Marlene Wilson - US grants

The benzodiazepines (BZs) are widely used for their anxiolytic, anticonvulsant, sedative and muscle relaxant properties, although their clinical usefulness is hampered by the development of tolerance to these various pharmacological effects during prolonged exposure. The actions of the benzodiazepines are mediated through binding to a high-affinity recognition site in the brain which alters the responsiveness of the inhibitory neurotransmitter GABA. Recently, several studies have demonstrated that steroid hormones or their derivatives can alter the GABA/BZ/ionophore complex and GABA- mediated responses. The proposed experiments are aimed at more closely examining the interactions between gonadal steroid hormones, the benzodiazepine receptor site and GABA responses in rats. I will examine the effects of gender, castration, hormone replacements and estrous cycle on the benzodiazepine recognition site and GABA modulation of this site. Several brain areas which are thought to mediate benzodiazepine responses and/or are target areas for steroid actions will be analyzed. Further, various hormonal manipulations will be tested for their ability to influence neuronal responses to GABA and/or benzodiazepine administration in rats. Using electrophysiological techniques, these studies will examine hormonal influences on neuronal activity and GABA sensitivity in two brain areas under differing GABAergic control; the substantia nigra pars reticulata and the dorsal raphe nucleus. Hormone effects on GABA responses will be analyzed in vivo and in brain slices to help elucidate the mechanisms by which steroid hormones can influence responses to prolonged benzodiazepine exposure in rats. These studies will examine if manipulating levels of gonadal hormones can alter some of the biochemical and physiological changes in GABAergic responses previously observed in male rats following prolonged benzodiazepine exposure. In addition,the effects of the steroid milieu on prolonged benzodiazepine exposure. In addition, the effects of the steroid milieu on anticonvulsant effects of the benzodiazepines and the development of tolerance to these effects will be studied. Understanding the effects of gonadal steroids on GABAergic systems, on responses to the benzodiazepines and on the development of benzodiazepine tolerance could lead to novel treatment regimens designed to modify certain benzodiazepine actions and/or the development of tolerance to these actions.

The benzodiazepines (BZs) are widely prescribed drugs, useful for their anxiolytic, anticonvulsant, sedative and muscle relaxant properties. However, the clinical usefulness of the benzodiazepines is hampered by the development of tolerance to their actions during prolonged exposure. Since the acute administration of benzodiazepines facilitates the inhibitory effects of gamma-aminobutyric acid (GABA) on neuronal activity, studies examining the neural adaptations underlying the development of benzodiazepine tolerance have focused on GABAergic systems. Both gonadal hormones and stress have been shown to modulate the: GABA/benzodiazepine system. In rats, in vivo fluctuations in gonadal hormones modulate GABAergic systems, but not the acute anticonvulsant actions of the benzodiazepines. However, gonadal status does modify both the apparent development of tolerance to the anticonvulsant effects of the benzodiazepines and the concomitant neural GABAergic adaptations following chronic exposure to benzodiazepines in rats. These gonad-related differences in GABA/BZ responses may be related to the dramatic sex differences in hormonal responses to stress in rodents. The proposed experiments will characterize the role of sexually dimorphic stress reactions in mediating gonadal influences on GABA/BZ receptors and their responses in rats, including the GABAergic adaptations associated with chronic benzodiazepine exposure. Studies will-investigate the mechanisms through which stress modulates the GABA/BZ system. These studies will determine if a) the sexually dimorphic, stress-induced release of peripheral hormones or b) the central activation of corticotropin releasing factor (CRF) mediate the stress-induced changes in GABA/BZ responses. A second goal is to determine if stress modulates neuronal responses to GABA and/or the benzodiazepines in a sexually dimorphic manner. Physiological responses to GABA and benzodiazepines will be measured in several brain areas of male and female rats using l) biochemical analysis of GABA-activated chloride influx, 2) electrophysiological determination of neuronal sensitivity to GABA and the benzodiazepines in brain slices, and 3) analysis of fluctuations in neuronal activity in awake, freely moving rats. Finally, we will assess the interaction between stress and gonadal factors in determining the adaptations associated with chronic benzodiazepine exposure and the associated development of benzodiazepine tolerance in rats. These studies should help elucidate the neural changes associated with tolerance to the benzodiazepines and indicate neural systems which might underlie gender- related influences on the etiology of anxiety or epileptic disorders.

The benzodiazepines (BZs) are widely prescribed drugs, useful for their anxiolytic, anticonvulsant, sedative and muscular relaxant properties. However, the clinical usefulness of the benzodiazepines is hampered by the development of tolerance to their actions during prolonged exposure. This RSDA will enhance the candidate's technical capabilities to analyze neural changes associated benzodiazepine tolerance from the level of gene expression through alterations in neuronal activity of distinct brain loci in freely moving animals. A reduction in formal teaching and service obligations will facilitate the development of new research skills by the candidate, including 1) molecular approaches initiated with in situ hybridization studies, and 2) electrophysiological analysis of neuronal activity in awake, freely moving animals. Since the acute administration of benzodiazepines facilitates the inhibitory effects of gamma-aminobutyric acid (GABA) on neuronal activity, studies examining the neural adaptations underlying the development of benzodiazepine tolerance will focus on GABAergic systems. Both gonadal hormones and stress have been shown to modulate the GABA/benzodiazepine system, including the apparent development of tolerance to the anticonvulsant effects of the benzodiazepines and the concomitant neural GABAergic adaptations following chronic exposure to benzodiazepines in rats. These gonad-related differences in GABA/BZ responses may be related to the dramatic sex differences in hormonal responses to stress in rodents. The proposed experiments will characterize the role of sexually dimorphic stress reactions in mediating gonadal influences on GABA/BZ receptors and their responses in rats, including the GABAergic adaptations associated with chronic benzodiazepine exposure. Studies will investigate the mechanisms through which stress modulates the GABA/BZ system. These studies will determine if a) the sexually dimorphic, stress-induced release of peripheral hormones or b) the central activation of corticotropin releasing factor (CFR) mediate the stress-induced changes in GABA/BZ responses. A second goal is to determine if stress modulates physiological responses to GABA and benzodiazepines in several brain areas of male and female rats using 1) biochemical analysis of GABA- activated chloride influx, 2) electrophysiological determination of neuronal sensitivity to GABA and the benzodiazepines in brain slices, and 3) analysis of fluctuations in neuronal activity in awake rats. Finally, we will assess the interaction between stress and gonadal factors in determining the adaptations associated with chronic benzodiazepine exposure and the associated development of benzodiazepine tolerance in rats. These studies should help elucidate the neural changes associated with tolerance to the benzodiazepines and indicate neural systems which might underlie gender-related influences on the etiology of anxiety or epileptic disorders. The institutional faculty and technological resources make this an ideal site for the candidate to begin molecular investigations of neuronal changes associated with hormones and benzodiazepine exposure using state-of-the-art anatomical resolution, and to ultimately assess the impact of these molecular alterations on neuronal activity in defined brain sites of freely moving animals.

DESCRIPTION: (Adapted From The Applicant's Abstract) The benzodiaepines (BZs) are widely prescribed for the treatment of anxiety-related disorders, although these compounds also have anticonvulsant, hypnotic, and sedative actions. The BZs act at specific receptor sites in the brain to potentiate the actions of the neurotransmitter gamma-aminobutyric acid (GABA). Evidence suggests that the GABA/BZ system in the amygdala may play a critical role in mediating the anxiety-reducing aspects of BZ agonists. Further evidence suggests that the endogenous opioid system also modulates the amygdalar GABAergic system underlying anxiety responses. The proposed studies employ herpes- mediated gene transfer techniques to alter expression of opioid peptides in the rat amygdala. The anxiety levels and the anxiety-reducing effects of BZs will be determined using the elevated plus maze and social interaction tests. Pilot studies using herpes virus vectors encoding either the bacterial protein Beta-galactosidase (virus SHZ.1) or human preproenkephalin (virus SHPE) in the amygdala have demonstrated that 1) we can induce high level expression of the transgenes encoded in these viral vectors in defined brain areas, 2) that viral expression in the amygdala does not have deleterious effects on the animal, 3) that overexpression of enkephalin in the amygdala enhances the anxiolytic effects of the BZs examined using the plus maze, and 4) that we can demonstrate viral expression of the encoded proteins. AIMS 1 and 2 will further characterize the ability of enkephalin overexpression in the amygdala to enhance the anxiety-reducing effects of BZ agonists (diazepam). This includes determining if enkephalin overexpression in amygdala enhances sensitivity to the anxiolytic actions of BZ agonists in another model of anxiety and insuring these effects are mediated through a BZ receptor-dependent mechanism (AIM 1). AIM 2 begins to address the mechanism through which enkephalin overexpression enhances the anxiolytic effects of BZs. Studies examine the opioid receptor subtype involved in this effect of enkephalin, begin to assess which neuronal population is affected in amygdala, and determine if changes in GABA/ BZ receptor sites are observed. Overall, this technique could provide a powerful tool to examine the role of neuropeptides in anxiety- related behaviors, and the anxiolytic action of the BZs. This approach has the marked advantage of altering neurotransmitter or receptor expression in defined brain regions of adult animals. It will thus help to elucidate the role(s) of specific, localized neurotransmitter functions in complex behaviors such as anxiety.

This proposal requests funds to sponsor a one-day symposium entitled "Gender differences in the brain and behavior". The satellite meeting will be held one day prior to the 1999 International Behavioral Neuroscience Society (IBNS) meeting, on June 23,1999 in Nancy, France. The association with this meeting of international behavioral neuroscientists will promote attendance, facilitate advertising, and promote research collaborations beyond US boundaries. The meeting is designed to bring together noted researchers analyzing sex and gender differences with biological, behavioral or cognitive outcomes. The focus of the meeting is on the neurochemical and/or neuroanatomical bases for these behavioral sex-related differences, with a particular focus on drugs of abuse (including alcohol). The emphasis for speaker selection was to facilitate discussion related to the similarities and differences between the sexes in neural systems which underlie behaviors relevant to drug abuse issues, and particularly the role of stress in affecting responses to drugs of abuse including psychostimulants, benzodiazepines, nicotine and alcohol. The symposium is organized around four major themes, including 1) Gender, anxiety, and anxiolytics (Benzodiazepines and alcohol) 2) Gender and stress-related hormones, 3)Gender and drugs of abuse, and 4) Effects of sex hormones on behavior. This will be an important venue through which these scientists can discuss their findings relevant to sex and gender differences, and provide a forum for interaction with the international neuroscience community. Although the symposium concentrates on the basic aspects of this topic, we have included one session focusing on the more clinical aspects of potential differences (session 4, organized by N. Ostrowski of Lilly). The main objective of this meeting is to discuss current findings relevant to sex differences in brain and behavior, and their implications for future directions in this important field. Given the recent emphasis on gender issues by NIH, the proposed symposium will provide an excellent opportunity at this critical junction to assess similarities and differences between genders, and the neurochemical or neuroanatomical systems which might be relevant to future basic and clinical research.

PROJECT SUMMARY (Fadel/Wilson): Anxiety and affective disorders represent an important clinical problem, yet our understanding of the disorders and the drugs used to treat them remains limited. Brain imaging studies show amygdala changes in patients with these disorders. The present studies use a multifaceted approach to elucidate how amygdalar opioid systems regulate anxiety and fear-related processes. These studies will enhance our understanding of amygdala circuits that control distinct aspects of anxiety and fear by comparing several anxiety-evoking stimuli, and elucidate the specific role of mu opioid receptors (MOR) in these different responses. Since our previous studies suggested that mu opioid receptors (MOR) and enkephalin in the amygdala can modulate basal anxiety responses and the actions of benzodiazepine anxiolytic drugs, the proposed studies will examine how MOR receptors modulate amygdalar circuitry to alter these anxiety-related responses. We hypothesize that distinct neuronal circuits are activated by different conditioned and unconditioned anxiety-evoking situations, and that presynaptic MOR receptors localized in specific amygdalar neurocircuits regulate changes in amygdala glutamate and GABA release to shift anxiety-related responses in a context-dependent manner. Four anxiety-evoking tests, including the elevated plus maze (unpredictable threat), predator odor-induced defensive burying (specific threat), restraint stress (psychogenic stimulus) and cue- conditioned freezing (learned fear), will be compared in these studies. Aim 1 uses virus-mediated gene transfer to examine if decreasing the expression of MOR in the amygdala alters anxiety-related behaviors and/or endocrine responses to restraint stress, and if selectively targeting these decreases to pyramidal neurons of the basolateral amygdala produces the same effects. Aim 2 uses cFos immunoreactivity to compare the cellular phenotype(s) activated by distinct anxiety-evoking situations, the localization of MOR in these activated neuron populations, and if activation patterns are altered by decreasing amygdala MOR expression. Aim 3 uses in vivo microdialysis in the amygdala to assess 1) if MOR activation alters GABA or glutamate efflux, 2) if anxiety-evoking situations induce release of enkephalin, GABA, or glutamate, and 3) if decreasing MOR expression modifies MOR-induced or anxiety-induced release of GABA or glutamate. The studies will enhance our understanding of how the amygdala and the opioid system regulate anxiety responses, and could provide novel therapeutic strategies for treating affective and anxiety-related disorders. Since opioid systems in the amygdala are modified during chronic pain states and altered by drugs of abuse, the results of these studies will also enhance our understanding of the neural basis of heightened anxiety states seen in chronic pain patients or during withdrawal from opiates, benzodiazepines, and alcohol. Relevance: Anxiety disorders are the most common mental illness and affect more than19 million US adults, yet our understanding of these disorders and the drugs used to treat them remains limited. The present studies use animal models to elucidate how the circuitry in the brain region underlying emotional behaviors, namely the amygdala, controls responses in three different anxiety-evoking situations. The focus on endogenous morphine-like chemicals (opioids) could lead to new treatment strategies for anxiety disorders, and increase our understanding of why chronic pain states or withdrawal from prescribed or abused opioid drugs lead to increased anxiety.

This symposium is part of the Pharmacology, Biochemistry and Behavior 5th International meeting being held in Mozine, France in January 4-9, 2004. Funding will enable trainees and scientists from the United States to maximize their interactions with European investigators interested in Behavioral Neuroscience and Neuroendocrinology. The program includes participants with expertise in neuroendocrinology, animal behavior, and developmental biology. The symposium will engage both senior and junior ranked scientists in discussions focusing on the pharmacology and biochemistry of reproductive behavior and cognitive function. Participants in this program will include undergraduate students, graduate students, post-doctoral fellows, and faculty. The symposium will engage participants in discussions regarding complex relationships between brain and behavior. Symposia, poster presentations, meet the professor lunches, and organized discussions on international research opportunities will encourage interactions between students, junior scientists, and senior scientists that are internationally recognized for their accomplishments. The organizing committee is committed to fostering the development of young scientists. Funds will help defray travel costs and registration fees for participants to attend the meeting. The impact of this will extend to numerous fields of study, such as neuroendocrinology, behavioral neuroscience, animal behavior, and population biology. Those trainees receiving travel awards will be asked to evaluate the effectiveness of their experience at this meeting and then these data will be published as an abstract and poster presentation that will take place during the education session at the 2004 Society for Neuroscience meeting.

[unreadable] DESCRIPTION (provided by applicant): This proposal explores differences in temperament (anxiety) and gene expression (NPY) patterns in the amygdala as examples of endophenotypes that may contribute to ethanol consumption. Alcohol abuse and dependence is a complex disorder resulting from gene-environment interactions, but the genes encoding neuropeptide Y (NPY) or its receptor appear to confer risk for alcohol dependence in clinical and preclinical studies. This proposal expands our earlier studies and will explore the causative relationship between gene expression of NPY in the amygdala, an anxious behavioral phenotype, and ethanol consumption using the inherent phenotypic variation in anxiety seen in a rat model. The amygdala plays a critical role in anxiety-related behaviors and the anxiolytic effects of NPY may be mediated through the amygdala. Our previous studies have shown that rats with an anxious or nonanxious phenotype, defined using exploration of the elevated plus maze, display differences in ethanol preference. Changing NPY expression in amygdala also shifts ethanol preference, but only in anxious rats. Given the evidence that amygdala NPY levels regulate anxiety-related responses, the present application examines the hypothesis that NPY gene expression levels in the amygdala determine the anxiety phenotype of an individual, and low NPY levels may predispose animals for greater ethanol consumption based on this anxious phenotype. Aim 1 examines if altered NPY expression in amygdala changes anxiety state and subsequently modifies ethanol consumption in a two bottle self-administration procedure. This aim will develop lentivirus-mediated gene transfer methodologies to more directly test if lowering NPY expression in the amygdala enhances anxiety and thereby promotes ethanol consumption and/or if enhanced amygdala NPY gene expression is anxiolytic and thereby reduces ethanol preference. This Aim also characterizes anxiety phenotypes using additional behavioral models. Aim 2 will examine ethanol consumption in anxious and non-anxious phenotypes using a limited access model. This limited access method will allow more efficient screening of gene targets for development of therapeutic strategies to combat alcohol dependence. Aim 3 will examine if the pattern of neuronal activation in the amygdala (and other regions) differs with elevated plus maze exposure in the anxious and non-anxious phenotypes, and if NPY neurons are activated by ethanol injection or ethanol consumption during a limited access period. Combined with viral vector methods characterized in this developmental R21 grant these studies will lead to better characterization of specific aspects of the anxiety phenotypes regulated by NPY gene expression in the amygdala, and a better understanding of how these differences in anxiety phenotype and NPY gene expression predict ethanol consumption. [unreadable] [unreadable] Public Health Relevance: Although stress and anxiety contribute to alcohol consumption and abuse, the brain systems that predispose certain individuals to abuse alcohol and how alcohol relieves anxiety states remains unknown. The present studies use animal models to elucidate how the genes expressed in the brain region underlying emotional behaviors, namely the amygdala, control individual responses in an anxiety-provoking situation and if these same processes contribute to the alcohol consumption. The studies will provide a better understanding of how individual differences in gene expression underlie the interaction between stress and alcohol abuse, and may lead to novel targets to combat the growing problem of alcohol consumption in adolescents and adult populations. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this project is to develop tools for cell-type-specific regulation of gene expression in the brain. The basolateral region of the amygdala (ABL) of the rat brain will be used as a model system. The project will develop lentiviral vectors to target transgene over-expression in identified subpopulations of ABL neurons and to selectively knock-down endogenous gene expression in these same cell groups. Aim 1- Tarqeted Over-expression: The proposed studies will test the hypothesis that lentiviral vectors can be constructed to restrict gene over-expression to defined subpopulations of ABL neurons by incorporating cell- type-specific promoters. Lentiviruses designed to target a marker gene or c-myc-tagged GABAA receptor a1 subunit to pyramidal cells (CAM kinase II promoter) or discreet populations of GABAergic interneurons expressing VIP and calretinin (calretinin promoter) or parvalbumin (pan/albumin promoter) will be constructed, tested and optimized for specificity of cell-type targeting. Aim 2-Targeted Knock-down of Expression: The proposed studies will test the hypothesis that lentiviruses incorporating cell-type-specific promoters can be used to selectively reduce gene expression in defined subpopulations of ABL neurons by expression of antisense RNA. After identifying antisense constructs that effectively reduce mRNA for the NMDA receptor subunit 1 and for GABAA receptor a1 subunit in cultured cells, the antisense constructs will be inserted into lentiviral vectors under control of the CaM kinase II, calretinin or parvalbumin promoters. These lentiviral vectors will be tested for cell-type specificity in knock-down of target gene expression following stereotaxic delivery to the ABL. Development of tools for cell-type selective manipulation of gene expression will greatly facilitate determining the roles of specific gene products in subpopulations of neurons participating in defined neuronal circuits of intact animals. This knowledge will, in turn, permit development of new drugs and genetic therapies for neurological and behavioral disorders. In the ABL, the development of these tools is particularly relevant to treatments for stress and anxiety disorders and for epilepsy. It is also expected that the vectors developed in this project will be useful to study synaptic transmission and the regulation of neuronal networks in other brain areas including the cortex and the hippocampus. These tools will have wide applicability in elucidating the functional anatomy of the central nervous system, particularly in determining the roles of individual proteins found in specific groups of brain cells in the behavior of the whole organism. Because of the brain region being studied, this work is of particular relevance to the understanding and treatment of stress, anxiety and epilepsy. It is expected that this work will ultimately lead to new drugs or genetic therapies for these and other disorders of the nervous system. [unreadable] [unreadable]