Rick A. Bevins - US grants

Current research uses sensation seeking and novelty seeking as predictive constructs of risk taking behaviors in humans. That research suggests that risk taking behaviors of high novelty seekers are maintained by some rewarding (appetitive) aspect of experiencing novel stimulation. From this perspective, developing a animal model to elucidate the behavioral and neural substrates of novelty reward would have implications for prevention/intervention strategies in such areas as sexually transmitted diseases, teenage pregnancy, and drug use. Results from previously studied model preparations of novelty seeking/reward with non-human animals are susceptible to other, non- reward, interpretations (e.g., stress). The place conditioning procedures that will be used int he present proposal are much less susceptible to these alternative accounts. Rats will receive repeated access to novelty (i.e., unfamiliar objects) in one of two distinct environments. A shift in preference toward the novelty-paired environment relative to within-subject and between subject control values will be taken as evidence for novelty reward. Specific Aim 1 will determine whether the objects must be novel to condition a preference. Related work published on novelty seeking has emphasized the importance of the dopamine neurotransmitter system. Thus, Specific Aim 2 will examine the role of the dopamine D1 and D2 receptor families by assessing the ability of specific dopamine receptor antagonists to block novelty place conditioning. Specific Aim 3 will examine the relationship between the present measure of novelty reward and a behavioral measure typically used to study the relationship between novelty and dopamine (novelty-induced activity). The experiments proposed in the present B/START application will establish the place conditioning preparation in my new laboratory and provide a firm foundation in which to develop a systematic research program identifying the behavioral and brain mechanisms involved the appetitive (rewarding) properties of novelty.

Conditioning (learning) processes involving stimulus-nicotine associations are believed to be a major factor contributing to the continued abuse of tobacco products. The present proposal will use an animal model to elucidate the processes involved in the acquisition and expression of a conditioned association between environmental stimuli and nicotine. In this preparation, a distinct environment that has been repeatedly paired with nicotine comes to elicit an enhancement of activity relative to control conditions. This enhanced activity is taken as evidence for an association between the environment and the locomotor stimulant effects of nicotine. Specific Aim 1 will assess competing accounts of the enhanced locomotor activity --- excitatory Pavlovian conditioning vs. inhibitory conditioning or novelty-induced activity. The goal of Specific Aim 2 will be to manipulate procedural variables found in other Pavlovian conditioning preparations to alter learning (e.g., number of conditioning trials, temporal relation between environment and nicotine). In doing so, more optimal conditioning procedures will be identified. Specific Aim 3 will investigate whether nicotine pre-exposure attenuates subsequent nicotine locomotor conditioning as predicted by theories based on research with traditional Pavlovian conditioning paradigms or whether nicotine pre-exposure facilitates the conditioned locomotor effects as characterized by a smaller set of literature in the drug conditioning field. This aim will also determine how the conditioned and unconditioned locomotor effects of nicotine vary as a function of different pre-exposure protocols (e.g., context, number, interdose interval, continuous vs. intermittent delivery). Elucidating the behavioral processes underlying acquisition and expression of Pavlovian drug conditioning will have important implications for prevention and intervention strategies.

DESCRIPTION (provided by applicant): For an individual, chronic tobacco use is associated with a significant increase in heart disease and many forms of cancer;the health of non-tobacco users is negatively affected by second- hand smoke. The potential benefits that would come from a better understanding of the etiology of nicotine dependence are enormous. Approaches to addiction that include Pavlovian conditioning processes have lead to important advances in our understanding and treatment of drug use in general, and nicotine dependence specifically. These approaches most often treat nicotine as an unconditioned stimulus that enters into an association with other stimuli (cigarette, situational cues) that reliably co-occur with its effects. Albeit less studied, also of import to the development and tenacity of nicotine dependence is the ability of the pharmacological effects of nicotine to serve as an interoceptive stimulus for other reinforcing events (peer acceptance, alcohol, work breaks, stress relief, etc.). In a preclinical model, rats that have such an appetitive learning history with nicotine will show evidence of conditioning controlled by the nicotine stimulus. This conditioning follows many of the known principles of conditioning and does not reflect state-dependent learning. The most provocative finding to date is that conditioning to the nicotine stimulus can be weakened by repeated non-reinforced presentation of an alternative drug (i.e., extinction) that shares interoceptive stimulus properties with nicotine. This "transfer of extinction" has been seen with varenicline (the smoking cessation aid Chantix(R)) and nornicotine (a potential smoking cessation aid that is a metabolite of nicotine). This competing renewal will build programmatically on the progress from the previous funding period by examining more closely extinction with nicotine and the transfer of extinction in a series of carefully designed experiments. These experiments will provide insight into the nature of the nicotine stimulus and related drugs such as varenicline, as well as identify basic processes underlying extinction learning with nicotine. RELEVANCE: A better understanding of excitatory associations involving the interoceptive stimulus effect nicotine and how the expression of such associations are affected by non-reinforcement, including non- reinforcement with pharmacologically related drugs will provide new an important insights into factors contributing to chronic tobacco use and the tenacity of nicotine dependence. Such insights can be used to design better behavioral and pharmacological treatments. PUBLIC HEALTH RELEVANCE: Tobacco use reflects a major health problem that results from an addiction to nicotine. With an estimated annual economic cost around $167 billion, this nicotine addiction is associated with a significant increase in heart disease and many forms of cancer;the health of non-tobacco users is also negatively affected by second-hand smoke. The potential benefits that would come from a better understanding of chronic tobacco use are enormous and this benefit is the primary reason the long-term goal of our research program is to elucidate potential associative learning processes contributing to nicotine dependence.

DESCRIPTION (provided by applicant): Research supports the assumption that associative learning processes contribute to acquisition, maintenance, and/or relapse of habitual drug-taking patterns. One preclinical animal model that has contributed importantly to understanding these conditioned associations is the cocaine place-conditioning preparation in rats. In a "standard" place-conditioning experiment, one distinct environment is repeatedly paired with cocaine administration; a second environment is equally experienced without cocaine. In a later drug-free test, rats given free access to both environments will spend more time in the cocaine-paired environment indicating that the paired environment has acquired rewarding value that controls approach/drug-seeking behaviors. Unfortunately, this widely used method is relatively insensitive to such variables as changes in cocaine dose (i.e., conditioned preference tends to be all-or-none) thus limiting its usefulness for a more detailed examination of processes mediating drug-seeking behavior. The main goal of the research in this proposal is to systematically assess the utility of a modified version of the standard protocol that overcomes some of these limitations. This new method, termed "reference-dose procedure," provides a known cocaine conditioning history in both environments (cf. standard protocol only conditions one environment). The following Aims will be used to obtain this goal. Specific Aim 1 will assess the change in choice behavior that occurs when varying doses of cocaine paired with one environment (e.g., 0.1, 0.3, 0.45, 0.6, 0.9, or 1.2) compete with another environment paired with a moderate or low reference-dose of cocaine (i.e., establish dose-effect curves). Specific Aim 2 will determine the extent to which the reference-dose method increases the sensitivity of the place conditioning procedure to antagonism using the dopamine D1 receptor antagonist SCH-23390. This research will provide the requisite foundation for this more sensitive reference-dose method. In doing so, new research into basic cognitive, behavioral, and neurobiological processes mediating drug-conditioned choice behavior will become possible. That research could translate into more effective intervention strategies.

DESCRIPTION (provided by applicant): Theoretical and empirical approaches to drug abuse that include associative learning processes have lead to important advances in our understanding and treatment of drug dependence. These approaches typically treat the drug as an unconditioned stimulus (US). That is, the biological effects of the drug (e.g., reward, psychomotor stimulation) enter into an association with stimuli that reliably co-occur with these effects (e.g., paraphernalia, situational cues). Recent research has shown that the pharmacological (interoceptive) effects of a drug also serve as a CS for other rewarding USs. Such an appetitive conditioning history with a drug state could speed the trajectory from experimentation to dependence or increase the tenacity of the addiction. The experiments proposed in this application will test an important prediction of this perspective using a preclinical animal model in which nicotine functions as the CS. This research builds programmatically on our past research with nicotine. More specifically, rats will receive intravenous (IV) nicotine paired with brief access to a sucrose solution (i.e., an appetitive US). In preliminary research, the nicotine CS comes to evoke an appetitive CR (anticipatory sucrose-seeking behavior). Our working hypothesis attributes this CR to the interoceptive nicotine CS acquiring additional appetitive properties by virtue of being reliably paired with an appetitive outcome. This hypothesis is consistent with most Pavlovian conditioning theories and suggests that nicotine's ability to serve as a reward may be modified by an appetitive conditioning history. Accordingly, the main goal of this application is to programmatically test this prediction with a widely used preclinical model of drug reward-place conditioning. The following Aims will be used to accomplish this goal. Specific Aim 1 will determine how conditioned responding varies as a function of IV nicotine dose (e.g., 0.005 to 0.06 mg/kg) used as the CS. In doing so, we will identify nicotine doses that have interoceptive stimulus properties that also acquired additional appetitive properties from this learning history. Aim 2 will determine whether having this appetitive conditioning history will alter nicotine's ability to then function as a reward (i.e., US) in an IV nicotine place conditioning task. We expect an appetitive learning history with nicotine to potentiate its later rewarding effects. PUBLIC HEALTH RELEVANCE: Tobacco use reflects a major health problem that results from an addiction to nicotine. With an estimated annual economic cost around $167 billion, this nicotine addiction is associated with a significant increase in heart disease and many forms of cancer;the health of non-tobacco users is also negatively affected by second-hand smoke. The potential benefits that would come from a better understanding of the etiology of tobacco dependence are enormous and is the reason the long-term goal of our research program is to elucidate potential associative learning processes contributing to nicotine dependence.

DESCRIPTION (provided by applicant): Chronic tobacco use is associated with a significant increase in heart disease and many forms of cancer; even the health of non-tobacco users is negatively affected by second-hand smoke. The potential benefits to the individual and society that would come from a better understanding of the factors that contribute to nicotine dependence are enormous. Conditioning is one important factor that contributes to the addiction cycle. Research and theory on drug conditioning most often treats nicotine as an unconditioned stimulus or reinforcer that enters into an association with other stimuli (cigarette, situational cues) that reliably co-occur with its physiological effects. An understudied area that is also important for the development and tenacity of nicotine dependence is the ability of the pharmacological effects of nicotine to serve as an interoceptive stimulus for other reinforcing events (peer acceptance, alcohol, work breaks, stress relief, etc.). Indeed, there has been a recent rise in investigators' and practitioners' awareness of the role of interoceptive conditionin to a wide range of psychopathologies and diseases-including addiction. We have developed a preclinical model to study interoceptive conditioning with nicotine. Rats that have an appetitive learning history with nicotine will show evidence of conditioning controlled by the nicotine stimulus; that is, anticipatory food-seeking. The most provocative finding to date is that conditioning to the nicotine stimulus can be weakened by repeated non- reinforced presentation of an alternative drug (i.e., extinction) that shares interoceptive stimulus effects with nicotine. This transfer of extinction has been seen with varenicline (the smoking cessation aid Chantix(®), bupropion (the smoking cessation aid Zyban®), and nornicotine (a potential smoking cessation aid that is a metabolite of nicotine and minor alkaloid in tobacco). The proposed research will build programmatically on this exciting finding by more closely examining novel approaches to deepening transfer of extinction, hence diminishing the control of nicotine over acquired appetitive behavior. In doing so, the experiments in this application introduce new methods for studying interoceptive stimuli to the field, begin to fill a significant gap in the scientific literature, advance our understanding of extinction processes involving nicotine, and elucidate the nature of the nicotine stimulus and treatment-relevant ligands.

? DESCRIPTION (provided by applicant): Smoking, ethamphetamine (meth), and alcohol use remain a significant health burden. Notably, clinical studies have identified sex differences in response and sensitivity to the drugs, as well as in abstinence and relapse rates. Interestingly, preclinical investigations on these topics have not been as prevalent. There is an extensive literature establishing that stimuli associated with drugs can be powerful modulators of drug-seeking behavior, and can promote relapse following a period of abstinence. One factor of importance to the development and tenacity of nicotine dependence is the ability of the pharmacological effects of nicotine to serve as an interoceptive stimulus for other reinforcing events (e.g., peer acceptance, alcohol, work breaks, stress relief, etc.). Consequently, over time a smoker has an opportunity to develop a rich appetitive conditioning history with the interoceptive stimulus effects of nicotine. An important prediction from theories of conditioned reinforcement and incentive motivation is that an excitatory conditioned stimulus, nicotine in our case, should be able to support other behavior. Using preclinical models, one goal of the present application is to directly test the hypothesis that following an appetitive interoceptive conditioning history with nicotine, the nicotine stimulus will acquire additional motivational valu that will exacerbate reinstatement of methamphetamine (Aim 1) and alcohol (Aim 2) seeking to a greater degree than not having this conditioning history. Another goal is to determine whether sex differences exist in these behaviors. The proposed studies will fill an important gap in the literature, as it is unknown whether interoceptive conditioning involving a drug stimulus can alter the extent of reinstatement that had been maintained by a different drug. This is an innovative conceptual framework by which to approach the subject of relapse in addiction. Accordingly, confirmation of this hypothesis will advance current theory as it reveals a potential new process that may serve as a risk factor in relapse. Opening a new area of scientific inquiry, advancement of theory, and improvement of animal models are consistent with the objectives of the R21 mechanism - Exploratory/Developmental Research Grant Award.

PROJECT ABSTRACT Nicotine is the primary addictive constituent in tobacco. For an individual, chronic tobacco use is associated with a significant increase in heart disease and many forms of cancer; even the health of non-tobacco users is negatively affected by second-hand smoke. There are some important sex differences in this addiction and its health consequences. Women on average take less time than men to become nicotine dependent, make fewer attempts to quit smoking, abstain for less time, relapse at higher rates, and benefit less from nicotine replacement therapy. Clearly, enormous gains to the individual and society would come from a better understanding of factors that contribute to nicotine dependence. Environmental stimuli associated with drugs such as nicotine can be powerful modulators of drug-seeking behavior. Another important factor to the development and tenacity of nicotine dependence is the pharmacological effects of nicotine serving as an interoceptive (internal) stimulus entering into a conditioned association with other reinforcers/unconditioned stimuli [USs (peer acceptance, alcohol, work breaks, stress relief)]. Consequently, over time, a smoker has an opportunity to develop a rich appetitive conditioning history with the stimulus effects of nicotine. This acquired reinforcing value likely contributes to the tenacity of the nicotine addiction. Using a newly developed preclinical animal model that innovatively merges interoceptive conditioning with nicotine self-administration, the present application will rigorously test the hypothesis that following an appetitive interoceptive conditioning history with nicotine, the nicotine stimulus will acquire conditioned reinforcing value that exacerbates the persistence of nicotine-taking behavior. To this end, the Specific Aims will examine how such enhancement of drug intake varies with the salience (dose) of the nicotine stimulus (Aim 1), assess the synergy between nicotine and ethanol by using ethanol or sucrose-ethanol solution as the US (Aim 2), and implement an innovative extinction protocol designed to weaken the acquired conditioned reinforcing value of nicotine to a greater extent than just simple non-reinforcement of the target stimulus (Aim 3) in female and male rats.

The abuse of the potent psychostimulant methamphetamine (meth) continues to pose a significant threat not just in the US but also globally. A significant attribute associated with chronic meth induced brain dysfunction is inflammation includes activation of glial cells such as astrocytes and microglia that play a crucial role in modulating inflammation including glutamate excitotoxicity at the synapse. Mounting evidence suggests that inflammation and alterations in glutamate neurotransmission are two novel pathways associated with the pathophysiology in mood disorders. Notably, this cross talk between neurons and glial cells is mediated by extracellular vesicles (EVs) which are emerging as key players in regulating brain function. Chronic meth dependent individuals, including those abstinent and then relapse, display significant behavioral (mood) alterations such as psychosis. A significant gap in knowledge is understanding how meth-induced inflammation perturbs glutamatergic physiology which subsequently impair EV dynamics and exacerbates relapse. Adding another layer of importance is emerging studies showing a role for sex differences with females progressing more quickly to regular use of meth as well as greater dependence and higher relapse rates. Given the lack of studies elucidating the role of EVs with meth relapse between the sexes, our proposed studies are well poised to address this important knowledge gap. Accordingly, the overarching goal of this proposal is to examine the role of EVs in the damaging effects of meth between the sexes using drug-triggered reinstatement (relapse) of extinguished intravenous meth self-administration in rats. Based on our recent preliminary studies with females showing higher inflammation, enhanced glutamatergic alterations, and significantly higher drug-triggered reinstatement (i.e., meth seeking), we hypothesize that EV biogenesis, release, and associated cargo are more impacted in females than males. Using an array of complementing behavioral, molecular, and biochemical approaches in a ?gold standard? preclinical model or reinstatement we will : determine how the anti-inflammatory drug ibudilast alters dynamics of EV release, biogenesis, and associated cargo between the sexes (Aim1); Characterize role of EVs isolated from the sexes on synaptodendritic damage (Aim 2 and) test meth induced changes in EV-associated surface proteins between the sexes (Aim 3). These studies will break new ground and importantly provide novel proof of concept studies which will further serve as a prelude to future basic research on developing EVs as sex-specific medication development for treating meth addiction. .!

PROJECT ABSTRACT The nature and challenge of illicit drug use in the United States continues to change rapidly, evolving in reaction to myriad social, economic, and local forces. While drug addiction affects every region of the country, most information about drug use comes from large urban areas. Emerging data on rural addiction and its harms justify greater attention. Record drug use, addiction, and overdose rates in rural states, coupled with a dearth of treatment facilities, point to a rapidly worsening health situation. While health sciences have made considerable progress in understanding the etiology of drug use and uncovering the link between drug use and its many associated harms, this promising scientific news has yet to translate to better rural health outcomes. The goal of this application is to create a sustainable, nationally recognized Center?the Rural Drug Addition Research Center (RDAR)?dedicated to understanding the causes and impacts of rural drug addiction and its related challenges and harms. Given the multifactorial nature of addiction, this research must unfold across a continuum of domains, ranging from the microscopic level of the synapse to larger social contexts. As such, RDAR will draw on senior investigators and existing resources from the University of Nebraska to mentor early career scientists in conducting cutting-edge research that addresses rural drug use challenges from multiple angles (i.e., from synapse to society). For example, initial research projects will focus on the neuroscience of addiction, cognitive implications of chronic drug use, the relationship between rural drug use and violence exposure, and spatial and structural simulation of drug-related disease epidemiology. To support these projects, RDAR will found the Longitudinal Networks Core (LNC), a research core built around cutting-edge cohort tracking and retention software developed by RDAR senior investigators to offer longitudinal cohort study capacity to RDAR projects. LNC will recruit and retain a cohort of rural drug users from which RDAR projects will regularly collect data. The Center will provide dedicated mentoring, professional development, and Center resources to enable early stage investigators engaged in rural drug addiction research to become independently funded researchers (Aim 1) while creating a sustainable environment for interdisciplinary research excellence on rural illicit drug use and its harms (Aim 2). This will lay the groundwork for long-term Center infrastructure (Aim 3) that allows RDAR researchers to lead efforts to address specific challenges raised by drug addiction in rural settings and develop appropriate interventions for those settings. RDAR will combine the resources of a nationally recognized research university (University of Nebraska-Lincoln) and a university-based medical center (University of Nebraska Medical Center) to create a sustainable research environment, capitalizing on existing collaborations with local practitioners and state policy and service organizations. Lastly, institutional support in the form of pilot project funding and new faculty hires in research areas important to RDAR will help ensure the long-term sustainability of the Center.