Mary M. Torregrossa, Ph.D. - US grants

DESCRIPTION (provided by applicant): Drug addiction is a devastating disease both for the addict and for society. Unfortunately there are few treatment options available that prevent relapse to drug use after a period of abstinence. Therefore, the overarching aim of this proposal is to extend our understanding of the neural circuitry underlying relapse behavior using in vivo microdialysis and the cocaine reinstatement model of relapse. The present proposal is based on the finding that cocaine-primed reinstatement is at least partially mediated by a decrease in GABA in the ventral pallidum (VP), and that the decrease in GABA is mediated by activation of mu opioid receptors. The mu antagonist CTAP infused into the VP blocked both the decrease in GABA and reinstatement behavior after a cocaine-priming injection. Therefore, the primary hypothesis of this proposal is that cocaine-priming after abstinence requires a decrease in GABA release in the VP to produce reinstatement, and interventions that increase GABA in the VP will prevent reinstatement. Therefore, several compounds that act on peptide receptors in the VP will be infused by reverse dialysis, and GABA concentrations will be measured using microdialysis and electrochemical detection. Compounds found to increase GABA will be tested in the cocaine-priming reinstatement model of drug relapse to determine if compounds that increase GABA in the VP will also block reinstatement. In addition, the finding that an opioid antagonist injected in the VP blocks reinstatement suggests that chronic cocaine and abstinence changes the way peptides like the endogenous opioids are released from the nucleus accumbens to the VP. In order to better understand how peptide release is altered after chronic cocaine in brain regions such as the VP, a reliable method needs to be developed to measure neuropeptides from microdialysis samples. Therefore, a second aim of this proposal is to develop a method for measuring peptides using mass spectrometry, which will ultimately be used to test the hypothesis that cocaine-priming causes a release of peptides, particularly the endogenous opioids, in the VP to produce relapse behavior. Ultimately, when the interactions between fast-acting neurotransmitters and peptides are fully understood, we will be able to find the best target for the prevention of relapse in drug addicts. This proposal is relevant to public health because it will increase our understanding of the changes that take place in the brain after chronic exposure to drugs of abuse that lead to such a strong propensity for an addict to relapse. Specific targets for the prevention of relapse will be determined and verified, leading to the development of potential pharmacotherapies for the prevention of drug relapse in addicted individuals.

DESCRIPTION (provided by applicant): Recently, researchers have been investigating the use of behavioral interventions aimed at disrupting drug- associated memories to treat addiction. One of these interventions is exposure or extinction therapy where cues associated with drugs are repeatedly presented without the drug, so that an addict learns that those cues no longer predict drug availability and they stop producing craving and relapse. Another intervention is to disrupt memory reconsolidation, where a drug cue is presented followed by an intervention designed to make the addict forget that the cue was associated with drug use. These methods have been used with some success to treat other psychiatric disorders. While both of these processes sound similar, there is evidence that they actually engage distinct neurobiological processes. For example, pharmacological agents that enhance extinction are also likely to enhance rather than inhibit reconsolidation. Likewise, agents that inhibit reconsolidation are also likely to inhibit extinction. In other words, the same treatment can potentially produce opposite effects on a drug memory depending on whether extinction or reconsolidation is occurring. Therefore, there is a need to find medications that can both enhance extinction and inhibit reconsolidation simultaneously, so that one does not unintentionally increase the likelihood of relapse. In this application, the candidate will conduct studies using proteomics technology to identify proteins that are differentially activated and inhibited by drug memory reconsolidation and extinction processes. The candidate has had substantial training in the behavioral pharmacology of drug addiction. The candidate has also published several papers investigating the neurobiology of both drug memory extinction and reconsolidation. However, the candidate has had little experience with the biochemical techniques required to do proteomic studies, so this proposal includes ample opportunity for structured training in proteomics, biochemistry, and molecular biology that are necessary to do these studies. The candidate's short-term career goal is to obtain training in these techniques and in the successful management of an independent laboratory. In the long-term, the candidate plans to obtain a position as a tenure-track professor at research institution to develop a broad-based, translational laboratory that will be focused on identifying and validating novel treatments for addiction. The training environment at Yale University is excellent both for the ability to complete the proposed experiments and for the training opportunities available to the candidate. Yale University has one of the leading proteomics core facilities in the nation, and experts in the latest technologies available for identifying and quantifying differences in protein phosphorylation. This is essential for the proposed experiments, as one critical component of the hypothesis is that differential activation of proteins by changes in phosphorylation state will allow the identification of novel targets for medication development to treat addiction. In addition, there are several experts within the candidate's division in the Department of Psychiatry who can provide training in biochemical and molecular biological techniques. Training at Yale University provides the advantage of several researchers in close proximity who are experts in the use of the proposed technologies in the field of addiction. The candidate will be mentored by Drs. Jane Taylor, Angus Nairn, Ralph DiLeone, and Erol Gulcicek who all provide a wealth of expertise in the research proposed. The university also provides all of the equipment and laboratory space necessary for completion of these experiments. Finally, the university provides the support, funding, and training opportunities, such as seminars and workshops, to learn new techniques and receive training in grantsmanship, lab management, mentoring, and the responsible conduct of research. The candidate proposes to use phospho-proteomics to identify novel signaling cascades that are differentially activated/inhibited by drug-cue memory reconsolidation and extinction processes. Once targets are identified, they will be validated using secondary confirmation methods including multiple reactions monitoring (MRM) and Western blotting. The targets will then be further validated in a behavioral animal model using known pharmacological agents or RNA interference via viral-mediated gene transfer. The candidate will also determine by Western blotting whether the activity of specific proteins known to be differentially activated/inhibited by reconsolidation and extinction of fear memories are also regulated by drug memories. Then, the candidate will test pharmacological or viral manipulations of these targets, which include cannabinoid receptor 1 agonists and viruses targeting inhibition of nuclear factor kappa B (NFkB) in the animal model. These experiments will provide the candidate with the opportunity to learn several new techniques and has the potential for obtaining novel information about drug memory processes that can lead to future grants as an independent scientist. The proposed experiments could also lead to the development of novel, more effective treatments for addiction that result in a long-term reduction in craving and relapse. The candidate will also spend significant time during the training period learning important skills for managing an independent laboratory, including grant writing, managing budgets, mentoring students and technicians, and training in the responsible conduct of research.

Project Summary/Abstract Marijuana (Cannabis sativa) is the most widely used illicit drug in the U.S. and use in adolescence is common. Onset of cannabis use in early adolescence is associated with temporary cognitive impairments, but the long- term neurobiological consequences are not well understood. In addition, medicinal use of cannabinoids in children and adolescents may be warranted for treating certain seizure-related disorders; therefore, an increased understanding of the costs vs. benefits of adolescent cannabinoid exposure are warranted to inform the ongoing development of policies regulating legal marijuana use. Rodent models provide the opportunity to perform controlled experiments on the timing, duration, and amount of cannabinoid exposure and eliminate potential confounds of human studies, such as pre-existing conditions and exposure to other drugs of abuse. In addition to determining the long-term behavioral consequences of cannabis exposure, it is also important to determine the effects on neural circuit activity that may produce more subtle effects on functional outcomes. Current technologies limit our ability to perform longitudinal recordings of neural activity in a cell-type specific manner across adolescent development, particularly in animals self-administering intravenous drugs of abuse and performing complex cognitive tasks. One solution is to perform in vivo imaging of ensemble neural activity using genetically-encoded calcium indicators (GECIs). In vivo Ca2+ imaging can resolve cellular activity in deep brain structures in awake behaving animals using integrated, head-mounted gradient refraction index (GRIN) lenses and miniaturized epifluorescent microscopes (microendoscopes). Currently, this technique has been primarily restricted to studies in adult mice. However, development of a transgenic rat expressing the latest generation GECI, GCaMP6f, will permit imaging of cortical activity over the course of adolescent development and make it feasible to compare neural circuit development in rodents that self-administer intravenous drugs of abuse, like cannabinoids, relative to controls. Thus, in this two-year project we propose to develop transgenic rats expressing GCaMP6f under the control of a neuronal promoter. We will assess neural activity patterns in prefrontal cortical regions responsible for working memory and cognitive performance, in rats that self-administer cannabinoids in adolescence. Adolescent self-administration groups will be compared to similarly trained adults and to food self-administering controls. We will assess working memory performance and associated cortical activity in these rats across development and in adulthood after a period of abstinence. The results of these studies will clarify the acute vs. long-term consequences of adolescent cannabinoid self- administration on working memory performance, and determine if the development of task appropriate neural activity patterns are affected by ongoing or prior cannabinoid self-administration. In addition, the GCaMP6f transgenic rat will be publicly available for broad use by the neuroscience community.

Abstract Drug addiction is a serious disorder that affects millions of people and produces a large burden on society. Many individuals try to abstain from use, but at least 60% of people relapse within one year. Thus, relapse prevention remains an important goal for addiction treatment research. One of the primary causes of relapse is exposure to the environmental cues (people, places, and things) that remind individuals of drug use and initiate craving. One potential treatment strategy is to reduce the strength of these drug-cue memories so that they are less able to cause renewed drug use. Clinically, this can be accomplished using exposure therapy, which is based on extinction learning and involves multiple presentations of drug-associated cues until the craving response is reduced. Unfortunately, this approach is only mildly effective. Thus, we have investigated the neurobiological mechanisms underlying the maintenance and extinction of cocaine memories, in order to identify potential strategies for improving the ability of cue exposure therapy to reduce relapse. We identified both neural circuit and molecular mechanisms that underlie cocaine memory formation and extinction. Specifically, we found that plasticity at synapses in the lateral amygdala (LA) that receive inputs from the medial geniculate nucleus of the thalamus (MGN) are strengthened after cocaine self-administration and that this plasticity is reversed by cue extinction training. We further found that we could induce long-term depression (LTD) at MGN-LA synapses to reduce relapse-like behavior in a manner that mimicked cue extinction training. Additionally, we found that modulating the activity of kinases and phosphatases in this region could either promote extinction learning or disrupt memory reconsolidation to reduce relapse and the strength of MGN-LA synapses. Thus, in this administrative supplement to support a NIDA Summer Research Intern, we aim to train the student in how to perform experiments investigating the regulation of cocaine- associated memories. The intern will be trained to perform IV drug self-administration experiments, including being given the opportunity to perform surgical procedures. The intern will be trained in proper care and handling of rodents and in the common behavioral methods used in the substance use disorder field. The intern will be assigned a specific experiment to investigate the role of GABAB receptors as a potential target for cocaine memory manipulation. The intern will test whether GABAB receptor agonists could be useful adjuncts to exposure therapy. We will determine if GABAB agonists enhance extinction, prevent reconsolidation, and if pharmacologically assisted exposure therapy is associated with changes in cue-associated neural activity across contexts. We will test if GABAB agonists are effective when infused directly into the LA, and if they are effective when given systemically. Overall, the proposed studies are designed to provide a rich training experience for an undergraduate fellow interested in substance use research.

Project Summary/Abstract Alcohol drinking is very common in the United States, with a majority of Americans having consumed alcohol within the past month. Roughly 7% of these individuals engage in heavy drinking and develop alcohol use disorders (AUDs), which produce significant economic and medical burdens on society. As problem drinking is characterized by consumption of intoxicating doses exceeding 80mg/dl (17.4mM), the majority of alcohol research targets blood alcohol concentrations (BACs) above this level. However, the effect of low concentrations of alcohol (<10mM) evident in early stages of drinking has not been well characterized. Identification of the neurobiological consequences of low-dose alcohol exposure can lead to a better understanding of the mechanisms involved in the initial reinforcing effects of alcohol and may ultimately lead to the development of better prevention efforts in mitigating subsequent alcohol misuse. We have shown that self- administration of low doses of ethanol in rats can lead to significant reinstatement of ethanol seeking, a model of ?craving?-like behavior. These doses of ethanol have also been shown to cause brain activation in regions known to be involved in reward. Importantly, self-administered ethanol produces unique patterns of neuronal activation in these brain areas that is not evident when ethanol is delivered non-contingently. We therefore propose to characterize the molecular targets of experimenter- versus self-administered ethanol at low doses using a phosphoproteomic approach. In Aim 1, we will use discovery-based quantitative label free mass spectrometry to identify differentially phosphorylated proteins in targeted brain regions known to be involved in ethanol reinforcement (nucleus accumbens, central, and basolateral nuclei of the amygdala, etc.) in male and female rats receiving chronic non-contingent injections of low (3mM), moderate (17.4mM), and high doses of ethanol (50mM) consistent with self-administration, binge drinking, and excessive drinking, respectively. Rats will then be euthanized to measure protein phosphorylation events, which are the major drivers of change in the functional activity of proteins, as a function of ethanol concentration. Aim 2 will extend these findings in rats trained to self-administer ethanol vs. yoked controls to address whether expectancy shifts the response of low- dose ethanol in the brain. We predict that ethanol will produce dose-dependent alterations in protein phosphorylation in terms of both magnitude and pattern of brain regions that are affected. Similarly, as female rats self-administer greater quantities of ethanol than males, we also expect to find sex-dependent patterns of protein regulation. Finally, we predict that self-administered ethanol will produce a unique pattern of neuronal activation compared to a similar low dose of experimenter-administered ethanol that may confer sensitivity to subsequent ethanol-motivated behavior. Overall, these experiments will help identify novel targets of low-dose ethanol that can be explored in future studies aimed at minimizing the risk of developing alcohol use disorders.

PROJECT SUMMARY Adolescence is a period of enhanced vulnerability to develop substance use disorders in part do to the ongoing development of neural circuits associated with reward and executive function (i.e., impulsivity, attention, reward sensitivity). In addition, adolescents experience a developmentally regulated shift in circadian rhythms to a more evening chronotype and have less perceived sleep drive. Thus, adolescents are biologically driven to stay up later at night and wake later in the morning. However, this natural shift in circadian rhythms is in conflict with societal norms, particularly early school start times, which can lead to a chronic state of circadian misalignment and insufficient sleep. The degree to which chronic circadian and sleep disturbances in adolescence impacts brain development and risk for drug abuse is not well understood. Moreover, there is a wide variation in the degree of circadian shift amongst adolescents, leading to the possibility that certain individuals are more at risk than others for circadian and sleep-associated dysfunction. Therefore, an increased understanding of the behavioral and neural consequences of sleep and circadian disturbances, and their interaction with individual differences in sleep and circadian preferences, is needed to inform new interventions and preventative strategies. Project 4 of the Center for Adolescent Reward, Rhythms, and Sleep (CARRS) aims to determine the effects individual differences in chronotype (Aim 1), circadian misalignment in the absence of sleep loss (Aim 2), and acute and chronic sleep disruption (Aim 3) on behavioral indices of addiction risk and corticolimbic neural activity in adolescent rats. Identification of individual differences in sleep and circadian preferences will be facilitated by using the heterogeneous stock (HS) outbred rats that produce more variability than standard outbred strains, and allow for precise genetic identification of trait differences. Rats will be phenotyped for circadian and sleep preferences in early adolescence by our Phenotyping and Bio-banking Core B. We will then examine how these phenotypes related to impulsivity and execute function on the 5-choice serial reaction time task (5-CSRTT) and if rats with extreme chronotypes (early vs. late) exhibit differences in nicotine or THC self-administration. Aims 2 and 3 will focus on how manipulations of circadian rhythms and sleep alter behavior on the 5-CSRTT and drug self-administration. In addition, we will test how corticolimbic activity is altered by circadian and sleep manipulations during behavior using in vivo fiber photometry. Results of these studies will be integrated with human neuroimaging data obtained in Projects 1 and 2, and with the molecular and ex vivo electrophysiological results obtained in Projects 3 and 5.

Abstract Sleep dysregulation is a hallmark of alcohol use disorders (AUDs) and disrupted sleep can contribute to relapse even after months of abstinence. Despite the well-recognized sleep-AUD interactions, few studies have investigated how sleep changes over the development of excessive drinking, and the mechanisms by which sleep disruptions exacerbate drinking and/or relapse liability are largely unknown. Based on several key findings from published studies and preliminary work, one hypothesis is that disrupted REM sleep induces maladaptive changes in the medial (M) and lateral habenula (LHb), which in turn contribute to escalated alcohol intake and promote relapse. The key findings are: 1. Specific REM sleep deficits at 2-3 weeks after alcohol withdrawal are a robust predicting factor for relapse in alcohol dependent patients; 2. Chronic, selective REM sleep fragmentation enhances medial Hb neuron tonic firing and negative affect; 3. Relapse to alcohol seeking is associated with increased activation of MHb and LHb neurons. 4. Sleep fragmentation precipitates drug craving during abstinence from cocaine, while selective consolidation of REM sleep reduces craving. Thus, it is predicted that chronic alcohol drinking and abstinence leads to disrupted REM sleep, which induces Hb hyperactivity, promoting a negative affective state that drives motivation for alcohol and relapse. Likewise, it is predicted that chronic REM sleep disruptions can initiate this cycle, leading to increased risk for developing AUDs. Therefore, aim 1 in this proposal will evaluate the effects of chronic and escalating alcohol drinking and abstinence on sleep architecture and Hb neural activity. The proposed experiments will use the intermittent access two-bottle choice ethanol drinking paradigm combined with chronic EEG/EMG recordings and ex vivo slice electrophysiology. It is expected that the baseline REM sleep features or the REM sleep deficits following alcohol drinking/abstinence may predict the amount of alcohol drinking as well as Hb neural activity. Aim 2 will determine if chronic REM sleep fragmentation promotes the escalation of alcohol intake in the intermittent access model and/or promotes relapse in an operant self-administration model. We will further determine if the REM sleep effects on alcohol intake/relapse are, in part, mediated by REM sleep fragmentation-induced hyperactivity of Hb neurons. Aim 3 will test the treatment potential of interventions that improve REM sleep. Utilizing recently developed REM sleep-selective manipulations, it will be determined if selective REM sleep consolidation can reduce alcohol drinking and relapse, and if this is associated with reduced Hb activity. Overall, the proposed studies will provide a critical assessment of the potential for developing REM sleep- focused therapeutics for preventing relapse, and provide novel mechanistic insight into the interactions between alcohol intake and sleep homeostasis in the context of habenula dysregulation. Thus, this proposal is consistent with the mission of NIAAA RFA-AA-19-006 to perform Mechanistic Studies on Chronic Alcohol Use and Sleep Homeostasis.

Project Summary/Abstract Historically, alcohol abuse and alcohol use disorders (AUDs) have been more common in men than women. However, recent epidemiological data in younger cohorts of women suggest that this gender gap is closing. Rates of risky/binge drinking in women are going up at a much faster rate than in men, women show a faster transition from social use to dependence, and women suffer greater alcohol-related health problems. Thus, identifying sex-specific neurobiological factors that underlie the development of problem drinking could lead to improved treatment approaches. In particular, women have been shown to have increased craving in response to alcohol-related cues and stressors relative to men, and neuroimaging studies show that this effect is associated with differential activation of brain circuits including the amygdala. We have found that female rats also show a greater relapse-like response to alcohol-associated cues, particularly when combined with an acute stressor, similar to the human studies. Moreover, we also have evidence for increased glutamatergic signaling in the basolateral amygdala (BLA) of female rats, raising the possibility that alcohol-associated memories may be more readily encoded in the BLA of females relative to males, and/or that the neurons encoding alcohol memories are more responsive to acute stress and able to drive activity in circuits controlling craving and relapse. Importantly, we have previously shown that differences in circulating gonadal hormones do not mediate the sex difference in behavior, suggesting that fundamental differences at the synaptic or circuit level underlie differential relapse-like behavior. For example, we have found that glutamatergic synapses from sensory thalamus to the BLA are critical for the encoding and extinction of cocaine-associated memories, and that depotentiating these synapses is sufficient to reduce cocaine relapse-like behavior. However, no one has previously investigated whether alcohol-cue memories are similarly encoded or if there are differences between males and females in the neural mechanisms mediating alcohol memory formation. Thus, in Aim 1 we will compare plasticity related mechanisms in the BLA regulating alcohol memory formation and extinction in males and females. In Aim 2, we will use genetically-encoded calcium indicators and miniature microendoscopes to image neural activity (calcium signals) in males and females when alcohol-cues are presented in the presence or absence of acute stressors. Finally, in Aim 3 we will use comprehensive cFos mapping to determine if males and females engage similar or different circuits during stress+/-alcohol-cue- induced relapse-like behavior. These results will inform if sex differences are due to altered circuit engagement that would point to sex specific neural targets for treatment. All together, these studies will provide a comprehensive analysis of how alcohol-cue memories are formed, modulated by stress, what other circuits are engaged, and if there are sex differences in any of these neurobiological factors that could explain the increasing vulnerability of females to alcohol-related disorders.