changes appear relevant to the altered behaviors. The same persistent alterations seen in CIE are also observed transiently after a single administration of an intoxicating dose of EtOH. In future we propose to study the molecular and cellular mechanisms whereby this GABAR plasticity develops and how it becomes persistent. In addition to acute and chronically EtOH-treated rats we will extend the model to mice to allow studies on genetically engineered animals with altered GABAR to help determine their role in developing dependence. We suggest that reduced GABAR function in ethanol-dependent individuals has profound effects on various emotional and intellectual aspects of brain activity. Finding the molecular mechanisms responsible may help in treatment of withdrawal symptoms and hopefully in reduction of ethanol dependence. This type of mammalian animal model seems to have great potential for uncovering important insights into abuse mechanisms. In addition, our studies on animal models of alcoholism will allow families and health professionals' better understanding of what environmental and genetic factors contribute to the susceptibility for alcohol abuse, of the behavioral changes of the alcohol abuser, and of possible behavioral modification and medications to consider in treating the disorder. PUBLIC HEALTH RELEVANCE: This project studies the cellular and molecular mechanisms of alcohol dependence in a rodent model in hopes of developing therapeutics for prevention and treatment of alcoholism. Rats and mice are given chronic intermittent ethanol (CIE) and studied for changes in inhibitory neurotransmission in brain involving receptors for the neurotransmitter 3-aminobutyric acid (GABA). The amounts, locations, and functions of the GABA receptors are related to the behavioral changes seen in alcohol dependence such as hyperexcitability, increased anxiety, sleep disturbances, and seizure susceptibility. [unreadable] [unreadable] [unreadable]
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1 |
2013 — 2019 |
Olsen, Richard W Wallner, Martin (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Unique Pharmacology of Ligand Sites On Delta Subunit-Containing Gaba-a Receptors @ University of California Los Angeles
DESCRIPTION (provided by applicant): Alcoholism is the most common form of substance abuse and has enormous economic burden on the United States and more than 100,000 deaths per year, with countless additional examples of contributory medical problems. Such tremendous costs have motivated intensive research efforts to understand how this drug affects brain function. Yet despite these efforts, there is no consensus as to how ethanol acts at a neuronal level, and no effective medical therapeutic treatment for alcohol abuse disorders is currently available. It is known that ethanol enhances the function of GABAA receptor-mediated signaling in brain and that plastic changes in GABAA receptor (GABARs) subunits (most notably the delta subunit) contribute to the behavioral alterations produced by chronic alcohol use and abuse leading to dependence. Previously evidence has been provided that delta GABARs, which give rise to the extrasynaptic or tonic inhibition, are sensitive to modulation by ethanol at low millimolar concentrations achieved in human social drinking. While these findings provide an explanation for decades of accumulated evidence that inhibitory GABARs are involved in mediating EtOH effects, there has been controversy over the unique of alcohol-sensitivity of these receptors in recombinant expression systems. Preliminary data show (for the first time) that recombinant receptors containing delta subunits are not only uniquely sensitive to EtOH and GABA but also that delta co-expression leads to receptors fractions with a rather dramatic (~1000-fold) increase in sensitivity for the GABA structural analogs THIP/gaboxadol and muscimol. The hypothesis is that in recombinant expression systems there are proteins missing that promote efficient expression surface expression of delta-GABARs. Therefore it is intended to use this property of high THIP sensitivity to screen for delta subunit-binding proteins identifid by a state of the art proteomic/mass spectroscopy approach using immune-affinity purified receptors protein, in order to find potential delta subunit partner proteins that promote cell surface and potentially modulate the function in other interesting ways. The fact that the azido group in the imidazobenzodiazepine EtOH antagonist Ro15-4513 is a photoaffinity will allow the identification of amino acid residues in the EtOH/Ro15-4513 binding site on delta GABA receptors. Based on a detailed structural model hypothesis, the exact amino acid residues that form the alcohol site on delta GABARs will be confirmed and verified using recombinant expression and mutagenesis. This work will help lead to a better understanding of how alcohol and sedative-hypnotic drugs affect brain function. The molecular level identification of ethanol targets is a prerequisite for the development of rational therapies to treat alcohol- related cognitive impairment and alcohol addiction.
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