Klaus A. Miczek - US grants

Previous work in our laboratory has explored the effects of alcohol on the aggressive behavior of mice, rats and squirrel monkeys. We have found an interesting interaction between testosterone and the effect of alcohol on behavior. We have also begun to analyse some of the social dynamics which modify alcohol response. There are three main objectives of the present proposal: (l) an analysis of the mechanism of testosterone's ability to alter differentially the aggression-heightening and -inhibiting effects of alcohol in males and females; (2) to investigate the role of social status and experience as determinants of alcohol's effect on aggressive behavior; (3) to assess the effects of self-administered compared to force-administered alcohol on social behavior. The use of quantitative ethological methodology in this proposal will provide a more reliable and complete analysis of several dimensions of behavior, including the sequencing and patterning of species-typical behavior as well as pathological behavior. This methodology will be used to further quantify the effect of alcohol in situations which reliably engender attack, threat, defense, submission, flight and other agonistic and non-agonistic behaviors. Three experiments will focus on objective l. They will investigate: a) the role of sexual differentiation in determining sensitivity to the effect of testosterone on alcohol response, b) the neural sites of the testosterone-alcohol interaction on aggressive behavior, and c) the importance of metabolism of testosterone to estradiol and/or dihydrotestosterone as part of its mechanism of action. Two experiments will focus on objective 2. The first will vary social status within groups of animals and determine possible changes in sensitivity to alcohol and corresponding changes in endocrine activity. The second will examine the effect of aggressive or defeat experience on the later behavioral and endocrine response to acohol. The final experiment will focus on objective 3 and study the effect of self-administration of low, aggression-heightening doses of alcohol on behavior. Our goal is to learn more about the effect of alcohol on the central nervous system and behavior. We believe that the study of alcohol, hormones and social behavior in animals may help clarify the mechanisms which control the effects of alcohol on aggression in humans.

DESCRIPTION: (Applicant's Abstract) The proposed research is driven by the major public health and criminal justice concern with the link between the abuse of psychomotor stimulants and opiates to violence and being the victim of violence. The proposed studies seek to determine the sources of individual sensitivities to salient social events that promote or retard the self-administration of opioids and psychomotor stimulants. A second objective focuses on the reciprocal: How do self-administered psychostimulants and opioids intensify or exaggerate aggressive and defensive behavior and disrupt social behavior. A vertical research strategy is implemented that integrates behavioral, physiological, and neurochemical levels of analysis in the intact behaving animal under unambiguously defined social conditions. Three sets of aims and experiments are proposed: (1) How do behavioral, physiological, and neurochemical characteristics of aggression promote psychostimulant and opioid self-administration? The focus is on individuals (a) who display "impulsive" aggression (i.e., low provocation threshold, short latency, injurious, non-ritualized, no inhibition from signals of submission) and who prefer an immediate small reinforcer vs. a large delayed reinforcer, (b) who readily entrain cardiovascular and core temperature activity to repeated aggressive confrontations, and (c) who show a unique pattern of increased dopamine (DA) and decreased serotonin (5-HT) release in mesocorticolimbic systems while engaged in aggressive behavior. Are these individuals more likely to engage in opioid- and stimulant-sensitized motor activity, self-administer opioids, cocaine and d-amphetamine in a binge-like pattern, withdraw more intensely, and relapse more readily? (2) How do social stress experiences that result from being the victim of aggression, impact on stimulant and opioid self-administration? Are individuals who (a) cope with being attacked by being passively submissive vs. actively defensive, (b) who show a large release of cortical DA release and inhibited striatal 5-HT activity, and (c) who readily entrain autonomic activity to recurrent confrontations, more likely to show sensitized motor activity, self-administer opioids, cocaine and d-amphetamine? (3) How do self-administered psychostimulants and opioids impact on aggressive behavior and responses to social stress? This aim is pursued in aggressive resident and defensive intruder animals, and the direct effects of specific doses of self-administered cocaine, d-amphetamine and morphine on aggressive and defensive behavior are assessed during social confrontations. Experiments are designed that determine how continuous access to psychostimulants and morphine disrupt social intercourse, how withdrawal from prolonged stimulant and opioid self-administration exaggerates defensive or aggressive behavior, and how autonomic and mesocorticolimbic DA and 5-HT mechanisms contribute to these effects.

The proposed work investigates several major determinants of alcohol effects on aggressive an social behavior. Two major possible mechanisms for these alcohol effects are explored, alcohol's interaction with gonadal steroid hormones and with benzodiazepine receptors. Specifically, we plan to manipulate critical behavioral experiences that will modulate testosterone activity and thereby alter the sensitivity to behavioral effects of alcohol. We will investigate how time- and dose- dependent effects of alcohol on aggressive and social behavior are modulated by endogeneously varying or experimentally manipulated testosterone activity. Pre- and postnatal presence of differential amounts of testosterone in females are expected to modulate alcohol effects on aggressive and reproductive behavior in adulthood. Seasonal rhythms in gonadal hormone levels are expected to be associated with distinctive patterns of alcohol effects on aggressive behavior. Past experiences with aggressive or submissive behavior produce divergent changes in gonadal activity, and these changes are expected to alter certain behavioral effects of alcohol. In a second set of experiments the role of benzodiazepine receptors in the behavioral effects of alcohol will be investigated. Direct measurements of benzodiazepine receptor binding, concentration of benzodiazepines and metabolites in blood and brain, and pharmacological of benzodiazepine receptors with agonists, inverse agonists and antagonists will give information on the patential significance of these receptors in the effects of alcohol on aggressive, defensive, social, reproductive and motoric activities. The use of quantitative ethological methods for the recording and analysis of social and agonistic as well as automated measuremenmts of motor functions and startle responses will ensure assessment of behavioral specificity of the pharmacological treatments. This research will further our understanding of how alcohol exerts its effects on aggressive behavior and the ways in which gonadal hormones and benzodiazepines can modulate those effects.

Stimulant abuse may engender explosive and seemingly unpredictable violent outbursts in certain individuals, whereas others withdraw entirely from social interactions. The specific aims of the proposed research are as follows: 1. How functionally specific or pervasive are the behavioral tolerance, sensitization and potentiation phenomena in the profile of opiate and psychomotor stimulant effects after distinctive experiences in situations of social conflict and affiliative behavior? 2. How do experience with certain social behaviors modulate the symptoms of opiate withdrawal? Which are the catecholaminergic mechanisms that mediate heightened aggressive and defensive reactions during opiate withdrawal? 3. How do experience with reproductive and aggressive behavior that are based on the hormonal status of the individual, particularly in females, influence the behavioral and physiological effects of opiates and psychomotor stimulants? 4. Are individual differences in sensitivity to amphetamine's and cocaine's acute and chronic behavioral effects linked to distinctive social experiences? We plan to conduct studies that will (1) parametrically determine the minimal and "optimal" social, aggressive, defensive and submissive behaviors that result in modulation of behavioral and physiological functions, (2) use biochemically identified selective ligands at different opiate receptor subtypes as well as dopaminergic and noradrenergic receptor subtypes as probes for functional assessments, and (3) quantitatively assess selected functions such as DA, DOPAC, NE, 5-HT, 5-HIAA in discrete fore- and midbrain regions, serum prolactin and corticosterone, telemetered heart rate, blood pressure, core temperature, pain and startle responses, motor activities, discriminative stimulus properties, maternal care behavior, sexual, social, aggressive, defensive, submissive and flight reactions. Additional experiments will investigate the effectiveness of acute morphine administration in comparison to incrementally longer morphine exposure to induce heightened aggressive behavior. Further experiments are designed to examine the receptor super- sensitivity proposal by challenging animals during opiate withdrawal with several selective dopaminergic and noradrenergic receptor agonists, first systemically, and subsequently by intra- cranial infusion into specific target regions. A final set of experiments will attempt to antagonize amphetamine and cocaine effects on social and aggressive behavior with prazosin and other drugs that interfere with noradrenergic and dopaminergic neurotransmission.

Alcohol is far more frequently associated with violent and aggressive behavior than all other drugs combined. The proposed research addresses our need to better understand the basic behavioral and neurobiological mechanisms of the alcohol-aggression link. Based on the discovery in the previous funding period that it is possible in several animal models to identify those individuals that are most prone to the aggression- heightening effects, the current proposal aims to delineate the behavioral, physiological and neurochemical characteristics of this subgroup of individuals. The marked individual differences in the aggression-heightening effects of alcohol and in the self-administration pattern for alcohol prompt focus on individual behavioral and physiological characteristics. Detailed investigations of GABA/A receptors using both pharmacological and endocrinological manipulations will examine this critical site of alcohol action in the modulation of aggressive behavior and related emotional expressions. Six series of experiments are proposed, the first focusing on the reciprocal relationship between aggression, social stress and alcohol self- administration, the second on a quantitative ethological analysis of the disruption of communicative social signals by alcohol and their neural modulation at GABA/A receptors, the third and fourth on the pharmacological and endocrinological influences on the benzodiazepine/GABA/A receptor complex for attenuating the aggression- heightening effects of alcohol, and the fifth and sixth on the behavioral, physiological and neurochemical characteristics of those individuals who are most prone to show aggression-heightening effects of alcohol. (1) Quantitative analysis of circadian rhythmicity of telemetered blood pressure, heart rate and core temperature, (2) autonomic response and adaptation to environmental, social and pharmacological challenges and (3) functional properties of GABA/A receptor populations are investigated for their predictive values to identify individuals with high propensity to become very aggressive after alcohol.

Funding is requested of offset the costs for U.S. scientists participating in symposia on drug abuse and dependence and drug discrimination at the European Behavioural Pharmacology Society Meeting in Berlin. Funding is requested for 20 students and 20 invited speakers. The conference is a 4 day meeting, and the symposia on drug abuse and dependence will form a major part of the meeting (9 out of 27 symposia). The symposia have been arranged to allow the participants to follow them as a single topic throughout the meeting. Symposia will specifically address different theoretical accounts of drug addiction; the factors that cause drugs to differ in their abuse liability; the problems of benzodiazepine abuse and dependence from the basis of animal, human experimental and clinical perspectives; and behavioral sensitization of psychomotor stimulants. In addition, a workshop on research methodologies in the drug abuse field has been organized. Additional free communication and poster sessions will allow young investigators to fully participate in the meeting.

Alcohol is far more frequently associated with violent and aggressive behavior than all other drugs combined. The proposed research addresses our need to better understand the basic behavioral and neurobiological mechanisms of the alcohol-aggression link. Based on the discovery in the previous funding period that it is possible in several animal models to identify those individuals that are most prone to the aggression- heightening effects, the current proposal aims to delineate the behavioral, physiological and neurochemical characteristics of this subgroup of individuals. The marked individual differences in the aggression-heightening effects of alcohol and in the self-administration pattern for alcohol prompt focus on individual behavioral and physiological characteristics. Detailed investigations of GABA/A receptors using both pharmacological and endocrinological manipulations will examine this critical site of alcohol action in the modulation of aggressive behavior and related emotional expressions. Six series of experiments are proposed, the first focusing on the reciprocal relationship between aggression, social stress and alcohol self- administration, the second on a quantitative ethological analysis of the disruption of communicative social signals by alcohol and their neural modulation at GABA/A receptors, the third and fourth on the pharmacological and endocrinological influences on the benzodiazepine/GABA/A receptor complex for attenuating the aggression- heightening effects of alcohol, and the fifth and sixth on the behavioral, physiological and neurochemical characteristics of those individuals who are most prone to show aggression-heightening effects of alcohol. (1) Quantitative analysis of circadian rhythmicity of telemetered blood pressure, heart rate and core temperature, (2) autonomic response and adaptation to environmental, social and pharmacological challenges and (3) functional properties of GABA/A receptor populations are investigated for their predictive values to identify individuals with high propensity to become very aggressive after alcohol.

DESCRIPTION (provided by applicant): Epidemiological and criminal statistics as well as neurobiology, and pharmacotherapy all provide converging evidence that suggests links between alcohol consumption, impulsivity, and pathological aggression. The proposed research aims to dissect these links at the behavioral and neurochemical level, with particular focus on the relative role and interactions between GABAA and serotonin systems. A first specific aim seeks to analyze how alcohol-heightened aggression is related to other forms of escalated aggression. Experiments are designed to test the hypothesis whether or not a common behavioral profile of varied forms of escalated aggression characterizes individuals who engage in alcohol-heightened aggression. The second and third aims focus on pharmacological tools that are employed to characterize the relative contribution of 5-HT1A, 5-HT1B, and GABAA receptors, their pre- versus post-synaptic sites in brainstem, and prefrontal cortical regions in animals that engage in alcohol-heightened aggression. The fourth aim examines how serotonergic modulation of GABAergic systems determines alcohol-heightened aggression. Inversely, how do neuropharmacological manipulations of the modulatory sites on the GABAA receptor, particularly via neurosteroids, enable 5-HT-mediated effects on aggressive behavior? Pharmacological experiments are designed to stimulate pre-synaptically 5-HT1A and 5-HT1B receptors or to neurotoxically lesion raphe nuclei in order to assess the importance of serotonergic inhibition on the activating effects of positive modulators like neurosteroids on alcohol-heightened aggression. A fifth aim is directed at the neural sites of the GABAergic and serotonergic mechanisms that are critical for the aggression-heightening effects of alcohol. Intracranial microinjections are used to determine whether activation of 5-HT receptors in the raphe nucleus or in terminal forebrain regions are the critical sites for reducing escalated fighting. Conversely, can blockade of the 5-HT autoreceptors in the raphe n. potentiate the aggression-heightening effects of alcohol and other positive modulators at GABAA receptors? Additional evidence will be obtained by in vivo microdialysis experiments, in order to learn whether behavioral changes are reflected in significant changes in the level of GABA and serotonin release in cortico-limbic and brainstem areas.

DESCRIPTION (provided by applicant): Epidemiological data, statistics on perpetrators of violent crimes and of victims of violence under the influence of drug as well as neurobiological evidence link social stress and drug use. The proposed research aims to characterize the common behavioral and neural features of individuals who experience salient types of social stress and who self-administer psychomotor stimulants and opioids in a compulsive-like manner, and to define the neural circuits for these apparently opposing activities. Ostensibly aversive events such as social defeat stress and euphorogenic effects such as those produced by cocaine or opioids share physiological and corticolimbic circuits. First, experiments are designed to identify the critical behavioral and mesocorticolimbic features of brief episodes of social defeat stress, chronic subordination stress, and impulsive aggressive behavior as potential determinants for the transition to compulsive-like intravenous self-administration of cocaine, d-amphetamine or morphine. We aim to characterize the transition from regulated to dysregulated drug taking by studying the long (>24 h) drug binge as a model. Secondly, the neurobiological mechanisms for the behavioral sensitization that results from repeated episodes of social defeat stress will be characterized with a focus on glutamatergic, GABAergic and serotonergic modulation of mesocorticolimbic dopaminergic pathway. In vivo microdialysis measurements aim to reveal the emerging neural sensitization in the microcircuit consisting of amgydaloid and cortical structures modulating the VTA DA system. Thirdly, neuropharmacological experiments are designed to examine the role of NMDA and AMPA glutamatergic receptos, GABAA receptors, and subtypes of the 5-HT receptor families in modulating activity in the mesocorticolimbic DA pathway for their relevance in sensitization to social stress and in the transition to intense dysregulated cocaine and morphine self-administration in stress-sensitized animals. It is anticipated that the sensitizing effects of social stress experiences can be reversed or protected against by targeting the modulatory influences on the VTA system. Reversal of cross-sensitization from stress may be a means by which to prevent the transition to out-of-control compulsive-like drug taking.

DESCRIPTION (provided by applicant): The proposed research will increase our understanding of the neural mechanisms via which alcohol escalates aggressive behavior in some individuals but not in others. Violent outbursts are one of the most costly, horrifying and damaging consequences of alcohol consumption, representing one of the most significant problems for the public health and criminal justice systems. The overarching hypothesis is to assess how escalated aggression, particularly under the influence of alcohol, is a function of dysregulation of serotonergic activity in the raphe cells by feedback via somatodendritic autoreceptors and by GABAergic and glutamatergic influences, especially by feedback from the prefrontal cortex, and by CRF input. We propose that the dysregulation of feedback control on serotonergic neurons projecting to prelimbic, infralimbic and orbitoventral regions of the prefrontal cortex characterizes those individuals who engage in escalated aggressive behavior after alcohol consumption. Specifically, experiments in mice and rats are designed to answer the following questions: (1) How is the activity of serotonergic projections from the dorsal raphe n (DRN) to the prefrontal cortex (PFC) regulated in individuals who engage in escalated aggressive behavior? To which extent is the expression of 5-HT receptor subtypes in the prefrontal cortex critical for escalated aggressive behavior? Is gene expression for the 5-HT1 and 5-HT2 receptor families in the prefrontal cortex suppressed in animals that engage in alcohol-heightened aggression? What is the respective role of presynaptic receptors in the PFC terminals relative to somatodendritic autoreceptors and SERT in gating serotonin transmission in highly aggressive individuals, particularly after alcohol consumption? (2) Are glutamatergic and GABAergic influences on the 5-HT cells in the DRN critical for the display of escalated aggressive behavior, particularly after alcohol self-administration? Do these signals originate from GABAergic interneurons? How significant is the glutamatergic feedback from the PFC? Which subunits in GABA-A receptors are essential for the aggression- heightening effects of alcohol? Are NMDA glutamate receptor subtypes more selective in their modulation of escalated aggression after alcohol self-administration than AMPA receptors? (3) How critical is the modulation by CRF of serotonergic projections to the PFC in individuals who engage in escalated aggressive behavior? Can the respective role of CRF 1 and 2 receptor subtypes be defined for the intensification or attenuation of alcohol-heightened aggressive behavior? Are the CRF receptors on serotonergic cells the critical population that is pivotal for escalated aggressive behavior after alcohol self-administration? The experimental work relies on quantitative ethological methodology for the analysis of species-normative and escalated forms of aggression, voluntary alcohol self-administration, real time PCR, in situ hybridization histochemistry, genetic point mutations, in vivo microdialysis and HPLC, and intracerebral microinfusions. The anticipated outcome will identify targets for therapeutic interventions. PUBLIC HEALTH RELEVANCE: The rationale for the proposed research is readily translated to one of the most significant problems for the public health and criminal justice system, namely to understand why alcohol escalates aggressive behavior in some individuals but not in others. Violent outbursts are one of the most costly, horrifying and destructive consequences of alcohol consumption. The proposed research will assess how escalated aggression, particularly under the influence of alcohol, is a function of dysregulation of serotonergic activity in the raphe cells by feedback via GABAergic, glutamatergic and CRF modulation.

DESCRIPTION (provided by applicant): The close link between social stress and drug use is based on reports from emergency rooms treating victims of violence and statistics from the criminal justice system on violent crimes committed by drug users as well as epidemiological evidence and neurobiological data. Some specific types of social stress can promote drug abuse and trigger relapse, whereas others do not, each stressor activating discrete neurobiological mechanisms. The present application focuses on the neuropeptide CRF, particularly receptor subtype 1 (CRF- R1), based on the growing evidence and our own preliminary data that implicate this system in the mechanisms of social stress leading to escalated drug intake. Specific Aim One tests the hypothesis that CRF-R1 modulation of discrete dopaminergic neurons is a critical mechanism for stress-escalated behavior, with a focus on different phases of cocaine self-administration (acquisition, maintenance, binge, relapse). The proposed research employs discrete intracerebral microinjections to stimulate and block CRF-R1 in discrete neural regions, in vivo microdialysis for sampling extraneuronal fluid, high performance liquid chromatography for assaying these samples to determine dopamine and other amines and behavioral measures as indices of neuroadaptive changes. Specific Aim Two tests the hypothesis that blockade of CRF-R1 in the VTA attenuates stress-escalated cocaine self-administration, whereas antagonism of CRF-R2 intensifies cocaine self-administration. Specific Aim Three tests the hypothesis that antagonists of CRF-R1 in the VTA will not only protect (Aim 1), but more importantly reverse social stress-induced behavioral sensitization and stress-escalated cocaine self- administration by modulating the activity VTA DA cells. Specific Aim Four tests the hypothesis that conditional CRF-R1 knockout mice will fail to show stress- induced psychomotor and neural sensitization in response to a cocaine challenge. We hypothesize that genetic prevention of CRF 1 receptor expression in forebrain structures alters behavioral, physiological and neurochemical responses to social stress. The proposed research on CRF-R1 in the ventral tegmental area promises to identify one critical target in the neurocircuitry of social stress for therapeutic intervention, especially in cases of intense binge-like cocaine intake.

DESCRIPTION (provided by applicant): Violent outbursts are one of the most costly, horrifying and damaging consequences of alcohol consumption, representing one of the most significant problems for the public health and criminal justice systems. The proposed research aims to increase our understanding of the neural mechanisms via which alcohol escalates aggressive behavior in some individuals but not in others. The proposal focuses on two types of aggression that are related to alcohol, escalated aggression after acute alcohol intoxication and aggression during withdrawal from intermittently accessible alcohol. The overarching hypothesis is to assess how escalated aggression, particularly under the influence of alcohol, is a function of dysregulation of two neurocircuits that are modulated by the CRF system. (1) We plan to functionally characterize ethanol-withdrawal aggression which appears defensive in nature, and contrast it with the aggression-heightening effects that follow the self- administration of an acut low alcohol dose, modeling the violence associated with acute alcohol intoxication. (2) The proposed studies will test the hypothesis that dysphoria and defensive aggression during the abstinence interval between consecutive access periods to alcohol is based on increased CRF activity in limbic forebrain structures, which in turn alters the adaptations in glutamate - GABA inputs to monoaminergic pathways. By contrast, we propose to test the hypothesis that antagonism of CRF-R1 modulates ascending serotonin impulse flow from the dorsal raph? nucleus to the prefrontal cortex and thus reduces the aggression- stimulating effects of acutely self-administered alcohol. (3) We aim to use molecular genetic tools to characterize the CRF and GABA receptor systems during escalated intermittent alcohol drinking and during the ensuing withdrawal aggression. We will use inducible knock-down technology in order to define the relative importance of GABAA receptor subtypes in the amygdala in alcohol-heightened aggression and alcohol- withdrawal aggression. The experimental work relies on quantitative ethological methodology for the analysis of species-normative and escalated forms of aggression, voluntary alcohol self-administration, real time PCR, in situ hybridization histochemistry, genetic manipulations, in vivo microdialysis and HPLC, and intracerebral microinfusions. The anticipated outcome will identify targets for therapeutic interventions.