Neil L. Harrison - US grants

The objective of this project is to understand the molecular basis of the modulation of the gamma-aminobutyric acidA (GABAA) receptor by commonly used inhaled anesthetics such as isoflurane ('Forane') and sevoflurane. The approach involves combining the techniques of electrophysiology and site-directed mutagenesis. The project proposes to consolidate and extend our recent findings concerning specific mutations within the human GABAA receptor that abolish receptor regulation by the anesthetic enflurane. Mutant GABAA receptors made up of of alpha2beta1 subunits will be studied, in which either alpha2 or beta1 subunits carry key mutations (e.g. alphaSer 270 His, alphaAla 291 Trp). Isoflurane and sevoflurane effects will be investigated in these mutant receptors. The role of the gamma2 subunit will then be studied, first by including this subunit with a mutant alphabeta combination, and then by mutation of the gamma2 subunit itself. The effect of varying the beta subunit isoform will then be studied by mutating the neuronally expressed beta2 and beta3 subunits at residues Asn265 and Met286, to compare with data previously obtained on the glial beta1 subunit. The role of Ser 270 and Ala 291 in the alpha2 subunit will then be further investigated by performing extensive mutagenesis of these residues to determine whether hydrophobicity, hydrogen bonding ability, or molecular size is a key feature of these critical amino acid residues. Finally, detailed information about other critical residues within TM2 and TM3 will be provided by site-directed mutagenesis of neighboring residues of Ser 270 and Ala 291. In all mutant receptors, both efficacy and potency of each of the inhaled anesthetics will be measured. Elucidation of the molecular site of action of inhaled anesthetics will lead to improved understanding of the pharmacology of these agents and the role of the GABAA receptor in achieving their desirable and undesirable effects.

Dr. Harrison plans to develop his present studies on the cellular pharmacology of general anesthetic interactions with gamma-aminobutyric acid (GABA)-A receptors, and to pursue exciting new avenues utilizing single channel and molecular biological methods to uncover the basic mechanisms by which general anesthetics modulate GABA-A receptors. Specifically, the aims of the proposed research project will be to determine whether a range of general anesthetics interact with the GABA-A receptors of hippocampal neurons, and to investigate the pharmacological correlation between the ability of various agents to enhance the function of GABA and their ability to induce surgical anesthesia. The project will proceed to study the effects of the anesthetics on the kinetics of inhibitory synaptic currents mediated by GABA, and attempt to explain such effects by investigations at the level of single GABA-activated chloride channels in outside-out membrane patches. Finally, the project will utilize some of the recent advances in the molecular cloning of GABA-A receptors, by utilizing a transient expression system to study the pharmacology of GABA-A receptors of known subunit composition.

The objective of this project is to understand the molecular basis of the modulation of the gamma-aminobutyric acid(A) (GABA/A) receptor by the volatile anesthetics, enflurane, halothane and isoflurane, using the techniques of electrophysiology and molecular biology. The project proposes to first extend pharmacological studies of anesthetic modulation of the GABA(A) receptors of hippocampal neurons, in order to evaluate the hypothesis that there exists a correlation between anesthetic potency and drug potency for modulation of hippocampal GABA(A) receptors. Non- anesthetic compounds will also be tested. Single channel studies of the mechanisms of volatile anesthetic modulation of the GABA(A) receptor will also be performed. The GABA(A) receptor is a member of the 'family' of ligand-gated chloride channels. To begin to investigate the molecular basis of GABA(A) receptor modulation by volatile anesthetics, the effects of these agents will be studied on recombinant human GABA(A) receptors of known subunit composition. We will study heterooligomeric receptors of alpha/Beta, alpha/Beta/gamma, alpha/gamma and, if possible, homomeric receptors consisting of alpha subunits. In addition, the possible modulation by volatile anesthetics of other members of the ligand-gated chloride channel 'family', including the structurally related human glycine receptor, the human p1 subunit ('GABA(C) receptor') will be investigated, to determine whether all members of the ligand-gated chloride channel family are university modulated by volatile anesthetics, i.e., whether there are specific structural requirements for volatile anesthetic action at the GABA(A) receptor. Chimeric receptors will be constructed in order to gain further information about the nature of the sites for volatile anesthetic action on the human GABA(A) and glycine receptor subunits. Finally, more detailed information will be provided by using the techniques of site-directed mutagenesis to alter individual amino acid residues within the ligand-gated chloride channel subunits. Analysis of the molecular site of action of volatile anesthetics may lead to the development of anesthetic 'antagonists' which would be invaluable to clinical practice.

DESCRIPTION: Central nervous system (CNS) depression caused by ethanol is a complex phenomenon which may involve several neurotransmitter systems. The present study aims to probe in detail the molecular mechanisms by which ethanol perturbs glycine receptor function. A strong case can now be made for the involvement of glycine and gamma-aminobutyric acid (GABA) in the CNS depressant actions of ethanol; enhancement of the actions of these inhibitory transmitters is consistent with the sedative and anesthetic effects of ethanol. The glycine and GABA receptors are related members of the 'superfamily' of ligand-gated ion channels. Homomeric glycine alpha1 or alpha2 receptors, like native glycine receptors, are quite sensitive to modulation by ethanol; the actions of glycine are potentiated by ethanol. The homomeric GABA receptor formed by the beta1 subunit is strongly inhibited by ethanol. This proposal aims to study the molecular mechanism of glycine receptor modulation by ethanol, by using a series of chimeric receptors by exchange of large or small domains of the human glycine receptor alpha2 and human GABA receptor gamma1 subunits. The hypotheses to be tested are: 1) that the human glycine receptor cc subunit possesses specific structural features which permit enhancement of agonist action by ethanol, and 2) that specific amino acid residues can be identified in the glycine receptor .2 subunit that are permissive for ethanol modulation. It is important to investigate the mechanisms of action of ethanol in order to have knowledge of the molecular mechanisms of this most widely abused drug.

DESCRIPTION: (Adapted from the applicant's abstract) The objective of this project is to understand the molecular basis of the modulation of the GABAA receptor by three clinical intravenous anesthetic agents--propofol, etomidate, and methohexital. The strategy is to employ recombinant human receptors expressed in human embryonic kidney (HEK 293) or immortalized quail fibroblast (QT6) cells and to use whole cell patch clamp to record responses to transmitter application. There are four specific aims: 1: To test the hypothesis that the a subunit is important for modulation of receptor function by examining drug effects in heterooligomers in which the b and g subunits are constant and the a isoform is varied. 2. To examine the actions of these agents on related members of the ligand-gated chloride channel superfamily, including the glycine receptor and the r1 (GABAC) receptor, in order to determine the structural requirements for anesthetic action. 3. To construct chimeric receptors with GABAA a or b and r1 receptor portions to refine the specific structural features required for anesthetic action to portions of the respective subunits. 4. To pursue the structural requirements to the single amino acid level by constructing point mutations of the GABAA receptor using site-directed mutagenesis.

genotype; biomedical facility; genetically modified animals; laboratory mouse; single strand conformation polymorphism; gene targeting; southern blotting; polymerase chain reaction; nucleic acid sequence; restriction fragment length polymorphism; karyotype;

DESCRIPTION (taken from application): The objective of this proposed program project is to advance understanding of the cellular and molecular mechanisms of action of general anesthetics at both pre- and postsynaptic sites, with particular emphasis on isoflurane and propofol. The program consists of four projects linked together by a Scientific and Administrative Core. In Project 1, quantitative structure-activity relations will be established among a group of propofol analogs for loss of righting reflex in Xenopus laevis tadpoles, and compared with that for the modulation of recombinant gamma-aminobutyric acidA (GABAA) receptors. In Project 2, the effects of isoflurane and propofol on presynaptic vesicle fusion and endocytosis will be studied in cultured hippocampal neurons, using both optical and biochemical techniques. In Project 3, interactions of isoflurane and propofol with specific amino acid residues in the GABAA receptor will be probed using the substituted cysteine accessibility method. The Scientific and Administrative Core will serve to organize and administer twice-yearly meetings of the group, together with a Scientific Advisory Board and guest speakers, synthesize novel analogs of propofol for study in Projects 1 and 3), and measure the concentrations of isoflurane and propofol present in the experimental salines at the time of the physiological measurements made in each project. The proposed program will focus on the molecular sites of action of general anesthetics at central synapses. Such information will aid in further characterization of the multiple targets underlying the complex in vivo pharmacology of general anesthetics, and may lead to the development of safer agents.

DESCRIPTION (provided by applicant): The objective of this research project is to study the regulation of expression of the a4 subunit of the GABAA receptor (GABAA-R), which shows a degree of plasticity that is remarkable among the family of GABAA-R subunits, with a striking increase in expression during a variety of hyper-excitability syndromes, such as withdrawal from chronic intermittent ethanol (CIE) exposure. The genetic elements responsible for the control of expression of the mouse GABAA-R a4 subunit will be studied. The specific aims of the present proposal are: 1) To clone the gene encoding the mouse GABAA-R a4 subunit (GABRA4), including the putative 5'-regulatory domain, and to characterize the gene structure and exon/intron boundaries. 2) To investigate the transcriptional start sites for mRNA species encoding the GABAA-R a4 subunit. 3) To investigate and characterize putative silencer, enhancer, initiator and/or positional regulator element/s in the mouse a4-subunit gene. Expression of the a4 subunit is limited to specific populations of neurons and is absent in non-neuronal cells - this may be due to the existence of one or more neuron-restrictive silencer elements in the 5'-regulatory domain of the gene. 4) To develop a cell line or primary neuronal culture system to study the regulation of expression of the GABAA-R a4 subunit, that will recapitulate key features of the induction of this gene in vivo in models of hyperexcitability. Repeated administration of alcohol, either to neurons in primary culture, or to a cell line model, results in induction of expression of the a4 subunit, and that this increased expression can be regulated by the inhibition of specific signaling pathways associated with transcriptional regulation. The completion of these Aims will increase our knowledge of a GABAA-R subunit that is associated with behavioral hyperexcitability and shows substantial plasticity. This knowledge will be useful in the study of transcriptional mechanisms involved in the induction of GABAA-R subunit expression, and potentially in designing interventions aimed at interrupting genomic mechanisms associated with withdrawal hyperexcitability.

[unreadable] DESCRIPTION (provided by applicant): The objective of this research project is to study the regulation of inhibition by alcohol in the mouse thalamus. The thalamus is well known to act as a relay of sensory information to the cortex and to play an important role in the regulation of sleep. Thalamocortical relay neurons in the ventrobasal (VB) complex exhibit a bi-stable pattern of excitability. In the "tonic firing" mode (when the neuron is depolarized), the neurons fire continuously, while in the "burst firing" mode (when the neuron is hyperpolarized), VB neurons fire brief rapid bursts of action potentials superimposed on a slow (delta) rhythm of about 3-5Hz that is a feature of slow wave sleep and absence epilepsy. VB neurons receive inhibitory inputs from GABAergic neurons in the peripheral reticular nucleus (RTN), which results in the activation of synaptic GABAA receptors (GABAA-R) and the generation of fast IPSPs in VB neurons. In addition to these 'phasic' inhibitory events, VB neurons also show 'tonic' inhibition, due to the persistent activation of extra-synaptic GABAA-R. This tonic inhibition generates a constant hyperpolarizing influence on the VB neurons. It has been hypothesized that both synaptic and tonic inhibition can have a strong influence on the timing and synchronization of "burst firing" in the relay neurons. We propose to carry out a comprehensive study of the interactions of alcohol with GABA in the thalamus. Although a variety of GABAAsubtypes exist in the thalamus, synaptic inhibition involves a, and y2 subunits in VB and cc3, f33 and Y2 subunits in RTN. Tonic inhibition in VB is generated by receptors containing ct4 and 5 subunits, a population of GABAA-Rs that is suggested to be highly sensitive to modulation by alcohol. The specific aims of this revised proposal are: 1) To study the effects of alcohol on GABA-mediated inhibition in VB and RTN neurons. 2) To study the effects of alcohol and GABAA-R antagonists on signal processing by VB neurons. 3) To investigate the mechanisms of the effects of alcohol on inhibition in VB and RTN neurons. Acute and chronic use of alcohol is known to disrupt sleep, so the detailed investigation of alcohol effects in the thalamus should provide insights that could direct future studies into sleep disorders in alcoholics and assist in the development of useful therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Drinking alcohol results in a variety of short-term behavioral changes and long-term physiological adaptations that can lead to tolerance and physical dependence. Chronic exposure to alcohol has been reported to produce loss of neurons and white matter within the brain. Alcohol is known to trigger the release of cytokines and reactive oxygen species (ROS) from astrocytes and may have similar effects in microglia (MCG), as well as decreasing the synthesis of myelin by oligodendrocytes (OLGs). Alcohol regulates gene expression, and distinct sets of alcohol-responsive genes (ARGs) are activated by acute alcohol. We recently discovered, in the Gabra4 gene, an 11 base pair sequence element essential for the induction of gene transcription by alcohol - the alcohol response element (ARE). Stimulation of Gabra4 by alcohol involves the heat shock pathway, causing heat shock factor 1 (HSF1) to bind to the ARE (Pignataro et al., 2007). The ARE is present in a large number of other neuronal ARGs. Alcohol also activates the heat shock pathway in astrocytes. We now propose to extend these studies to OLG and MCG cells. Our overall hypothesis is that alcohol alters the expression of genes critical for the function of glial cells via activation of the heat shock pathway. The specific aims are: Aim 1) To study the activation of the heat shock pathway by alcohol in astrocytes. We will investigate activation of the heat shock pathway by alcohol in a mouse astrocyte cell line and in primary cultures of mouse astrocytes. We will measure the effects of alcohol on the expression of heat shock proteins (Hsps) and study the translocation of HSF1, and the interaction between HSF1 and the ARE in astrocyte nuclei. Aim 2) To identify alcohol-responsive genes in astrocytes. We will use a combination of bioinformatics and gene microarrays to identify novel ARGs in astrocytes, and then examine the subset of ARGs dependent on the ARE via the heat shock pathway. We will use pharmacological inhibitors of the heat shock pathway and RNAi to determine the involvement of HSF1. Aim 3) To study the effects of alcohol on gene expression in oligodendrocytes. We will study effects of alcohol on the expression of myelin- associated genes in an OLG cell line, investigate the effects of alcohol on expression of Hsps in OLGs and use pharmacological inhibitors to study the role of of HSF1 in the regulation of ARGs in OLGs. We will use bioinformatics, microarrays and Q-PCR to identify ARGs in OLGs. Aim 4) To study the effects of alcohol on gene expression in microglia. We will study the effects of alcohol on the expression of cytokine IL-1b (Il1b), the cytokine receptors, IL-1R1 (Il1r1) and TLR4 (Tlr4) and the enzymes iNOS (Nos2) and COX-2 (COX2) in a MCG cell line. We will investigate the effects of alcohol on the expression of Hsps in MCG and use pharmacological inhibitors to assess the role of HSF1. PUBLIC HEALTH RELEVANCE: Alcoholism is a major public health problem in the US. In the long-term, chronic drinking results in physiological changes that are detrimental to human health and have societal consequences. The brains of alcoholics become "rewired", and a part of this process results from changes in gene expression that occur in response to alcohol. We have shown that a cellular defense mechanism known as the heat shock pathway is activated by alcohol. In this proposal we plan to extend this work to study the changes within cells known as glia, the brain cells that support the activity of nerves within the brain. Astrocytes control the chemical environment of the nerve cells, and form a barrier between the brain and the blood. Oligodendrocytes form a sheath around nerve cells that speeds signal conduction within the brain. Microglia are the immune component of the brain and eliminate foreign invaders. Alcohol may alter the activity of all three types of glial cell by changing gene expression, and in doing so may harm nerve cells.

DESCRIPTION (provided by applicant): Drinking alcohol results in short-term behavioral changes and longer-term adaptations that lead to abuse and dependence. These adaptations are not understood, but a consensus suggests that, in accordance with the mechanism of all addictive drugs, an increase in the release of dopamine (DA) is associated with behavioral sensitization to alcohol and is related to the abuse liability of the drug. Although alcohol is known to enhance DA release in the nucleus accumbens (nAc), the mechanism is not known. In this proposal we will evaluate competing hypotheses for the action of alcohol on the DA system. The Specific Aims are: 1) to investigate the effects of alcohol on dopaminergic and GABAergic neurons in the VTA. The release of DA in the nucleus accumbens is activated by acute alcohol exposure in vivo. One possible explanation for this is that alcohol directly or indirectly increases the firing rate of DAergic neurons within the VTA, as is the case with other drugs of abuse, such as the opiates. We will investigate the effects of alcohol on identified populations of DAergic projection neurons and local GABAergic neurons in the VTA using GAD67-GFP and TH-GFP mice. The hypothesis to be tested is that acute alcohol activates the VTA, either by disinhibition (as with opiates) or by direct activation of DA neurons, which subsequently results in increased release of DA within the nAc. 2) To study the interactions between alcohol and dopamine release in the nucleus accumbens (nAc) We will use the newly developed technology of fluorescent false neurotransmitters (FFNs) to image individual DAergic terminals, and combined with the use of physiologically relevant stimulation parameters, compare these results with those using conventional cyclic voltammetry. The hypothesis to be tested is that alcohol increases DA release from a subset of DAergic neurons terminating within the nAc, possibly due to the differential presence of presynaptic GABAergic and/or cholinergic receptors. 3) To study the effects of alcohol on the activity of a population of cholinergic interneurons the in nAc and medium spiny neurons in the nAc. More than 90% of the cells in the ventral striatum are medium spiny neurons (MSNs), but several classes of interneurons also exist, including a population of cholinergic neurons, the activity of which strongly regulates evoked DA release in the nAc. The hypothesis to be tested is that alcohol enhances cholinergic neuron activity in the nAc.

DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Inhibition in the CNS as part of the Gordon Research Conference series to be held in Waterville, Maine, U.S.A. July 24-29, 2011. The broad and long-term goal of the Conference is to increase our understanding of the fundamental mechanisms of inhibitory synaptic transmission, the role of interneurons in regulating excitability and network activity. A significant emphasis is placed on interneuron diversity and how neuronal stem cells differentiate into interneurons, as well as the mechanisms by which drugs of abuse, including opiates and alcohol, influence inhibition in the CNS. The specific aims of this meeting will be to convene 40 speakers that represent critical areas of inhibition research with a total of ~150 participants for a five day conference in a relatively isolated setting. The program will have a keynote address and eight sessions that broadly address current issues in the biology of interneurons. Sessions are typically devoted to the anatomy and physiology of a special subset of neurons in the brain containing the inhibitory transmitter GABA, the function and regulation of inhibitory synapses, and the role these specific interneurons play in regulating and synchronizing the activity of neuronal networks, as well as the cell biology of the receptors gated by the inhibitory neurotransmitters GABA and glycine, and a session devoted to the development of interneurons from stem cells. Speakers will be diverse, with multiple nations featured, and younger investigators and women well represented. In addition, two evening poster sessions will permit all of the participants to interact and contribute to these topics. The significance of this application is that the Gordon Research conference on Inhibition in the CNS is a critical component of the yearly series of conferences that propel research in the international community of inhibition researchers. The health relatedness of this application is that the discussions at this meeting will define the questions that require experimental resolution in areas that affect human development, drug abuse liability and cognitive function. PUBLIC HEALTH RELEVANCE: The Gordon Conference on Inhibition in the CNS is a scientific meeting that is held on the campus of Colby College in Maine. 150 participants from around the world will gather for a five-day meeting, sleeping in dormitories and eating college dorm food, while spending time discussing their scientific work in an isolated setting that promotes conversations among experts who might not otherwise meet. About 40 lectures will be given, and junior scientists are encouraged to attend and to lead the discussion of the work presented. This year's meeting will highlight ground-breaking areas of brain science such as the modeling of human brain wave activity by computer simulations, the actions of drugs such as alcohol and sleeping pills on the brain, and the applications of stem cells for the treatment of human brain disorders. Many new and interesting scientific developments begin as informal conversations between scientists from different parts of the world, who have the unique opportunity at these small meetings to discuss their experiments together in a secure and protected environment that allows for imaginative and creative thought, far away from their university teaching and fund- raising responsibilities.

DESCRIPTION (provided by applicant): The consequences of alcohol abuse on the American public are profound, both in terms of individual well-being and impact on the family structure, as well as the enormous cost to society in terms of lost productivity and associated health care expenses. Despite increasing efforts, our understanding of the neurobiological mechanisms that underlie the effects of alcohol and the development of alcohol use disorders (AUD) remains incomplete. Epidemiological research has pointed to adolescence as a critical period in the development of alcohol disorders. The prefrontal cortex (PFC) is a brain region that is not yet mature at the onset of human adolescence and continues to develop during this period, during which some individuals may be highly susceptible to the effects of alcohol. The PFC mediates control over goal-directed behaviors and dysfunction of the PFC is thought to underlie compulsive drug-taking and relapse in substance abusers. Binge drinking is highly prevalent in adolescents, and episodes of high alcohol intake have been associated with decreased PFC activity and function (hypofrontality). Imaging studies suggest that hypofrontality persists in chronic alcohol abusers, and may therefore be a contributory factor in the development of AUDs and behavioral pathologies in adulthood. The underlying mechanisms of this PFC hypoactivity are unknown, and the development of robust animal models would therefore be useful in investigating the underlying changes in neuronal excitability. A better understanding of these changes would enable possible molecular and therapeutic interventions in order to prevent the development of alcoholism. One plausible mechanism for hypofrontality involves the depression of persistent activity, a mode of firing that can be observed in recordings from pyramidal neurons in the PFC of rodents. This type of activity is seen at more depolarized membrane potentials and is associated with performance in working memory tasks, and is dependent on the Ih current, which is mediated by a family of hyperpolarization-activated and cyclic nucleotide modulated (HCN) channels. We propose that chronic changes in persistent firing might result from prolonged alcohol exposure. To date there have been few detailed studies of excitability in the PFC after drinking and none in adolescent rodents. In three separate but integrated aims, we plan to test the overall hypothesis that the HCN1 channel that contributes to the Ih current in PFC PNs is important for the regulation of alcohol drinking, and specifically that (a) binge drinking of alcohol during adolescence inhibits persistent firing and excitability in the PFC via reduction of Ih and (b) reduction in HCN1 channel activity in layer 5 of PFC can mimic the effects of alcohol consumption during adolescence, while (c) activation or over-expression of HCN1 channels can restore persistent activity and normal levels of excitability in PFC of binge drinking adolescent animals.