Lori A. Knackstedt, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The specific aims of the proposed experiments are to examine the potential differences between rats allowed extended access (6 hours) to cocaine self-administration and rats given the traditional short access (1 hour); in 1) locomotor sensitization, 2) cocaine- and cue-induced reinstatement of cocaine-seeking after withdrawal, 3) the differences in basal and stimulated glutamate and dopamine levels, 4) molecular changes that accompany cocaine self-administration, namely in glutamate transmission-related proteins such as AGS3, PSD-95, mGluR1/5, xCT, GluR1, GluR2/3, and the Homer proteins. Rats trained to self-administer cocaine will experience two weeks of withdrawal and will then be challenged with three doses of cocaine (3, 10, and 30 mg/kg IP) to examine both locomotor sensitization and the ability of cues and cocaine to elicit the reinstatement of cocaine-seeking. Two weeks after the last self-administration session, the basal levels of glutamate and the stimulated levels of glutamate and dopamine in the nucleus accumbens will be measured via microdialysis. At the same time point that these behavioral and neurochemical changes will be monitored, we will also look for changes in known molecular markers of addiction via immunoblots. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Relapse to cocaine-seeking in the animal model of reinstatement is associated with altered glutamate homeostasis in the nucleus accumbens core. Relative to control animals, basal levels of extracellular glutamate are reduced in withdrawal while reinstatement is accompanied by increased glutamate levels. System xc-, which exchanges one extracellular cystine molecule for one intracellular glutamate molecule, has been found to account for the majority of basal extracellular glutamate levels in the nucleus accumbens and its activity is significantly down-regulated after chronic cocaine. The nutritional supplement N-acetylcysteine (NAC) restores the function of the exchanger and basal levels of glutamate. NAC treatment attenuates relapse to cocaine seeking in both animal models and in a human clinical pilot study. Preliminary data presented here demonstrates that glutamate re-uptake, mediated primarily by the glial transporter GLT-1, is also compromised after cocaine self-administration. We propose that akin to restoring glutamate homeostasis with NAC, normalizing glial glutamate transport via GLT-1 up-regulation will inhibit relapse. It is known that beta-lactam antibiotics such as ceftriaxone increase the expression and activity of GLT-1. Experiments within the current proposal are designed to study the co-regulation of system xc- and GLT-1 following cocaine self-administration and treatment with NAC and ceftriaxone. The ability of ceftriaxone to block reuptake and the increase in extracellular glutamate that accompanies it will also be examined. Metabotropic glutamate receptors (mGluRs) are found in the extrasynaptic space and are likely to be influenced by the function of both system xc- and transporters, and current evidence points to the down-regulation of both Group I and II mGluRs in the nucleus accumbens following cocaine self-administration. We will examine the ability of both NAC and ceftriaxone to restore function of group I mGluR in the nucleus accumbens. Completion of the experiments proposed here will further our knowledge regarding the mechanisms by which cocaine addiction alters glutamate homeostasis and mGluR function, and potentially provide further evidence that targeting glutamate transporters is an effective method of treating cocaine relapse. PUBLIC HEALTH RELEVANCE: Cocaine addiction remains a substantial public health problem in the United States today. It is widely recognized that a high risk of relapse exists even after long periods of abstinence and this relapse represents one of the key challenges in the successful treatment of cocaine addiction. This goal of the current proposal, entitled "Striatal Glutamate Homeostasis and Cocaine Relapse," is to test the efficacy of an FDA-approved compound at attenuating relapse in an animal model. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Decreased basal levels of extracellular glutamate have been observed in the nucleus accumbens following withdrawal from cocaine self-administration. Cocaine-primed relapse to cocaine-seeking in the animal model of reinstatement is associated with enhanced glutamate release relative to drug-naive animals. System xC-, which exchanges one extracellular cystine molecule for one intracellular glutamate molecule, has been found to account for the majority of basal extracellular glutamate levels in the nucleus accumbens and its activity is significantly down-regulated after chronic cocaine, as is that of the major glial glutamate transporter, GLT-1. The beta-lactam antibiotic ceftriaxone has been found to increase the expression and function of both GLT-1 and system xC-. We have previously reported that ceftriaxone prevents relapse to cocaine seeking in the extinction-reinstatement animal model of relapse. Here we propose to assess the relative importance of increasing expression of GLT-1 and xCT (the catalytic subunit of system xC-) in mediating the therapeutic effects of ceftriaxone. Completion of the experiments proposed here will further our knowledge regarding the relative importance of GLT-1 and system xC- in mediating reinstatement probability and maintaining glutamate homeostasis in the nucleus accumbens core. PUBLIC HEALTH RELEVANCE: Cocaine addiction remains a substantial public health problem in the United States today and it is widely recognized that a high risk of relapse exists even after long periods of abstinence from the drug. We have previously shown that the FDA-approved antibiotic ceftriaxone reduces cocaine relapse in the animal model of reinstatement. The goal of the current proposal, entitled Glutamate transporters and cocaine seeking is to investigate the mechanism of action by which ceftriaxone works to attenuate relapse in order to further our knowledge regarding the neurobiology of this complex behavior.

Project Abstract Cocaine use disorder remains a significant public health problem in the US today, and there is a high risk of relapse even after long periods of abstinence. The current translational pipeline relies on animal models such as the extinction-reinstatement model to screen potential therapies for efficacy at attenuating relapse. While many pharmacological agents successfully reduce cocaine-seeking in this model, these agents show little clinical efficacy, and none have been FDA-approved for cocaine use disorder. One explanation for the failure of these agents to translate to the clinic may lie in the fact that most cocaine users engage in poly-substance use (PSU), while existing animal models of cocaine addiction involve self-administration of cocaine alone. Such PSU likely engages different behavioral and neural mechanisms compared to cocaine alone. Indeed, our preliminary data from a rat model of combined cocaine and alcohol use show that alcohol co-consumption significantly changes the neurobiology supporting cocaine relapse, and renders potential pharmacotherapies ineffective. These data and others highlight the need for a better understanding of PSU. Progress in this area is hampered, however, by a paucity of information regarding how substance (particularly cocaine) users actually engage in PSU. The long- term goal of this project is to determine the unique consequences of PSU on behavior and neurobiology underlying cocaine-seeking. The objectives of the current proposal, which represent the first steps toward our long-term goal, are to 1) develop and validate a survey instrument for evaluating detailed temporal patterns of PSU in cocaine users; 2) determine in a cocaine-using population the most common temporal patterns of alcohol and cannabis use (which are the most frequently used substances in combination with cocaine); 3) back- translate these data to develop rat models of cocaine+alcohol and cocaine+cannabis use; and 4) determine their consequences on neurobiological measures relevant for relapse (glutamate signaling and D2/3 dopamine receptor expression in the nucleus accumbens). Our rationale is that rat models which more closely mimic actual patterns of human substance use should better yield the underlying neuroadaptations present in humans, and should thus serve as better platforms for therapeutic discovery. As such, our central, unified hypothesis is that cocaine users will display high rates of comorbid cocaine+alcohol or cocaine+cannabis use in unique patterns that can be translated into rat models, in which the neurobiology underlying relapse to cocaine-seeking will be altered by such alcohol or cannabis use.

Project Summary (Supplement) This proposal is for a Diversity Supplement for a PhD student, Javier Mesa. This supplement will allow Javier, who is an underrepresented minority in the biomedical sciences, to work under the parent grant DA045140. During the course of the supplement, Javier will be trained on cutting-edge techniques in neuroscience and expand his conceptual knowledge on translational rodent models of drug seeking. The mentoring team is composed of the MPIs of the parent grant, Drs. Lori Knackstedt and Linda Cottler, and co-I?s Drs. Marek Schwendt and Barry Setlow. Additionally, Dr. Jesse Dallery, a member of Javier?s dissertation committee, will contribute to mentoring. All mentors are current or past NIDA grantees, and have expertise ranging from the use of preclinical rodent models of drug-seeking to uncover neurobiological changes (Drs. Knackstedt, Schwendt and Setlow) to assessing human patterns of drug-taking (Dr. Cottler) and using behavioral strategies to reduce drug seeking (Dr. Dallery). Javier will contribute to data collection for parent grant aims, while also pursuing his own independent aims that he has constructed. The parent grant aims to back-translate human patterns of cocaine-alcohol and cocaine-cannabis polysubstance use (PSU) into rodent models. One such model has been in use in the Knackstedt lab, with two publications finding that sequential self-administration of cocaine and alcohol leads to unique NA core glutamate adaptations differing from that of cocaine self-administration alone. Specifically, we found that during a both a cocaine- and a cue+cocaine-primed reinstatement test, NA core glutamate efflux increases in rats that consumed cocaine alone, but not in those with a history of sequential cocaine and alcohol consumption, despite equivalent levels of cocaine-seeking during the reinstatement test. This glutamate efflux drives the reinstatement of cocaine-seeking, as blockade of NA core glutamate receptors attenuates such reinstatement. We also found that reduced reinstatement-induced Fos expression in the NA core and the PFC occurred in the PSU group relative to the cocaine-only group, consistent with the idea that PFC-NA core pathways do not mediate reinstatement of cocaine seeking in the PSU condition. Conversely, we observed increased Fos expression in the BLA of cocaine-alcohol PSU rats relative to cocaine-only rats. Here, Javier proposes to use this model of sequential cocaine+alcohol consumption to investigate the novel neurocircuitry underlying relapse to cocaine seeking in the PSU model. His aims are designed to test the hypotheses that 1) inactivation of the BLA will suppress the reinstatement of cocaine seeking in rats in PSU rats, possibly to a greater extent than in cocaine-only rats; and 2) inactivation of the PFC-NA core pathway will suppress the reinstatement of cocaine seeking in rats that self-administered only cocaine and not in PSU rats. These aims will provide valuable information regarding the presence of distinct vs. overlapping neurocircuitries mediating cocaine seeking in cocaine monosubstance use vs. cocaine-alcohol polysubstance use.