Michael T. Bardo - US grants

Some behavioral effects of opiates are thought to be mediated by opiate receptors which modulate the neurotransmission of dopamine within the nigrostriatal and mesolimbic systems. The number of opiate receptors may be increased following chronic treatment with opiate antagonists such as naloxone and naltrexone. If chronic antagonist treatment increases the number of brain opiate receptors, then an enhanced effect of morphine on dopamine activity and behavior may also be evident. The present proposal examines that hypothesis. Rats will be implanted with a slow-release pellet of naltrexone for 10 days and then the pellet will be removed. At one of the various different intervals after pellet removal, animals will be sacrificed and different regions of the nigrostriatal and mesolimbic systems will be examined for changes in mu, delta and kappa binding sites. Some animals will also be challenged with an injection of morphine in order to determine whether naltrexone pretreatment alters the functional activity of dopamine neurons within the nigrostriatal and mesolimbic systems. Functional activity will be examined in vivo by measuring turnover of DA and by measuring the spontaneous firing rate of neurons in these dopaminergic subsystems. Correlated with these neurochemical and electrophysiologic changes, morphine-induced changes in antinociception, locomotor activity, and conditioned reinforcement will also be examined. While the results from these experiments may have some potentially significant clinical implications for the treatment of human opiate addiction with opiate antagonists, the long-term objective of the project is to advance our knowledge about the role which opiate receptors normally play in various neurobehavioral processes.

People who have a high "drive" for novel and arousing sensory stimulation (i.e., high sensation-seekers) are known to use drugs more frequently than low sensation-seekers. One interpretation of this relationship is that high sensation-seekers are predisposed biologically to find both novel stimuli and drugs to be more rewarding than low sensation-seekers. The present research will determine if novelty activates the mesolimbic DA system in a manner analogous to amphetamine, and will examine the potential neurochemical connection between novelty- and drug-seeking behaviors. Specifically, the role of D1 and D2 DA receptors in novelty-seeking will be evaluated in rats. Other experiments will be conducted to explore the possibility that reactivity to stress may influence novelty preference behavior. In separate experiments, rats will be categorized as either "high" or "low" in novelty preference behavior based upon their approach towards a novel environmental stimulus. These animals will then be tested for their sensitivity to the locomotor-activating and rewarding effects of amphetamine using the conditioned place preference paradigm. In addition to amphetamine, another psychostimulant (cocaine), as well as other drug classes (diazepam and morphine), will be evaluated in order to assess the generality of the findings. A parallel set of neurochemical experiments will assess whether any behavioral differences reflect a change in the functional activity of the mesolimbic DA system. In another set of experiments, rats will be raised from 21 days of age in isolation cages (1 per cage), group cages (3 per cage) or in an enriched environment with novel toys (12 per cage). At 50-60 days of age, the animals will be tested for their preference for a novel environment, as well as their behavioral reactivity to amphetamine and other drugs of abuse. In addition, isolated-, grouped- and enriched-reared rats will be examined neurochemically to determine the functional activity of the mesolimbic DA system when challenged with novel stimuli or drugs of abuse.

Studies which have investigated the classical conditioning of various drug effects have almost exclusively employed a contextual or environmental cue as the conditioned stimulus (CS). One exception to this has been the use of gustatory cues in examining the aversive properties of drugs. The objective of this project is to investigate the ability of a gustatory cue to also serve as a CS when paired with the analgesic and locomotor effects of morphine. Rats will be given repeated presentations of a saccharin taste cue paired with morphine and then they will be tested for pain responsivity or locomotor activity following exposure to the taste cue alone. Pain sensitivity will be measured using a hot plate test and locomotion will be measured by counting photobeam interruptions in a chamber. The conditioned response (CR) will be characterized in different experiments by varying the drug dose used, the number of conditioning trials, and the delay between CS exposure and the behavioral test. Control experiments will be conducted in order to assess the possible influence of nonassociative mechanisms, as well as nongustatory CS elements, in the response obtained on the test day. This project will fill our gap in knowledge about the selective associations that a gustatory CS can make with different properties of a drug such as morphine.

There is now considerable evidence indicating that people who have a high need for novel and arousing stimulation (i.e., high sensation-seekers) use drugs more frequently than low sensation seekers. One interpretation of this relationship is that high sensation-seekers are predisposed biologically to find both novel stimuli and drugs to be more rewarding than low sensation-seekers. The major long-term objective of the present application is to determine if novelty-seeking and drug-seeking behaviors share a common overlapping mechanism in the brain. If these two behaviors are linked biologically, then perhaps novel stimulation may substitute for drug reward in high sensation-seekers. Using an animal model, rats will be evaluated for novelty-seeking behavior in three different behavioral measures: (1) activity in an inescapable novel compartment; (2) novelty-induced place preference; and (3) approach to a novel object. High and low novelty-seekers will then be evaluated for their behavioral response to amphetamine, cocaine and agonist drugs selective for either D1, D2, or D3 dopamine receptors. Parallel experiments will determine the functional activity of the mesolimbic dopamine system in both high and low novelty-seeking animals. Experiments will also be conducted to evaluate the role of environmental and genetic factors in determining novelty- and drug-seeking behaviors. The role of the environment will be examined by raising rats in one of three different conditions varying in stimulus novelty. They will then be examined for their response to drugs, as well as for differences in functional activity of the mesolimbic dopamine system. The role of genetics will be examined by screening a recombinant series of mouse strains for both novelty- and drug-seeking behaviors in order to determine if these behaviors are associated with common chromosomal loci in the mouse genome.

DESCRIPTION (provided by applicant): Evidence indicates that repeated early life exposure to an enriched environment produces changes in response to novelty and response to drugs of abuse later in life. The overall working hypothesis of this proposal is that exposure to novel environmental stimulation during development decreases responding for nondrug and drug reinforcers during adulthood and that this behavioral change is due, at least in part, to enhanced basal DA activity in the mesocorticolimbic reward system. The specific aims of the proposed experiments are to determine if environmental enrichment alters: (1) extinction or reinstatement of amphetamine seeking behavior; (2) the ability of nondrug reinforcers to decrease amphetamine self-administration; (3) neuronal processing in the nucleus accumbens (Nacc) and medial prefrontal cortex (mPFC) in response to nondrug reinforcers; and (4) activity of the DA transporter (DAT) and vesicular monoamine transporter (VMAT2) in mPFC. In the proposed experiments, rats will be raised from 21 days of age in either an enriched condition (EC), social condition (SC) or imporverished condition (IC). At 50 days of age, rats from each condition will be trained to self-administer amphetamine at one of various doses. Extinction and reinstatement of responding for amphetamine will be examined, and the ability of a concurrent alternative nondrug reinforcer (sweet solution or visual novelty) to reduce amphetamine self-administration will be examined in EC, SC and IC rats. To identify the critical neural mechanisms that underlie these enrichment-induced behavioral changes, separate groups of EC and IC rats will be examined for single-unit neuronal activity in the mPFC, Nacc core and Nacc shell while having access to sweet solution or novelty. Other experiments will use in vivo voltammetry or in vitro binding/uptake to access potential changes in DAT or VMAT2 in EC and IC rats. To the extent that both novelty and drugs of abuse activate the same mesocorticolimbic DA circuitry, this would suggest that novel stimulation might substitute for drug reward. Novelty-induced reductions in amphetamine self-administration would provide the impetus for examining the effectiveness of presenting enriching stimulation in drug abuse prevention interventions in humans prone to abuse drugs. The long-term objective of our work is to design biologically relevant, behavioral treatment and prevention strategies that can be evaluated in a controlled human study.

DESCRIPTION: (provided by applicant) The Center for Prevention Research at the University of Kentucky is distinguished by thematic integration around the construct of novelty/sensation (N/S) seeking. Over the past 12 years, we have evaluated N/S seeking behavior at a number of complementary levels of analysis and have made considerable progress in our understanding of the onset and development of drug abuse, and in applying this information to the development of more focused and effective prevention efforts. Our work has clearly benefited from the strong interdisciplinary collaboration among distinct yet complementary laboratories, both within the Center and with other ongoing projects on the University of Kentucky campus. The three specific aims of the Center Core are (1) Scientific: To generate and publish high quality prevention science that is recognized as state-of-the-art research across relevant disciplines; (2) Organizational: To provide an intellectually stimulating, creative, and productive environment in which research questions and issues are examined from a multilevel, interdisciplinary perspective; and (3) Leadership: To serve as a resource, advocate and leader for drug abuse and prevention science, in part by training the next generation of drug abuse prevention researchers. The substantive theme that consistently links scientists and research projects together in our Center is a broad and in-depth focus on N/S seeking behavior in the onset and development of drug abuse, and in applying this information for the development of more focused and effective prevention efforts. The three projects proposed in the current application continue and extend the Center?s on-going evaluations of the effects of N/S seeking on drug use and abuse. (1) Animal Laboratory: Individual Differences in Drug Response: An Animal Model (Mike Bardo, PI). This project will use an animal model to study the relationship between N/S seeking and amphetamine reward; the guiding hypothesis is that novelty, similar to stimulant drugs of abuse, activates the mesolimbic dopamine reward system. (2) Human Laboratory: Drug Abuse Liability and Sensation Seeking Status (Tom Kelly, PI). This project will expand the research team?s prior work on N/S seeking as a moderator of the relationship between drug dose and the discriminative, reinforcing and other behavioral effects and abuse liability of marijuana, nicotine, alcohol and methylphenidate. (3) Community Laboratory: Comprehensive Drug Prevention for Youth (Dick Clayton, PI). This project will continue a long-standing Center tradition of evaluations of school-based, curriculum-driven prevention programs with the evaluation of a comprehensive prevention program featuring both universal and selective high sensation value components.

DESCRIPTION (provided by applicant): Drug abuse is a significant health-related problem and novel preventive intervention strategies are needed to address this problem. The Center for Drug Abuse Translation (CDART) is a prevention center responding to the NIDA Center of Excellence (PA-10-189). CDART proposes to determine the biologically-based facets of personality that increase risk for drug abuse and to test different preventive interventions that target these multiple risk-related facets. The central theme of CDART is that: (1) sensation seeking involves the reward relevant dopamine system and is most closely related to initiation of use; (2) disinhibition involves the inhibition-relevant prefrontal cortex and is most closely relatd to escalation of use; and (3) urgency, or mood-based rash action, involves the stress-relevant amygdala and is most closely related to problem use. CDART hypothesizes that prevention interventions may be maximally effective if they are tailored specifically to these risk-related facets. Three integrated research projects are proposed; one will use laboratory rats and two will use college students transitioning from adolescence to young adulthood. Using neurochemical and electrophysiological techniques in rats, as well as neuroimaging techniques in humans, the basic neural mechanisms that mediate these different risk-related facets will be assessed. Humans varying in risk-related facets also will be tested for their response to drugs of abuse under controlled laboratory conditions. This basic research will be translated into the application of prevention in small-scale efficacy trials. An especially innovative aspect of the proposed work is that it will test the hypothesis that individuals high in urgency will be more responsive to an intervention that promotes emotional self-regulation (i.e., mindfulness training) relative to an intervention tailored to high sensation seekers. In addition to the specific researc proposed in the 3 research projects, an administrative core, a statistics/psychometrics core and a training/pilot program core will further synergize the projects. An outreach component also is proposed to enhance the bi-directional transfer of information between basic scientists and prevention practitioners. The long-range goal is to improve the design and implementation of targeted anti-drug preventive interventions.

The current renewal application for the Center for Drug Abuse Research Translation (CDART) consists of a core and four projects that are linked around the central theme that individual differences in personality are important predictors of drug abuse vulnerability during adolescence and young adulthood. Previous work in our center and other laboratories has identified sensation seeking as a critical personality construct associated strongly with drug use, especially use of illicit drugs. In the current proposal, sensationseeking will be divided into two separate constructs, namely, reward seeking and inhibition, both of which are thought to be associatedwith drug use. In Project 1 (Bardo, PI), a laboratory rat model will be used to determine if individual differences in reward seeking and inhibition are associated with stimulant drug self-administration and mesocorticolimbic cellular processes. In Project 2 (Kelly, PI), human volunteers will be used to assess if individual differences in reward seeking and inhibition are associated with differences in drug self- administration and neural function as measured by fMRI and EEC. In Project 3 (Lynam, PI), a cohort of students transitioning from high school to college will be used to determine if individual differences in reward seeking and inhibition are associated with the initiation, escalation and/or cessation of drug use in a longitudinal design. In Project 4 (Zimmerman, PI), students will be used in experimental reserch designed to evaluate if individuals who vary in reward seeking and inhibition show a differential response to televised anti-drug public service announcements. There are three specific aims in the Core: one scientific, one organizational, and one that involves continuing and expanding leadership in translating the prevention science and drug abuse research communities. In relation to the four projects, some key functions of the Core are to: (1) provide thematic scientific integration of the different projects around the constructs of reward seeking and inhibition; (2) create synergy across the four proposed projects and other ongoing projects on campus; (3) facilitate interdisciplinary collaboration among basic and prevention sciences using both neurobehavioral and psychosocial levels of analysis; and (4) oversee the long-range goal of translating our basic research into practice by designing more effective anti-drug public service announements that target at-risk young adults.

Recent theories describe drug abuse as a disorder involving not only positive incentive motivation, but as a disorder also charactered by a loss of inhibitory control. In this project, we will use a laboratory animal model to determine the critical neural mechanisms that underlie the association between risk-related traits and drug abuse, questions that cannot be addressed fully using human subjects. Our working hypothesis is that individual differences in the risk-related traits, reward seeking and inhibition, play a role in drug abuse vulnerability and that individual differences in these two behavioral constructs are mediated by dissociable neural systems. While reward seeking and inhibition undoubtedly involve complex and overlapping neural circuits, our current knowledge suggests that reward seeking is subserved by ascending mesocorticolimbic dopamine (DA) systems, whereas inhibition is subserved by frontal cortical regions involving both DA and serotonin (5-HT). Anatomical studies have also revealed that the medial prefrontal cortex (mPFC) is implicated in both reward seeking and inhibition, whereas the orbitofrontal cortex (OFC) is implicated in inhibition. These neural systems will be examined to determine their role in the association of reward seeking and inhibition with drug reward. Rats will be assessedfor individual differences in reward seeking and inhibition using a variety of behavioral tests and then will assessed for amphetamine reward, DA and 5- HT transporter function, and neural activity assessed by in vivo voltammetry and electrophysiological studies. We predict that rats that are high in reward seeking and low in inhibition will be most vulnerable to the rewarding effect of amphetamine and related drugs. Also, we predict that rats that are high in reward seeking and low in inhibition will be most sensitive to nondrug alternative reinforcers that compete with drug reward, which may have important translational implications for the design of effective drug abuse prevention intervention strategies in humans. Finally, we predict that individual differences in reward seeking and inhibition will be associatedwith differences in DA and 5-HT systems within the nucleus accumbens, mPFC and/or OFC. These results, combined with the human behavioral pharmacology and neuroimaging results in Project 2, will provide a more comprehensive understanding the neural systems involved in risk-related traits relevant to the design of tailored anti-drug prevention intervention messages.

The central theme of CDART is that multiple facets of impulsivity defined by the UPPS scale of Whiteside and Lynam (2001) are related to different paths to drug use and that these paths are associated with different neurobiological mechanisms. To the extent that we can link specific behavioral tasks to these different self-report facets, animal models can be used to identify critical neural mechanisms that undedie the association between risk-related facets and drug use. A detailed understanding of the mechanisms that mediate the link between different facets of impulsivity and drug use will provide information about designing preventive interventions that target these risk-related facets. The overall goal of this preclinical project is to test two overarching hypotheses: (1) individual differences in the facets termed urgency, disinhibition and sensation seeking are related most strongly to problem use, escalation and initiation respectively, as operationally defined within an animal model of drug self- administration; and (2) individual differences in urgency, disinhibition and sensation seeking are related most strongly to differences in amygdala, prefrontal cortex and nucleus accumbens respectively, as measured by in vitro neurochemistry and in vivo electrophysiology. The specific aims are to: Aim 1: Develop an animal model of urgency using a task similar to that developed in humans. Aim 2: Determine if individual differences in urgency, disinhibition and sensation seeking predict problem use, escalation and/or initiation of amphetamine self-administration, respectively. Aim 3: Determine if individual differences in facets of impulsivity differentially predict the ability of nondrug interventions to reduce use, escalation and/or initiation of amphetamine self-administration. Aim 4: Determine if individual differences in these risk-related facets of impulsivity are related to alterations in monoamine transporter function in amygdala, prefrontal cortex and/or nucleus accumbens. Aim 5: Determine if individual differences in these risk-related facets of impulsivity are related to altered patterns of neuronal activity in amygdala, prefrontal cortex and/or nucleus accumbens. RELEVANCE (See instructions): Anti-drug preventive interventions may be most effective if they are designed to target those at highest nsk. The current project will explore the basic neurobehavioral mechanisms that mediate risk. The long- term goal is to translate these basic research findings into the design of targeted biologically-relevant preventive interventions that can be evaluated in field studies.

PROJECT SUMMARY One of the most difficult problems facing drug abuse practitioners is the continuing high rate of relapse following treatment. While there are multiple internal states and external cues that may trigger relapse, one common factor is to re-associate with drug-using social peers. Although some recent preclinical work has examined the role of social influence on acquisition and maintenance of drug self-administration, there is essentially no information about the role of social cues in reinstatement of drug seeking. The overall hypothesis of this project is that self-administering social peers will serve as a cue to induce drug seeking in a controlled preclinical laboratory setting. Separate from discrete cues (light, tone) and contextual cues (tactile and visual cues, time of day), these studies will determine if social peers also are able to reinstate extinguished responding for drug. In addition, these studies will provide definitive information about the potential interactive effects of social and contextual cues in reinstatement. Two specific aims are proposed. First, we will determine if there are sex differences in discriminative social cue-induced reinstatement and if social cues potentiate the ability of contextual cues to reinstate drug seeking. Second, in these of behavioral experiments, c-Fos protein immunoreactive cells will be quantitated in various brain regions to determine if neural activation following social cues differs between males and females or if activation by social cues differs from contextual cues. If social cues prove to be a trigger for drug seeking, this would provide evidence that informs psychosocial treatments which emphasize skill-building to avoid deviant peers.

Project Summary/Abstract Misuse of opioids represents a substantial public and economic burden in the US and worldwide. The existing pharmacological approaches to treatment of opioid use disorder (OUD) are most efficacious when coupled with behavioral therapies that target individual triggers to reduce or eliminate excessive drug consumption. While individual variability in opioid use have been acknowledged repeatedly in animal behavioral models, the genomic markers linked to neurobiological adaptations underlying such variability are not well understood. We argue that understanding the molecular background of individual differences in behavioral vulnerability to opioid use is critical for development of personalized pharmacogenomic approaches for OUD that may replicate clinical success of personalized cancer treatments. In line with this argument, we hypothesize that individual behavioral variability in escalation of fentanyl use is linked to systems level variability of genomic and functional networks within in the nucleus accumbens and prefrontal cortex. Escalation of drug intake is a central component of OUD diagnosis that can be modeled in animals trained to self-administer opioids under extended access conditions. Based on the published literature and preliminary data, we propose three complementary Aims to monitor development of escalated intake at behavioral, functional cellular/network, and genomic levels of analysis. Our Aim 1 hypothesizes that escalation of fentanyl intake emerges on the background of individual differences in sensitivity to non-drug (sucrose) reward. Finding evidence to support this aim has the potential to identify vulnerable individuals prior to initiation of opioid use. Aim 2 examines neuronal outcomes associated with escalated fentanyl intake. Specifically, we will evaluate whether individual profiles of escalated intake reflect altered regulation of cell excitability by four potassium channel families and the impact on neuronal output at single cell and network levels. The data collected as part of this aim will establish functional, neuronal drivers of vulnerability to escalated intake. Finally, Aim 3 compares the genomic landscape underlying variable fentanyl escalation in laboratory animals (rats) to human opioid use databases. In this aim, we take advantage of cell-type specific RNA sequencing to evaluate both neuronal and non-neuronal mechanisms of escalated intake in the nucleus accumbens and prefrontal cortex. This aim is expected to identify novel molecular pathways linked to fentanyl escalation and test the translational relevance of our preclinical findings to a human sample. To characterize interactions at the behavioral, functional, and genomic levels of analysis, a unifying statistical framework is developed based on linear mixed models to examine the strength of bi-directional relationships between behavioral escalation of intake and molecular outcomes.