Wendy J. Lynch - US grants

DESCRIPTION: (Applicant's Abstract) Drug research involving both humans and animals has focused predominately on the study of males. However, it is now apparent that men and women differ on several characteristics of drug addiction. These differences may be due to a real gender difference, or to sociocultural factors. Animal models that mimic the different phases of the addiction process might be useful in addressing this question. This project will focus on differences between male and female rats in three phases of the addiction process: acquisition, maintenance, and relapse. Additionally, it will investigate the contribution of the estrous cycle and ovarian hormones to any differences that are obtained. Acquisition of IV cocaine, heroin, and nicotine self-administration will be investigated using an autoshaping procedure. The criteria for acquisition of drug self-administration will be standardized across subjects and adjusted for dose and drug available. Regulation of cocaine, heroin, and nicotine intake will be-investigated using a two-lever drug self-administration procedure that allows the subject to increase and decrease their dose in discrete steps throughout an experimental session. The correlation between interdose interval and preceding dose size will be determined daily for each subject as a measure of regulation. Relapse to cocaine self-administration will be investigated using a priming model of drug reinstatement in which lever pressing for drug is extinguished by replacing drug infusions with saline infusions. Reinstatement of responding is then tested by administering a priming injection of drug. Specific aims are to compare in male and female rats: (1) the rate of acquisition of cocaine, heroin, and nicotine self-administration, (2) the effects of ovarian hormones on acquisition of cocaine, heroin, and nicotine self-administration, (3) regulation of cocaine, heroin, and nicotine intake, (4) regulation of cocaine, heroin, and nicotine intake across stages in the estrous cycle (in female rats) and, (5) relapse to cocaine self-administration as a function of priming dose of cocaine.

DESCRIPTION (provided by applicant): Despite the prevalence of cocaine addiction among women, few investigators have included females as subjects in cocaine self-administration experiments. Recent reports indicate gender differences in all phases of the addiction process including initiation and prevalence of use, patterns and levels of use, the progression to addiction, withdrawal, and relapse. While similar sex differences have also been observed in animals, few studies have investigated sex differences during the transition to addiction or the mechanisms that may underlie sex differences. Specific Aim 1 will characterize sex differences in cocaine self-administration behavior using a 24-hr access procedure that leads to high levels of intake and dysregulated patterns of use, features that may reflect compulsive aspects of addiction. An additional goal of Aim 1 is to explore the molecular and hormonal mechanisms that may underlie sex differences under high cocaine access conditions. Aim 2 will characterize the effects of high access to cocaine on subsequent motivational and behavioral changes and how these changes may differ by sex and ovarian hormone status. Specifically, we will investigate motivation to obtain cocaine as measured by responding under the progressive ratio schedule during early withdrawal (i.e., 0-72 hr) and after an extended drug-free period (i.e., 10-days). Additionally, since previous work has shown that chronic cocaine exposure produces not only motivational changes but is also associated with deficits in impulse control, Aim 3 will investigate the effects of high cocaine access on measures of impulsivity using a delay-discounting task. These experiments are of significance for drug abuse research and women's health research since they may help to identify molecular adaptations that occur following chronic cocaine self-administration and hormonal mechanisms that may underlie sex differences in vulnerability to cocaine addiction. Thus, these studies may provide a basis for the development of novel treatment strategies for cocaine abuse/dependence, particularly in women.

DESCRIPTION (provided by applicant): Drug addiction is the leading cause of preventable death in the United States. Effective treatments are critically needed, but developing treatments is challenging because the underlying neurobiology of addiction varies over time as the disease progresses. Our work over the past 15 years has revealed important sex and hormone effects in the different phases of addiction suggesting that its underlying neurobiology may also vary between males and females. In the last 5-yr grant period we expanded our behavioral work to include studies of the neurobiology underlying sex differences. Now, in this competitive renewal, we turn to characterization of sex-specific behavioral and neurobiological changes that underlie the development of addiction. This is important because the vast majority of data on the neurobiology of cocaine addiction is based on short access self-administration studies conducted in males. Short access conditions (1-2 hr/day), however, by virtue of their stability, do not capture critical features of addiction in humans, including an enhanced motivation to use the drug and enhanced relapse vulnerability. Extended access procedures (6-24 hr/day) have been developed that produce these characteristics and reveal that the underlying neurobiology is different from that observed under short access procedures. An enhanced motivation for cocaine, as measured under a progressive-ratio schedule, has been used to define the development of addiction in rats. This characteristic develops over an abstinence period following extended, but not short access self- administration, and develops sooner in females compared to males. We recently showed that while dopamine (DA) D1 receptor signaling in the nucleus accumbens (NAc) is a critical mediator of motivation for cocaine following short access self-administration, once addiction is established, its role is diminished and glutamate AMPA/KA receptor signaling appears to be critically involved. In females, estradiol appears to be necessary for the development of an addicted phenotype and the shift to a diminished role for DA. We hypothesize that a shift from NAc DA to AMPA transmission underlies the development of addiction, and that in females, this shift is accelerated, and estradiol is necessary for it to occur. To address this hypothesis, in Aim 1 we will determine the time-point during abstinence for this shift to determine if it underlies the development of addiction and the accelerated time-course in females. In Aim 2, we will pin-point the dopamine versus glutamate receptors involved, and in Aim 3 we will evaluate the requirement of estradiol in its development. These results will greatly contribute to our understanding of not only the neurobiological processes relevant for the development and expression of addiction, but also how sex and ovarian hormones influence these processes.

DESCRIPTION (provided by applicant): Neuromodulation of cortico-mesolimbic dopamine (CMDA) function may be more effective for the treatment of alcohol dependence than DA receptor blockade, and consistent with this hypothesis, we have shown that ondansetron a serotonin-3 antagonist that modulates CMDA primarily in the ventral tegmental area and nucleus accumbens (NAc), is efficacious treatment for a subtype of alcohol dependent humans with high biological loading for the disease. We have also demonstrated that topiramate, a GABA/glutamate (GLU) modulator that is believe to produce widespread suppression of CMDA, is an efficacious treatment for a heterogeneous group of alcohol dependent individuals. Because ondansetron and topiramate manifest their effects through different neuronal processes, both resulting in modulation of CMDA function, it is reasonable to hypothesize that their combination shall be more efficacious than either alone in the treatment of alcohol dependence. Therefore, we propose to characterize the mechanistic process by which ondansetron and topiramate, both alone and in combination, exert their behavioral and neurochemical effects using different rat self-administration models and strains. In Aim 1 and 2 we will examine the prediction that both ondansetron and topiramate alone and in combination will reduce ethanol reinforcement, and in Aim 2 and 3 we will examine the prediction that both ondansetron and topiramate alone and in combination will reduce ethanol reinstatement. The combination is expected to be associated with added or synergistic decreases in ethanol reinforcement and reinstatement as well as associated modulation in CMDA and GLU concentrations. This pharmaco-behavioral response pattern or finger-print will enable us to identify future promising putative medications with similar effects as well as enable us to understand which of their properties can be harnessed to develop even more potent medications. PUBLIC HEALTH RELEVANCE Our study is significant because it focuses on determining the biological basis for the effects of two potential medications, ondansetron and topiramate, for treating alcohol dependence. The overall goals of this basic science work is to helping to identify populations that may be ideally suited for treating alcohol dependence with these two medications, and to provide information that may guide the development of even more effective medications.

? DESCRIPTION (provided by applicant): Long-term neuroadaptive and epigenetic changes that develop following chronic drug exposure are believed to underlie persistent behavioral changes that characterize addiction. Thus, one strategy for treating addiction is to focus on interventions that can reverse these changes, which would theoretically reverse the addiction process back to a level where the individual is no longer compulsively seeking drugs. We recently demonstrated that exercise during abstinence is one type of intervention that is able to reverse/block drug- induced neuroadaptations associated with relapse vulnerability. The goal of this R01 proposal is to determine the mechanism for the efficacy of exercise as an intervention for cocaine addiction focusing on epigenetic regulation of brain-derived neurotrophic factor (BDNF) in the prefrontal cortex (PFC). BDNF is one of the few markers that positively associates with relapse vulnerability in both humans and animal models. Evidence indicates that increasing BDNF during early cocaine abstinence, when levels are low, prevents its subsequent increase as well as the increase in relapse vulnerability. Exercise is known to both increase BDNF and to attenuate cocaine-seeking, with evidence to suggest that it exerts these effects through epigenetic regulation of Bdnf exon IV. Notably, we showed that wheel running, an animal model of exercise, beginning during early abstinence dose-dependently reduced subsequent cocaine-seeking and Bdnf exon IV expression in the PFC. Our recent data also show time-dependent effects of exercise where during early abstinence, but not during later abstinence, it effectively reduced subsequent cocaine-seeking. Therefore, we hypothesize that exercise, through epigenetic regulation, blocks cocaine-induced decreases in BDNF that are observed during early abstinence, thus preventing compensatory neuroadaptations that lead to enhanced cocaine-seeking. Based our recent findings showing that the efficacy of exercise at reducing cocaine-seeking differs between males and females and in females at different hormonal states, we further hypothesize that sex and hormone-specific differences in the effects of exercise on BDNF signaling and remodeling underlie its efficacy. These hypotheses will be tested using an animal model of cocaine relapse wherein following chronic cocaine self-administration, cocaine-seeking increases, or incubates, over protracted abstinence. To accomplish our goals, in Aim 1 we will first determine the markers of BDNF signaling and remodeling that are associated with the incubation effect in males and in females at different hormonal states, and then in Aim 2 we will determine the effects of exercise on these changes. Finally, in order to establish a causal mechanism for the efficacy of exercise, in Aim 3 we will assess the effects of site-specific manipulation of PFC BDNF alone and in combination with exercise. This is an understudied area of research and the results will greatly contribute to our understanding of not only exercise as a sex-specific intervention for cocaine addiction, but also how sex and ovarian hormones influence the process of addiction and recovery.

Abstract This new program is a marriage of three important research fields; Drug Addiction, Sex Differences, and Environmental Health. Sex differences in drug-related behaviors have been well documented and attributed to actions of estrogens and progesterone in adult females. Here we move beyond this hypothesis and examine two other factors both of which have been implicated in reward via their actions on the dopamine system. Endocrine disruptors (EDCs) are ubiquitous in the environment and are known to alter sex differences in brain and behavior. EDC exposure is particularly potent when it occurs during neonatal development. As such it is a model for fetal origins of adult disease. This two-year grant will test the hypothesis that exposure to Bisphenol A (BPA), one of the well-studied and abundant EDCs in the environment, can increase vulnerability to cocaine. To further dissect the role of gender on drug vulnerability the four core genotype (FCG) mouse model will be used. The FCG allows separate analysis of gonadal hormones and sex chromosome complement (XX vs. XY). In aim one a dose-response study will be conducted with BPA exposure during gestation. In the next aim the FCG mice will be tested. Adult mice are fitted with chronic indwelling jugular cannula which allow them to self-administer cocaine. Fixed and progressive ratio schedules are used to examine self-administration and measure both acquisition and maintenance. The long term objectives of this work are to develop a research program to test the effects of environmental endocrine disrupting compounds in combination with gender, on motivated drug seeking and taking behavior.

ABSTRACT Relapse to drug seeking after abstinence is a major challenge in treating substance use disorder. Abstinent drug users remain at risk of relapse even after extended drug-free periods. Exposure to drug-associated cues or stress during abstinence can trigger intense craving and precipitate relapse. New and more effective anti-relapse interventions are critically needed, particularly for cocaine use disorder since no effective pharmacological intervention is available. We discovered that a nutritional supplement we developed for clinical use, as part of a nutritional approach for managing substance use disorder, had the benefit of being a potentially robust anti-relapse therapy. This supplement, SMAASH-C, contains a combination of vitamins, omega-3 fatty acids, and minerals, as well as tyrosine and other amino acids that are known to be depleted by chronic cocaine exposure. We originally learned of its potential therapeutic value through anecdotal reports from our patients of reduced drug craving, reduced stress and anxiety, and less frequent relapses. As a follow-up, we screened SMAASH-C in a rat model of relapse, and confirmed its potential efficacy as an anti-relapse intervention. Specifically, we found that 15-days of SMAASH-C treatment during abstinence, via a clinically relevant oral route (mixed in food), resulted in a 50% reduction in extinction and cue-induced cocaine-seeking as compared to control treatment (non-supplemented food). Notably, SMAASH-C did not affect body weight, food intake, or non-specific responding during the cocaine-seeking tests indicating its potential as a safe and selective intervention. In fact, results from toxicology analyses of a 12-week SMAASH-C treatment regime in saline and cocaine-withdrawn rats suggests that it offsets markers of cocaine-induced toxicity (e.g., oxidative stress, hepatic injury). The overall objective of this R21 application is to provide preclinical proof-of-concept data for the use of SMAASH-C as an effective, safe, and selective anti-cocaine relapse intervention. To do so, we will determine its efficacy at reducing cocaine-seeking following extended access self-administration and protracted abstinence, and in response to two of the most common triggers of craving and relapse in humans: drug-associated cues (Aim 1) and stress (Aim 2). Selectivity will be assessed by comparing effects on cocaine- versus sucrose-seeking. Both males and females will be included given literature highlighting the need for sex-specific interventions. As a first step toward understanding the mechanisms underlying its efficacy, we will examine markers of dopaminergic and glutamatergic signaling in the reward pathway following relapse testing using qPCR and Western Blot analysis. Our overall hypothesis is that SMAASH-C will robustly (>50%), selectively (no effect on sucrose-seeking), and dose-dependently decrease relapse vulnerability and associated neuroadaptations in both males and females. Our long-term goal is to bring SMAASH-C into clinical trials as a single or adjunct intervention for cocaine relapse. This application is a major step toward this goal since the results will provide the necessary preclinical proof-of-concept data.

Abstract Sex differences in drug-related behaviors have been well documented and attributed primarily to differences in levels of estrogens in adult men versus women. This program advances the field beyond this hypothesis to examine another important factor, sex chromosome complement, with a focus on X-chromosome genes that escape X-inactivation which are expressed in greater levels in female versus male brains. The four core genotype (FCG) mouse model allows separate analysis of gonadal hormones and sex chromosome complement (XX vs. XY). In Aim 1 these mice will be used to examine combined and separate actions of estradiol and sex chromosome complement on vulnerability to cocaine addiction using intravenous self- administration. Rates of acquisition and levels of motivation to obtain the drug will be evaluated. In Aim 2, we will define the role of two X-chromosome genes that escape inactivation in mouse and human brain, Utx and Smcx. These genes code for enzymes that demethylate histone modifications, regulating chromatin accessibility and transcription on a wide basis. This work will be conducted with two lines of inducible, tissue- specific knockout mice that lack the expression of either gene (Utx or Smcx) in CaMK2? in forebrain, including neurons in the nucleus accumbens. The mice will be tested as in Aim 1 for cocaine vulnerability and motivation. In Aim 3 brain tissue from the nucleus accumbens of mice tested in Aims 1 and 2 will be used for epigenetic analysis. A combination of ATAC (Assay for Transposase Accessible Chromatin-), Pro (Precision Nuclear Run-On-) and ChIP (Chromatin Immuno-Precipitation-) Sequencing will be used to identify transcriptionally active and repressed genes associated with chromatin restructuring and important transcription factors. The long term objective of this work is to reveal new mechanisms that underlie sex differences in vulnerability to addiction that help clinicians design sex-specific preventions/interventions.

ABSTRACT Opioid use disorder (OUD) is a major epidemic in the US. Drug craving and relapse are major challenges in treating OUD as abstinent drug users remain at risk of relapse even after extended drug-free periods, and well after the acute physical withdrawal symptoms have subsided. Once abstinent, long-lasting negative affective symptoms contribute to increased drug craving, and following exposure to drug-associated cues or stress, an increased risk of relapse which further intensifies over abstinence. New treatments are critically needed to reduce craving and prevent relapse for OUD as current approaches result in ~50% relapse. One promising anti-relapse intervention is kratom, Mitragyna speciosa, a medicinal herb used for centuries in Southeast Asia and more recently in the US, as a self-treatment for pain, depression and anxiety, and opioid withdrawal, craving, and relapse. There are an estimated 3 to 5 million users of kratom in the US, and despite a high prevalence of kratom use as a self-treatment for OUD, its efficacy has yet to be established in humans or animal models. While kratom itself has not yet been examined in the context of addiction, there have been a few studies conducted with alkaloid-rich extracts of kratom and several with mitragynine (MG), its most abundant alkaloid. Findings demonstrate that while both compounds induce positive reinforcing effects, their abuse liability is relatively low. Importantly, as with reports in humans, in animals, kratom (extract) and MG reduce opioid withdrawal symptoms, and these effects are believed to occur via agonistic effects at mu-opioid receptors (MOR). However, unlike classical opioids, MG does not recruit ?-arrestin signaling, and thus the beneficial effects associated with MOR activation (analgesic, anxiolytic, and antidepressant effects) can be obtained with less risk of dangerous side effects (i.e. respiratory depression). Together, these data provide compelling support for the overall objective of this R21 application: to determine in a rat model the potential utility of kratom as an anti- relapse intervention. To do so, we will first determine the composition of commercially-purchased kratom powder with respect to MG as well as 7-hydroxymitragynine (7-HMG), a structurally-related alkaloid that also has agonistic effects at MORs (Aim 1). Although less research has been conducted with 7-HMG since it constitutes significantly less of kratom's total alkaloid content (approximately 2% versus 66% for MG), it has a higher affinity and potency for MORs as compared to MG. Next, using characterized samples, we will determine the efficacy of kratom at reducing relapse vulnerability in response to two of the most common triggers of craving in humans, drug-associated cues (Aim 2) and stress (Aim 3). Effects will be determined in both males and females and correlational analyses will be conducted to determine how the composition of kratom, and specifically, levels of MG and 7-HMG, impacts its efficacy. The question posed in our application is translationally relevant and the results will provide science-based evidence that may influence the scheduling of kratom and help guide its development as a medically-sanctioned treatment for OUD.