Ruth E. Siegel - US grants

The benzodiazepines have found wide therapeutic use in the treatment of anxiety and other clinical disorders. While neuronal benzodiazepine receptors have been described, much remains to be learned concerning this receptor and its mechanism of action. To study the benzodiazepine receptor, cDNAs that direct its expression have been identified. In this proposal, the characteristics and expression of a cloned cDNA and its use in understanding benzodiazepine receptor structure and function will be explored. Initial experiments will be aimed at ensuring that the cDNA encodes a central benzodiazepine receptor. The binding properties and size of the protein expressed in transfected cells will be compared with those of the benzodiazepine receptor in brain membranes. In addition, the tissue distribution of mRNAs complementary to the cDNA will be determined by Northern blot analysis and in situ hybridization histochemistry. This will establish that the mRNA is present in appropriate brain regions. Once the identity of the cDNA has been established, it will be characterized in detail. By analyzing its DNA sequence, the complete amino acid sequence of the receptor protein will be determined. This information will provide a basis from which to examine receptor structure and function. Further studies using Southern and Northern blot analysis will determine the number of genes and mRNAs directing receptor biosynthesis. These studies will provide insight into the basis of receptor heterogeneity in the central nervous system and probe the relationship between central and peripheral receptor types. In this proposal, cellular and molecular biological approaches will be used to characterize the benzodiazepine receptor. These studies will enhance our understanding of drugs of great clinical importance.

biomedical equipment purchase; freezing; ultracentrifugation; biomedical automation;

The brain is composed of many different nerve cells (neurons) that interact through an intricate series of circuits. Proper functioning of the brain requires appropriate cellular wiring and precise cell-to-cell interactions. This communication between cells is achieved by the release of a transmitter chemical from one neuron that acts at a specific site, the receptor, on the target neuron. The distribution of several different transmitters and their corresponding receptors have been mapped in the central nervous system. One important receptor in the brain mediates the actions of gaba- aminobutyric acid (GABA). GABA is the major inhibitory transmitter in the brain, and the binding of GABA to its receptor decreases the activity of the target neuron. Although the receptor is present on at least 30-40% of all neurons in the brain, its exact structure and regulation remain unknown. Recent studies indicate that it is composed of multiple protein subunits, each of which is actually a family of closely-related proteins. These studies suggest that multiple GABA receptors, composed of different subunit subtypes, may exist in different brain regions. To gain a better understanding of GABA receptor structure and function, a number of studies are being performed. First, studies on the adult brain will determine whether all GABA receptors are identical or whether receptors having different subunit compositions are localized at different sites. Second, studies on the developing brain will investigate whether receptor composition changes as the nervous system matures. Finally, additional studies will investigate how cell-cell interactions regulate GABA receptor composition. Knowledge of this complex and prominent receptor system will enhance our understanding of normal brain function.

This proposal will examine GABA/A receptor subunit composition and assembly in cerebellar neurons in vivo and in culture. the GABA/A receptor is a multisubunit, ligand-gated ion channel that mediates the actions of gamma-aminobutyric acid, the major inhibitory neurotransmitter in the central nervous system. Recent studies have shown that the receptor is composed of several subunits, most of which are encoded by families of genes. Each of the subunit genes exhibits a distinct cellular and temporal pattern of expression in the cerebellum. While only one isoform of a specific subunit is expressed in some cerebellar populations, multiple subunit isoforms are present in other cell types. These results, coupled with electrophysiological findings, raise the possibility that cerebellar neurons express multiple receptor subtypes having diverse subunit compositions and functional properties. However, the subunit composition of native receptors and the process by which receptors are assembled remain unknown. To elucidate GABA/A receptor subunit composition and assembly in identified cerebellar neurons, a number of studies will be performed. First, receptor subunit expression in cerebellar cells in vivo and in culture will be examined using subunit-specific antisera. changes in subunit levels during postnatal development will be assessed by Western blot analysis. In addition, subunit distribution in granule and Purkinje neurons will be examined by immunohistochemistry. These studies will begin to define GABA/A receptor subunit composition in identified cell populations and characterize developmental changes in receptor expression. Second, the subunit composition of cerebellar GABA/A receptors will be determined using immunoprecipitation with receptor subunit-specific antisera. These studies will elucidate which subunits from different classes and which subunit isoforms from a single class are coassembled into a receptor complex. Finally, the assembly of GABA/A receptors in cultured cerebellar granule neurons will be examined using metabolic labeling followed by immunoprecipitation with subunit-specific antisera. These studies will determine whether receptor subunits exhibit comparable rates of biosynthesis and will examine the process of subunit assembly into oligomeric receptor complexes. Together, these studies will yield important new information concerning GABA/A receptor subunit composition and assembly in identified cerebellar cell populations. Since the different subunits confer distinct physiological properties to the receptor, knowledge of subunit composition is essential for an understanding of GABA/A receptor function throughout the central nervous system.

This proposal will examine GABA/A receptor subunit composition and assembly in cerebellar neurons in vivo and in culture. the GABA/A receptor is a multisubunit, ligand-gated ion channel that mediates the actions of gamma-aminobutyric acid, the major inhibitory neurotransmitter in the central nervous system. Recent studies have shown that the receptor is composed of several subunits, most of which are encoded by families of genes. Each of the subunit genes exhibits a distinct cellular and temporal pattern of expression in the cerebellum. While only one isoform of a specific subunit is expressed in some cerebellar populations, multiple subunit isoforms are present in other cell types. These results, coupled with electrophysiological findings, raise the possibility that cerebellar neurons express multiple receptor subtypes having diverse subunit compositions and functional properties. However, the subunit composition of native receptors and the process by which receptors are assembled remain unknown. To elucidate GABA/A receptor subunit composition and assembly in identified cerebellar neurons, a number of studies will be performed. First, receptor subunit expression in cerebellar cells in vivo and in culture will be examined using subunit-specific antisera. changes in subunit levels during postnatal development will be assessed by Western blot analysis. In addition, subunit distribution in granule and Purkinje neurons will be examined by immunohistochemistry. These studies will begin to define GABA/A receptor subunit composition in identified cell populations and characterize developmental changes in receptor expression. Second, the subunit composition of cerebellar GABA/A receptors will be determined using immunoprecipitation with receptor subunit-specific antisera. These studies will elucidate which subunits from different classes and which subunit isoforms from a single class are coassembled into a receptor complex. Finally, the assembly of GABA/A receptors in cultured cerebellar granule neurons will be examined using metabolic labeling followed by immunoprecipitation with subunit-specific antisera. These studies will determine whether receptor subunits exhibit comparable rates of biosynthesis and will examine the process of subunit assembly into oligomeric receptor complexes. Together, these studies will yield important new information concerning GABA/A receptor subunit composition and assembly in identified cerebellar cell populations. Since the different subunits confer distinct physiological properties to the receptor, knowledge of subunit composition is essential for an understanding of GABA/A receptor function throughout the central nervous system.

DESCRIPTION (provided by applicant): The long term goal of our studies is to gain insight into mechanisms mediating the effects of alcohol. Alcoholism is a chronic disorder that afflicts at least 1 million Americans and exacts a cost of over 166 million dollars per year (NIAAA data) in alcohol-related expenses. Despite its prevalence and cost to society, the molecular mechanisms underlying the actions of ethanol have not been fully elucidated. Recent studies suggest that ethanol produces many of its acute and chronic effects in the CNS through the 5HT3 and GABA-A ligand-gated neurotransmifter receptors. Although each of these receptors alone can mediate some of the actions of ethanol, both receptors are expressed in some neuronal populations. The possibility that cross-talk between these receptors mediates some of the actions of ethanol has not yet been studied. Although much is known about the GABAA receptor and its responses to ethanol, little is known about the 5-HT3 receptor or how it modulates the actions of ethanol. To investigate the importance of 5HT3 receptors in mediating the actions of ethanol, information concerning its subunit composition, distribution, function, and interactions is essential. To determine receptor subunit composition and function, studies will be performed on transfected cells expressing the 5HT3A subunit in the presence and absence of the newly discovered 5HT36 subunit. These studies will determine whether subunit coexpression alters receptor properties and will provide insight into the role of the B subunit. Next, the possibility that the 5HT3 receptor subunits can interact with GABA-A receptor subunits in transfected cells will be determined. Finally, since the hippocampus is involved in mediating the cognitive effects of alcohol, and 5HT3 and GABA-A receptors are coexpressed in this region, studies will be performed to characterize receptor subunit distribution, colocalization and interactions in hippocampal neurons in culture. Together, these studies will provide novel information concerning 5HT3 receptor composition and will provide tools for future research to elucidate its function in mediating the actions of alcohol.

DESCRIPTION (provided by applicant): Our long term goal is to understand how plasticity of the GABAA receptor, a pentameric ligand-gated ion channel, participates in maintaining CNS function. The proposed studies will test the hypothesis that changes in expression of two GABAA receptor subunits, ?6 and ?4, participate in mediating the response to reduced oxygen. In previous studies on adult rats we found that sustained hypobaric hypoxia selectively triggers de novo ?6 subunit mRNA and protein expression and increases ?4 and ? subunit mRNA levels in the pons, a brainstem region involved in maintaining homeostasis. Induction of ?6 expression is notable because this subunit normally is found only in the postnatal cerebellum. More importantly, ?6 or ?4, in other brain regions, coassemble with ? in extrasynaptic GABAA receptors that mediate the response to basal levels of GABA, i.e., tonic inhibition. To investigate the role of GABAA receptor plasticity in the response to sustained hypoxia, molecular, histological, and physiologic studies on wild-type and ?6 or ?4 subunit-deficient mice are proposed to: 1) Determine how sustained hypobaric hypoxia alters GABAA receptor subunit mRNA expression in the brainstem of mice maintained in control or hypoxic conditions using qRT-PCR;2) Locate cells that express the ?6 and ?4 subunit polypeptides and determine whether changes in subunit levels alter receptor number in the brainstem following sustained hypoxia using immunohistochemical and biochemical approaches;and 3) Determine the importance of ?4 or ?6 GABAA receptor subunit expression for the ventilatory response to sustained hypoxia using whole-body plethysmography. Findings from these studies will begin to define molecular mechanisms used in adapting to reduced oxygen, a stress that occurs physiologically at high altitude and in several pathological conditions, including chronic obstructive pulmonary diseases. Specifically, the proposed studies will provide insight into the contribution of GABAA receptor plasticity. Our findings will lay the groundwork for studies aimed at understanding the circuitry involved in maintaining brain function. Identifying the molecular machinery involved in the response to sustained hypoxia is an essential first step for creating novel therapeutic approaches to combat a variety of potentially life-threatening conditions. PUBLIC HEALTH RELEVANCE The ability of the mature brain to adapt to environmental stress is required for survival. Our recent findings raise the possibility that plasticity of the GABAA receptor, which mediates the actions of the major inhibitory neurotransmitter in the brain, participates in mediating adaptation to hypoxia. Oxygen deprivation is a stress that occurs during many physiological and pathological conditions, including life at high altitude and chronic obstructive pulmonary diseases. The goal of our proposed studies is to demonstrate that changes in the expression of specific GABAA receptor subunits are required for adaptation to hypoxia. Identifying the molecular machinery involved in the response to hypoxia is an essential first step for creating novel therapeutic approaches to combat a variety of potentially life-threatening situations.