Richard Olsen - US grants

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.

This proposal involves studies to develop methods for the total synthesis of the didemnin depsipeptides, a group of antibiotics isolated from Caribbean tunicates. The antibiotics are of interest due to their significant antiviral and antitumor activities at low dosage levels. The antibiotics are characterized by a 23-membered ring composed of L-threonine, the novel amino acid epistatine, a 4-hydroxy-3-keto-2, 5-dimethylhexanoic acid of unknown configuration, L-leucine, L-proline, and N,O-dimethyl-1-tyrosine. Various amino acid or peptide units attached to the alpha-amino group of L-threonine constitute the didemnins A, B, and C. The project will focus on the preparation, by methods that afford stereochemical control, of the novel 4-hydroxy-3-keto-2,5-dimethylhexanoic acid common to the antibiotics. The incorporation of this unit, along with the novel amino acid epistatine, into a linear depsipeptide corresponding to the cyclic portion of the antibiotic will be studied. Cyclization of the linear precursor to provide the cyclic component of the didemnins will be investigated. The cyclic depsipeptide will contain protection at the amino function of L-threonine, which upon deprotection will allow incorpoation of amino acid or dipeptide units at this position to furnish didemnins A, B, and C or analogues thereof. The synthetic didemnins will be evaluated for antitumor and antiviral activities.

The objectives of this program are to characterize the immunosuppressive properties of a retroviral envelope protein (p15E) and to determine its role in retroviral disease. Earlier, we demonstrated that FeLV p15E suppressed lymphocyte blastogenesis and lymphocyte membrane mobility (receptor capping) of cat, human, and dog peripheral blood lymphocytes (PBL). Recently, the protein was found to suppress the one-way mixed lymphocyte reaction and to inhibit proliferation of erythroid colony-forming cells but not granulocyte/macrophage colony-forming cells in vitro. Attempts to determine which specific cell types are affected have indicated that both B lymphocytes and macrophages are resistant, whereas T-cell functions appear sensitive. FeLV p15E did not interfere with B-cell immunoglobulin capping or macrophage production of interleukin 1, whereas it significantly affected Con A capping and interleukin 2 simulation, both T cell-dependent functions. The mechanism of the action of FeLV p15E is unknown; however, research with the prostaglandin and cyclic nucleotide systems has led to a possible answer. Using sensitive radioimmune precipitation assays to measure prostaglandin and cyclic nucleotide levels, we found that prostaglandin synthesis (PGE2, PGI2, PGF2, and TXB2) was not affected by FeLV p15E, whereas cyclic nucleotide (cAMP) levels were altered. Specific inhibitors of prostaglandin synthesis, indomethacin and nordihydroguaiaretic acid, reversed the FeLV p15E-induced suppression of lymphocyte blastogenesis and capping. Neither inhibitor had any effect on normal lymphocyte capping or blastogenesis. Indomethacin also increased capping and blastogenesis of lymphocytes from FeLV-infected (imunosuppressed) cats. Using other specific inhibitors of prostaglandin synthesis, we found that inhibitors of thromboxane synthesis and prostacyclin synthesis had no augmenting capabilities. Research to date indicates that modulation of AMP levels by FeLV p15E may mediate its immunosuppressive effect. This effect may be of secondary importance, in that altered cAMP levels may reflect changes caused by FeLV p15E at the level of the cell membrane. (IS)

The objective of this proposal is to determine the feasibility of developing an HTLV-I vaccine using methods which were successfully used in the feline leukemia virus system. The approach will be to use a HTLV infected cell product as the vaccine material. Tissue culture fluids from HTLV infected cells grown under appropriate conditions will be collected and concentrated. The concentrate will be tested for antigenicity, first in rabbits and then in subhuman primates. Antibody titers to HTLV-I will be measured in these animals and used as a criterion for determining the vaccine potency. Vaccination and challenge studies will be conducted in subhuman primates to determine immunoprophylaxis.

The objective of this project is to investigate the hypothesis that an alteration in inhibitory synaptic transmission mediated by Gamma-aminobutyric acid (GABA) receptors may contribute to the pathophysiology of some types of epilepsy. Human brain tissue from temporal lobectomies performed on focal epilepsy patients and frozen autopsy material from epileptic patients, especially those with genetic factors, will be compared to non-epileptic tissue. In addition, three genetic animal models will be studied, the seizure-suceptible gerbil, the audiogenic seizure-sensitive mouse, and the tottering mouse mutant. The affinities and densities of receptors for GABA and associated modulatory drug receptors for benzodiazepines and for barbiturates/convulsants will be quantitatively determined by appropriate radioactive ligand binding in membrane homogenates of finely dissected brain regions or in tissue slices by autoradiography. Quisqualate/AMPA-sensitive glutamate receptor binding will be assayed in parallel. Preliminary results show a deficit in levels of benzodiazepine receptor binding sites in the substantia nigra and mid-brain periaqueductal gray regions of seizure-sensitive gerbils, suggesting an impairment of GABA-mediated inhibition in these midbrain regions. Since the substantia nigra GABAergic system has been implicated in controlling seizures of various kinds, a deficit in GABA receptors could contribute to seizure susceptibility. This finding will be further analyzed in gerbils as a function of seizure severity and ontogenic development of seizures. Midbrain areas, especially s. nigra, will be emphasized in the studies on other animal models and on human generalized seizure patients, where available. Findings by others of altered GABA nerve endings (gutamate decarboxylase immunoreactive) and GABA receptor binding in human and animal focal epilepsy suggest that studies on GABA receptors in human temporal lobe epilepsy also should be worthwhile. Parallel studies of the PI on the purification of the GABA receptor complex will likely lead to the early development of antibodies for radioimmune assay and immunocytochemical localization of these receptors, as well as development of tools of modern molecular biology for noninvasive analysis of gene expression for GABA receptors in human clinical disorders including the epilepsies. Mammals are needed for this study to produce any reasona- ble approximation to epilepsy in man. Animals at UCLA are cared for according to NIH Guidelines.

Feline leukemia pathogenesis presents a well characterized model of retrovirus-mediated disease resulting in either a progressive chronic viremia with immune suppression and malignancy or a regressive disease without apparent retroviral damage. Factors other than virus inoculum, strain and age related susceptibility which lead to viral persistence in the host are obscure. In this multifaceted study, we propose to examine the critical immunological and nonspecific events which predispose the host to disease progression. The specific aims are: to examine the role of cell membrane FeLV-induced changes on cell function and cell-cell recognition interactions necessary for developing adequate host protection; to compare the immune response in progressor and regressor cats by studying phagocytic and intracellular killing mechanisms, production of immune modulators such as interferon and prostaglandins, and examining the role of immune complex formation on pathogenesis and immunomodulation; and last to ascertain whether transient or latent undetectable FeLV infection in cats produces long term hemolymphatic defects which may become evident during subsequent concomitant disease or immunosuppressive drug treatment. Methods for studying these parameters include: Percoll separation of hemolymphatic cells; antibody-ELISA on cells for identification or retroviral-induced cell membrane changes; cell affinity chromatographic separation of altered cells for functional studies; detection of Ia-like antigen expression and subsequent changes after infection by use of the feline MLR; titration of prostaglandin production and cellular levels of cAMP and cGMP by RIA; C1q ELISA assay for detection of circulating immune complexes; and the use of Staph Protein A columns for isolation of CIC for analysis and in vitro cell function assay, lymphocyte blast transformation and phagocytosis assays.

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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.

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.

The Diels-Alder cycloaddition reaction will be applied as a synthetic route for preparation of diaryl ethers. A major objective will be the preparation of optically active beta-(diaryl ether)-alpha-amino acids, such as isodityrosine and related amino acid units as present in the cyclic peptide antibiotics K-13, OF4949, bouvardin, and vancomycin. Enone and eynone dienophiles will be prepared from L- or D-serine derivatives. Cycloaddition with aryloxy functionalized 1,4-dienes and subsequent aromatization will provide the target diaryl ether amino acids. Model studies have demonstrated the potential of this method for the synthesis of isodityrosine derivatives in optically pure form. This approach also has the potential to lead to the synthesis of beta-hydroxy tyrosine derivatives, as present in the vancomycin family, by stereoselective reduction of an intermediate beta-keto tyrosine derivative. The synthesis of the peptide antibiotics K-13, OF4949-III, and the 14-membered cyclo(isodityrosine) unit of the bouvardins will be studied in which the cyclization step will involve a Diels-Alder cycloaddition to form the diaryl ether unit. These studies will involve preparation of the required tri- or dipeptide units containing in the N- and C-terminal side chains the diene and dienophile components needed for the intramolecular Diels-Alder reaction. Two routes to the synthesis of optically active phenylglycines by Diels-Alder methodology will be studied. These will involve (1) cycloaddition of 1,3-diaryloxy dienes with a serine-derived a, 6-unsaturated ester followed by a reaction sequence involving a Barton decarboxylation-phenylselenation leading to aromatic product, and (2) an inverse electron demand Diels-Alder method using a serine-derived a-pyrone and an aryloxy-substituted alkene. These methods will be applied for a synthesis of the core triaryl diether tripeptide unit of vancomycin.

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.

The Diels-Alder cycloaddition reaction will be applied as a synthetic route for preparation of diaryl ethers. A major objective will be the preparation of optically active beta-(diaryl ether)-alpha-amino acids, such as isodityrosine and related amino acid units as present in the cyclic peptide antibiotics K-13, OF4949, bouvardin, and vancomycin. Enone and eynone dienophiles will be prepared from L- or D-serine derivatives. Cycloaddition with aryloxy functionalized 1,4-dienes and subsequent aromatization will provide the target diaryl ether amino acids. Model studies have demonstrated the potential of this method for the synthesis of isodityrosine derivatives in optically pure form. This approach also has the potential to lead to the synthesis of beta-hydroxy tyrosine derivatives, as present in the vancomycin family, by stereoselective reduction of an intermediate beta-keto tyrosine derivative. The synthesis of the peptide antibiotics K-13, OF4949-III, and the 14-membered cyclo(isodityrosine) unit of the bouvardins will be studied in which the cyclization step will involve a Diels-Alder cycloaddition to form the diaryl ether unit. These studies will involve preparation of the required tri- or dipeptide units containing in the N- and C-terminal side chains the diene and dienophile components needed for the intramolecular Diels-Alder reaction. Two routes to the synthesis of optically active phenylglycines by Diels-Alder methodology will be studied. These will involve (1) cycloaddition of 1,3-diaryloxy dienes with a serine-derived a, 6-unsaturated ester followed by a reaction sequence involving a Barton decarboxylation-phenylselenation leading to aromatic product, and (2) an inverse electron demand Diels-Alder method using a serine-derived a-pyrone and an aryloxy-substituted alkene. These methods will be applied for a synthesis of the core triaryl diether tripeptide unit of vancomycin.

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.

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.

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.

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.

ABSTRACT NOT PROVIDED

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.

[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]

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.