A Leslie Morrow - US grants

Several lines of evidence have suggested that GABAA receptor activity may be involved, at least in part, in the pharmacologic and behavioral actions of ethanol. The purpose of the present proposal is to examine the role of ethanol in modulating the expression of GABAA receptor subunits in the rat brain. Measurement of GABAA receptor mRNA levels is the most sensitive method for detecting alterations in the expression of receptor subunits. The first goal is to ascertain if acute or chronic ethanol administration alters the level of GABAA receptor subunit mRNAs in the cerebral cortex. Preliminary evidence suggests that alpha1 subunit mRNA levels are modulated by acute as well as chronic ethanol exposure. Prolonged ethanol inhalation selectively reduces the levels of GABAA receptor alpha1 and alpha2, but not alpha3 subunit mRNAs in the rat cerebral cortex. This is a specific effect of ethanol since there is no change in the levels of total RNA, ribosomal RNA, polyadenylated RNA or mRNA levels for glutamate decarboxylase or beta- actin. The next goal is to study the effects of acute and chronic ethanol administration on GABAA receptor mRNAs in various brain regions where the composition of GABAA receptor subunits differ from the cerebral cortex. If ethanol administration has differential effects on the various GABAA isoreceptors, there may be regional differences in the effect of ethanol on GABAA receptor subunit mRNA levels. The third goal is to determine whether the effects of ethanol on GABAA receptor alpha subunit mRNA levels are mediated by the receptor. These studies will test whether the effect of ethanol in rat cerebral cortex is dose-dependent and reversible by a GABA receptor antagonist, bicuculline or the benzodiazepine receptor inverse agonist and ethanol antagonist, Ro15-4513. Further, we will investigate whether alterations in intracellular Ca++ or cAMP are involved in the rapid reduction in mRNA levels. The final goal of these studies is to determine whether genetic differences in ethanol withdrawal seizure sensitivity are correlated with an alteration in a) the expression of GABAA receptor subunit mRNAs or b) the effect of ethanol on the levels of GABAA receptor subunit mRNAs. These experiments will test the hypothesis that the structure or the regulation of the GABAA receptor is different in ethanol withdrawal seizure prone and withdrawal seizure resistant rats. These studies will provide the foundation for a systematic investigation of the genetic basis for differences in behavioral sensitivity and preference for ethanol at the level of gene regulation. The results of these studies should have important implications for our understanding of the neurobiology of ethanol tolerance and withdrawal and may lead to an understanding of the causes of alcoholism.

DESCRIPTION: We have shown that ethanol dependence is associated with a reduction in GABAA receptor-mediated chloride channel function that may contribute to the development of physical dependence upon ethanol. The mechanism of these functional effects does not appear to involve a reduction in the number of GABA receptor-gated chloride channels. Prolonged ethanol exposure selectively and differentially alters the expression of several GABAA receptor subunit mRNAs and polypeptides in cortex and cerebellum. We have proposed that alterations in GABAA receptor subunit composition may be an important mechanism that underlies the reduction in GABAA receptor function and the development of dependence upon ethanol. Our first aim is to determine if chronic ethanol administration alters the subunit assembly of GABAA receptors using a combination of quantitative RT-PCR analysis of mRNA levels, Western blot analysis of polypeptide levels and immunoprecipitation studies with selective antibodies. Nuclear run-off experiments will be conducted to determine if changes in gene expression can be attributed to alterations in transcription rates. The next aim is to study the effects of chronic ethanol administration on GABAA receptor subunit expression in various brain regions where GABAA receptors differ in their sensitivity to ethanol. Studies of GABAA receptor expression at the cellular level are proposed using immunobinding assays on rat brain tissue sections. Our third goal is to investigate whether genetic differences in ethanol withdrawal seizure sensitivity are correlated with an alteration in a) the expression of GABAA receptor subunits in ethanol-naive mice or b) the effect of chronic ethanol administration on GABAA receptor subunit expression. Differences in GABAA receptor expression between the selected lines will then be subjected to genetic analysis to determine if the changes segregate with withdrawal seizure sensitivity. Our final goal is to investigate whether ethanol-induced alterations in GABAA receptor subunit expression are responsible for alterations in GABAA receptor function and CNS excitability. We plan to use selective antisense oligonucleotides to determine whether specific alterations in GABAA receptor expression in vivo are associated with the same alterations in GABAA receptor function and behavioral sensitivity that are observed with ethanol dependence. Future studies will address the same question in recombinant expression systems where the receptor subunit composition can be more precisely controlled. The results of these studies should have important implications for our understanding of the neurobiology of ethanol dependence and withdrawal.

The objective of this proposal is to explore the potential role of endogenous neuroactive steroids in the development of ethanol dependence. We propose to investigate whether the potent GABA/A receptor neuroactive steroid, 3alpha-hydroxy-5alpha pregnan-20-one (3alpha-5alpha-THP) prevents the development of the negative symptoms associated with ethanol withdrawal and thereby alters the development of ethanol dependence. Preliminary evidence from our lab has demonstrated that 3alpha-5alpha-THP protects against bicuculline-induced seizures in ethanol-dependent rats. 3alpha-5alpha-THP may influence the development of the negative symptoms of chronic ethanol intake which may alter the development of ethanol dependence. 3alpha-5alpha-THP is present in brain of male and female rats (107,110). The levels of 3alpha-5alpha-THP in brain during acute stress in male rats (110) and during proestrus and estrus in female rats (101) are sufficient to modulate GABA/A receptor function in brain. Epidemiological studies have shown that the men have substantially higher rates of alcohol dependence than women between the ages of 20-50 (17,50,51). Higher levels of neurosteroids in female brain may play a role in the development of alcoholism and explain why the incidence of alcoholism in women is significantly lower than men during the years of normal menstruation. The first goal of this proposal will be to determine whether chronic ethanol administration causes sensitization to the effects of 3alpha-5alpha-THP in vivo and in vitro. We propose to investigate whether 1) 3alpha-5alpha-THP alters bicuculline-induced seizure susceptibility in ethanol-dependent rats; 2) chronic ethanol administration alters 3alpha-5alpha-THP potentiation of GABA/A receptor function and binding to GABA/A recognition sites in cerebral cortex and 3) chronic ethanol administration alters endogenous 3alpha-5alpha-THP levels in blood and brain. The second goal will be to determine whether 3alpha- 5alpha-THP and ethanol co-administration alters the development of ethanol dependence. The effect of chronic co-administration of 3alpha-5alpha-THP levels in blood and brain will be measured at the termination of each experiment. These studies will test the hypotheses that chronic ethanol administration sensitizes rats to the anti-dependence. The results of this investigation will extend our knowledge of the potential role of neurosteroids in ethanol dependence and may identify new factors which are involved in the etiology of alcoholism.

The purpose of this component of the ARC is to investigate the role of GABAergic neurotransmission, including neurosteroid modulation, in the initiation and maintenance of ethanol self-administration in rats. There is increasing behavioral evidence that mesolimbic GABA systems are involved in voluntary ethanol self-administration. However, there is a significant gap in our knowledge of the molecular mechanisms in brain that underlie alcohol self-administration and preference. Since we have previously found evidence for alterations in GABAa receptor sensitivity and expression in cortex following prolonged ethanol consumption, we propose to investigate the GABAergic mechanisms involved in mesolimbic brain regions following prolonged ethanol self-administration. These studies will delineate the role of GABAa, receptor function and gene expression in the control of ethanol self-administration in the nucleus accumbens, ventral tegmental area, amygdala and prefrontal cortex. We also propose to elucidate the role of neurosteroid modulators in ethanol self-administration. We plan to determine if the initiation and/or maintenance of ethanol self-administration alters endogenous neurosteroid synthesis or metabolism and if direct neurosteriod administration to the lateral ventricle, or mesolimbic brain sites alters ethanol self- administration. Previous studies have demonstrated gender differences in ethanol self-administration in the rat that could be related to gender differences in neurosteroid levels. We have also found differential effects on cerebral cortical GABAa receptor alpha1 subunit expression in female versus male rats. We propose to determine whether gender modulate GABAa receptor function, expression or neurosteriod metabolism in the GABAergic mesolimbic circuitry during initiation or maintenance of voluntary ethanol consumption. Finally, the effects of alterations in GABAa receptor expression on ethanol self-administration will be measured using vector-mediated gene delivery. This component is designed to test the overall hypothesis that ethanol self-administration is influence by differences in GABAa receptor function and/or neurosteroid levels in the nuclear accumbens, amygdala, ventral tegmental area and prefrontal cortex. We predict that ethanol self- administration will alter GABAa receptor sensitivity and neurosteroid levels in the mesolimbic circuitry of brain. These alterations will, in turn, regulate ethanol self-administration and contribute to the development of ethanol addiction. We predict that gender related differences in these systems will correlate with neurosteroid modulation of GAMAa receptors. Using vector-mediated gene transfer, we expect to be able to manipulate ethanol self-administration behavior, showing promise for new therapeutic approaches to alcoholism. These studies will make significant contributions to our understanding of etiology and pathogenesis of alcoholism.

The objective of this proposal is to explore the potential role of endogenous 3a-hydroxy-5a-pregnan-20-one (3a,5a-THP) in the electrophysiological and behavioral actions of ethanol, including the development of ethanpl tolerance. We recently discovered that ethanol administration to rats produces an elevation in plasma and brain levels of the potent GABAA receptor neuroactive steroid. 3a,5a-THP. to pharmacologically relevant concentrations. The effects of ethanol on 3a,5a-THP levels in cerebral cortex are time and dose dependent and sufficient to potentiate GABAA receptor function. Moreover, there is a strong correlation between ethanol sleep time and cerebral cortical levels of 3a.5a-THP. In contrast, brain levels of 3a,5a-THP are not altered by acute ethanol challenge in ethanol dependent rats, therefore, tolerance may develop to the effect of ethanol on the induction of 3a,5a-THP. The loss of ethanol induction of 3a,5a-THP levels may underlie tolerance to the pharmacological effects of ethanol. Therefore, we propose to test the overall hypothesis that 3a,5cc-THP mediates pharmacological effects of ethanol in vivo. The first goal is to investigate the role of 3a,5a-THP in the behavioral and neurophysiological effects of ethanol. 3a,5cc-THP formation will be prevented by pretreatment steroid biosynthesis inhibitors and the effects of ethanol on neuronal firing rates, GABAA receptor-mediated inhibition of spontaneous neuronal activity, intoxication, aerial righting reflex and seizure thresholds will be measured. The role of 3a,5a-THP will also be investigated in conditional knock-out mice that lack 3a-hydroxysteroid dehydrogenase - the final step in 3a,5a- THP formation. The second aim will determine if 3a,5oc-THP plays a role in the development of tolerance to ethanol using both knock out mice and steroid biosynthesis inhibitors. The third aim will focus on the mechanisms of 3a,5cc-THP accumulation following ethanol administration. Studies will be conducted to determine if ethanol directly alters the activity of the 3a,5cc-THP biosynthetic enzymes. Preliminary results suggest that ethanol may increase 3a,5a-THP biosynthesis, while high dose ethanol may release or uncover a "store" of 3a,5oc-THP. The effect of ethanol on 3oc,5a-THP release from cultured astrocytes will be measured. These studies will address the effects of ethanol at rapid time points (seconds to minutes) that may be relevant to the electrophysiological actions of ethanol. These studies may elucidate a new mechanism of ethanol action in the CNS and explain why the effects of ethanol on GABAergic neurotransmission could not be adequately explained by the direct action of ethanol at GABA.4 receptors. The results of this investigation will extend our knowledge of the potential role of neurosteroids in ethanol action and ethanol tolerance and may identify new factors involved in the etiology of alcoholism.

The overall goal of this proposal is to elucidate the molecular mechanisms that underlie alterations in gamma-aminobutyric acid (GABA)A receptor adaptations that influence the development of ethanol dependence. Ethanol has several sites of action in the brain, but direct or indirect modulation of GABA/A receptors may behavioral actions of ethanol. Moreover, prolonged ethanol consumption results in the development of tolerance and dependence upon ethanol. Withdrawal from ethanol, and particularly repeated withdrawals from ethanol, produce marked increases in CNS excitability and anxiety. Substantial evidence suggests that these behavioral and neural adaptations involve marked adaptations in the pharmacological properties of GABA/A receptors. Furthermore, research over the previous funding period has established that GABA/A receptor submit adaptations accompany these changes and differ markedly across brain regions. We plan to focus on the role of PKCgamma and PKCepsilon in mediating GABA/A receptor adaptations. We hypothesize that PKC interactions with GABA/A receptors may determine receptor subunit adaptations and may underlie the regional differences in these differences in these adaptations. Specific Aim 1 will determine if ethanol dependence alters the association of GABA/A receptors with PKC isozymes in alter ethanol-induced adaptatins in GABA/A receptor function and seizure susceptibility. Specific Aim 2 will utilize these mice to determine if PKCgamma and PKCepsilon differentially alter the effects of ethanol on membrane expression and internalization to alter specific GABA/A receptors. The final aim will investigate the role of PKCgamma and PKCepsilon in the phosphorylation state of GABA/A receptors, again using mutant mouse models. Vector-mediated gene delivery for tissue specific rescue of PKCgamma or PKCepsilon in vivo will be used to establish a cause and effect relationship between the alterations in PKC and subsequent effects on receptor membrane expression, internalization and function. Tissue specific rescue of PKCgamma or PKCepsilon as well as pharmacological challenge with PKC antagonists will also be used to control for the possibility that adaptations of other proteins contribute to the effects of genetic deletion of PKCgamma or PKCepsilon. We predict that these experiments will delineate specific GABA/A receptor adaptations involved in ethanol dependence-induced enhancement of seizure susceptibility (bicuculline seizure threshold) and ethanol self-administration (in collaboration with Clyde Hodge). These studies will provide important mechanistic information on the molecular basis of ethanol-induced adaptations in GABA/A receptors that influence the development of ethanol tolerance and dependence.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this proposal is to explore the relationship between stress response, alcohol drinking and neuroactive steroid levels in primate plasma. A large body of evidence in rodent models suggests that GABAergic neuroactive steroids contribute to ethanol sensitivity, tolerance, protection against dependence and reduces excessive alcohol consumption. Primates synthesize different steroid precursors and exhibit 5preductase activity that necessitates the development of a highly sensitive GCMS assay to measure both 3

DESCRIPTION (provided by applicant): The goal of this project is to systematically delineate the effects of chronic intermittent ethanol (CIE) exposure on GABAergic neuroactive steroids and their regulation in limbic brain areas after stress challenge. Both neuronal and extracellular neuroactive steroid levels will be examined by immunohistochemistry as well as gas chromatography-mass spectroscopy (GC-MS) analysis of microdialysates and/or tissue specimens, allowing investigation of limbic brain regions for the first time. We propose to test the hypotheses that ethanol exposure dysregulates basal or stress-induced levels of these steroids and these changes are related to ethanol consumption. We will further explore genetic contributions to adaptations in neurosteroid levels or responses. The first aim will determine the effects of CIE on neuronal vs. extracellular 3a,5a-THP levels in limbic/reward brain areas, including the prefrontal cortex, nucleus accumbens, amygdala, and bed nucleus of the stria terminalis of C57BL/6J mice. Aim 2 will delineate the effect of acute stress challenge on neuronal vs. extracellular 3a,5a-THP in limbic brain areas and the effect of stress challenge following five cycles of CIE in C57BL/6J mice. Aim 3 will determine if basal or CIE-related neuroactive steroid levels in limbic/reward brain areas are correlated with ethanol preference drinking across BXD mouse strains. Aim 4 will extend and compare our studies on neuroactive steroid adaptations to CIE in mice to primates. We will delineate the effects of prolonged ethanol drinking on 3a,5a-THP levels in limbic brain areas of rhesus monkeys. Our results will be integrated with changes in neuronal function, neurochemistry, gene expression and ethanol drinking behavior studies by our collaborators under the same experimental conditions. These studies will elucidate the effects of ethanol on GABAergic neuroactive steroids in limbic regions of brain and provide novel insights into the role of basal and stress-induced neuroactive steroids in various allostatic adaptations to chronic intermittent ethanol exposure.

The pathological consequences of ethanol dependence that involve adaptations of GABA-A receptors include heightened CNS excitability, seizures, tremor, anxiety, and cognitive deficits. We have shown that the GABA-A receptor adaptations involved are dependent upon PKC gamma and moderated by protein kinase A (PKA). The goal of this project is to systematically delineate the protective role of PKA in ethanol dependence pethology mediated by GABA-A receptors and to determine the cellular localization of GABA-A receptor adaptations in cortical circuits. The first aim will examine PKA regulation of GABA-A receptor trafficking and synaptic function in cultured cortical neurons. We will investigate whether pharmacological activation of PKA or inhibition using siRNA alters ethanol-induced internalization of GABA-A al receptors and increased surface expression of a4 receptors. The second aim will determine if PKA modulates the ethanol dependence phenotypes of GABA-A receptor trafficking, increased seizure susceptibility or withdrawal-induced anxiety-like behavior in vivo. PKARlip mutant mice (PKARlip-/-) will be utilized since they exhibit complete loss of PKARlip expression and 50% loss of the A kinase anchoring protein (AKAP150), producing phenotypes with GABA-A receptor dysfunction. We will examine receptor surface expression, seizure thresholds and withdrawal-induced anxiety-like behavior. The final aim will identify the cortical neurons that exhibit ethanol-induced alterations in the trafficking and function of GABA-A al and a4 receptors and test their sensitivity to PKA activity. We plan to determine the effects of chronic ethanol exposure on GABA-A al and a4 receptors using specific markers for GABAergic interneurons (parvalbumin) or pyramidal projection neurons (Cam Kinase II) via dual label confocal microscopy. Electrophysiological responses will be determined by voltage clamp recording within slices and the effects of PKA activation will be determined in these cells. These studies will delineate the role of PKA in GABA-A receptor adaptations associated with ethanol dependence and identify intracortical pathology that results from GABA-A receptor adaptations to chronic ethanol exposure. This work will elucidate specific targets for reversal of ethanol dependence pathopathology.

Project Summary Animal and human studies suggest that elevation of neuroactive steroids may address many of the behavioral pathologies associated with alcohol use disorders. The goal of this project is to evaluate the hypotheses that elevated steroidogenesis in the ventral tegmental area will reduce operant ethanol self-administration and the escalation of voluntary drinking following deprivation in male and female alcohol preferring (P) rats. Endogenous neuroactive steroids will be elevated by viral vector-mediated gene delivery of the biosynthetic enzyme P450scc that converts cholesterol to pregnenolone. Our recent studies demonstrate that vector-mediated delivery of P450scc to the VTA reduces ethanol self-administration and increases local expression of (3?,5?)-3-hydroxypregnan-20-one (3?,5?-THP, allopregnanolone) (Cook et al., 2014). We now propose to extend these studies by examining effects in both male and female P rats, probing effects on deprivation-induced drinking and targeting the vector to tyrosine hydroxylase (TH) neurons in the VTA. We will examine vector and behavioral specificity as well as the persistence of effects. Aim 1 will investigate if elevation of steroidogenesis by gene delivery of P450scc to VTA alters A) operant ethanol self-administration in non?dependent male and female P rats or B) deprivation-induced drinking in ethanol dependent male and female P rats. Aim 2 will examine whether TH neuron-specific elevation of steroidogenesis in VTA alters A) operant ethanol self-administration in non?dependent male and female rats or B) deprivation-induced drinking in ethanol dependent male and female P rats. These studies will increase our understanding of the role of VTA neuroactive steroids in ethanol reinforcement, anxiety-like behavior and escalated drinking following ethanol deprivation.

Numerous studies have implicated adaptations in GABA signaling as a major factor in the pathogenesis of alcohol dependence. Despite the established actions of ethanol on inhibitory control and GABAA receptors, little is known about ethanol adaptations in GABAA receptor expression and signaling that occur in the medial prefrontal cortex (mPFC) and central amygdala (CeA), interconnected brain regions that are implicated in the development of alcohol dependence. Previous work has demonstrated significant adaptations in GABAA receptor expression in cerebral cortex, cultured cortical neurons and CeA neurons, however the impact of these changes on local microcircuitry and functional connectivity of the mPFC and CeA remain unclear. The overarching goal of this component is to examine the role of GABAA receptor adaptations in overall circuit function of mPFC and CeA following chronic ethanol exposure using electrophysiological, biochemical, and molecular methods. We will further investigate the impact of histone deacetylases (HDACs) as the underlying mechanism governing the GABAA receptor adaptations to determine the utility of HDAC inhibition as a potential therapeutic target to selectively reverse pathological changes in GABAA receptor expression and circuit function. We will also examine the effect of chronic ethanol exposure on functional connectivity between mPFC and CeA using resting state functional magnetic resonance imaging (fMRI). Collectively, the proposed studies will delineate molecular and cellular mechanisms of ethanol dependence in local mPFC and amygdalar microcircuits as well as the mPFC projection to amygdalar subregions. These studies may lead to microcircuit-specific molecular targets for reversal of ethanol dependence pathology.