Stephanie M. Groman - US grants

DESCRIPTION (provided by applicant): The economic and social burdens on society that are a direct result of substance dependence are enormous. As such, determining the neural mechanisms of phenotypes that confer risk for the development of this disorder is crucial for advancing scientifically-based prevention and treatment strategies. Associations between variation in cognitive processes and substance dependence liability have been demonstrated, and the corticostriatal dopamine system has been implicated in these relationships. Further, recent evidence has demonstrated an important role of the dopamine D2 receptor in controlling aspects of impulsivity, cognitive control and drug abuse. Therefore, the D2 receptor may represent a mechanistic link between cognitive control processes and risk for the development of substance dependence. Because the D2 receptor can exist in two conformational states - a state in which dopamine displays high affinity for the receptor and has greater functional activity, and a state in which dopamine displays less affinity for the receptor and is less functionally active - determining whether variation in receptor availability detected with standard neuroimaging techniques (i.e. PET) reflect variation in receptor state (high versus low) is critical to understanding the relationship between cognitive processes and risk for substance dependence. Because we have found an association between a naturally occurring variation of trait impulsivity and D2 radioligand binding in vivo, this proposal will attempt to examine this question using behavioral pharmacological techniques to probe D2-like receptor states (Aim 1) and using this information to examine a possible neural mechanism by which variation in receptor state may influence global brain function (Aim 2). These results will provide evidence for a mechanism by which variation within a biological system can influence higher- order processes that have direct relevance to substance dependence. The advancements in the characterization of the link between the D2 receptor system and impulsivity offered in this proposal will provide an important step in understanding how manipulations of the dopamine system may be utilized to enhance cognitive control and therefore mitigate risk for the development of substance dependence.

Project summary and abstract One of the most profound changes that occurs during gestational development is the rapid increase in the connections between neurons. Synaptogenesis, or the formation of synapses, that occurs in utero is critical for subsequent neural development, and several mental and developmental disorders are believed to be the resultant of alterations in prenatal synaptogenesis. Synaptic quantification, until recently, has required post- mortem tissue, so direct evidence linking prenatal synaptic disruptions with postnatal neural and behavioral development has been limited. We have recently developed a novel positron emission tomography (PET) radioligand ([11C]UCB-J) that targets synaptic vesicle glycoprotein 2A (SV2A), a protein expressed in almost all synaptic vesicles, and have demonstrated that [11C]UCB-J is able to detect subtle alterations in human disorders, such as Alzheimer?s disease. We have conducted a pilot study using [11C]UCB-J to quantify synaptic development throughout prenatal and postnatal development in non-human primates and have found striking changes in [11C]UCB-J binding that parallel post-mortem measures of synaptogenesis. Therefore, longitudinally characterization of synaptic density with [11C]UCB-J has the potential to provide unprecedented insight into the developmental mechanisms that underlie mental illness. This proposal will use [11C]UCB-J to quantify synapses throughout gestation and the first nine postnatal months in non-human primates to determine the predictive relationship between prenatal synaptic development and postnatal development of the brain and cognition. In the first aim, we will quantify SV2A during gestation (post-conception days 120 and 150) and following birth (postnatal days 15, 30, 60, 120 and 240) to determine if development trajectories of SV2A co-vary with changes in reward learning, working memory and response inhibition, which are cognitive domains known to be altered in mental illness. The second aim will determine the relationship between prenatal SV2A density and magnetic resonance (MR) measures in order to identify a putative MR biomarker of prenatal synaptic density that could serve as a noninvasive diagnostic tool for use in human infants. Measures of brain morphometry, myelination and functional connectivity will be longitudinally assessed in the same infant non-human primates used in Aim 1 following birth (postnatal days 30, 60, 120 and 240) and related to synaptic and cognitive development trajectories of the same monkey. These studies of normal development in non- human primates will provide the preliminary data for future studies examining SV2A in animal models of mental illness, and ultimately lead to the identification of noninvasive MR imaging biomarker of prenatal synaptic density that can be safely acquired in human infants and children and serve as a diagnostic tool.

PROJECT SUMMARY / ABSTRACT The transition from drug use to abuse and, eventually, to dependence may be mediated by biological factors that are present prior to drug use. Although chronic exposure to drugs of abuse is known to disrupt many signaling pathways, little is known about the molecular mechanisms that mediate addiction susceptibility. There is considerable interest in identifying early biomarkers for addiction susceptibility to improve addiction prevention strategies. Most studies have been conducted in substance-dependent individuals where the dissociation between ?susceptibility? and ?consequence? is ambiguous. I have recently identified a behavioral phenotype in rats that reliably predicts future drug-taking behaviors and identified three proteins as potential mediators of addiction susceptibility: sorting nexin 1 (SNX1), ryanodine receptor 2 (RYR2), and ataxin 2-like (ATXN2L). These proteins are involved in intracellular trafficking, calcium signaling and cytoskeleton reorganization, and have been previously linked to addiction. Precisely how differences in expression of these proteins impact the signaling cascades underlying addiction susceptibility is not known. The overarching goal of this proposal is to determine the functional and molecular role of proteins that mediate addiction susceptibility and investigate how these factors are mechanistically linked to genetic and/or environmental components of risk for addiction. To accomplish this goal, the proposed research will combine sophisticated behavioral and computational assessments with viral, proteomic and bioinformatic approaches. In Aim 1 I will use an inducible and reversible viral construct to bi-directionally manipulate expression of SNX1, RYR2, and ATXN2L and also determine how changes in expression of SNX1, RYR2, and ATXN2L alter methamphetamine self-administration and protein signaling mechanisms. In Aim 2 I will determine if variation in the expression of SNX1, RYR2, and ATXN2L is altered in a model of genetic addiction susceptibility and is associated with increased addiction risk. In Aim 3 I will determine if variation in expression of SNX1, RYR2, and ATXN2L is altered in a model of environmental addiction susceptibility and is associated with increased addiction risk. Completion of these aims will generate new insights into the signaling mechanisms of addiction susceptibility that could identify early biological markers of risk for addiction and improve current strategies for addiction prevention. The Principal Investigator will receive mentorship and technical training in viral and proteomic technologies by experts in cell signaling and cellular mechanisms, and viral technologies in motivated behaviors. Yale University and the Department Psychiatry provide exceptional facilities and resources for completing the proposed experiments, as well as having an exceptional reputation and track record for mentoring and transitioning early-stage investigators in to independent investigators. The proposed training, education and research will provide the PI with the technical and professional training to become a successful, independent addiction investigator.

PROJECT SUMMARY / ABSTRACT The transition from opioid use to abuse and, eventually, to dependence may be governed by distinct genetic mechanisms that alter the molecular pathways that mediate opioid use disorder (OUD). The identity of these genetic/genomic mechanisms is unknown, in part, because the majority of OUD studies have been done in substance-dependent individuals where the dissociation between susceptibility and consequence is ambiguous and genetic/environmental factors are equivocal. One potential strategy for investigating the genetic mechanisms underlying differences in opiate-use trajectories that we have been pursuing in rats is to examine the neurobiology of complex behavioral phenotypes that are associated with different phases of drug use. Our work has identified a decision-making phenotype (e.g., ¨+ parameter) that predicts opiate-taking behaviors in rats, which differs from the decision-making phenotype that is affected by opiate use (e.g., ¨0 parameter). We have found that these distinct phenotypes are controlled by different orbitofrontal circuits that involve the amygdala and nucleus accumbens, and our preliminary proteomic data indicates that these phenotypes are mediated by divergent signaling pathways. We posit, therefore, that these computationally-derived phenotypes could serve as a powerful tool for dissociating the genomic/genetic mechanisms of opioid use susceptibility from those that are consequential to drug use. Here, we propose to use state-of-the-art genomic approaches to identify genes that mediate susceptibility to opiate-taking behaviors and those that mediate drug-induced behavioral changes. In Aim 1, we will investigate the genomic mechanisms underlying susceptibility to oxycodone use. Decision making will be assessed in rats (N=300) to identify individuals who either have low or high ¨+ parameter (N=60/group) that predicts susceptibility to opiate-taking behaviors. RNA sequencing will be performed on tissue from the orbitofrontal cortex, nucleus accumbens and amygdala to identify genes whose expression differs between rats with a low or high ¨+ parameter. We will then perform ATAC sequencing to identify the open chromatin regions associated with the genes that differ between rats with a low or high ¨+ parameter. In Aim 2, we will investigate the genomic mechanisms underlying the decision-making consequences of oxycodone self- administration. Decision making will be assessed in rats (N=300) before and after they self-administer oxycodone to identify individuals who either have low or high ¨0 parameter (N=60/group) following drug exposure. Tissue collected from the orbitofrontal cortex, nucleus accumbens and amygdala will be sequenced using next- generation RNA sequence to identify genes whose expression differs between rats with a low or high ¨0 parameter. We will then perform ATAC sequencing to identify the open chromatin regions associated with the differentially expressed genes. Our results ? integrating genomic approaches with human-based computational phenotypes in rats ? will provide critical insights into the genomic mechanisms associated with distinct stages along the OUD trajectory and develop a translational platform for future studies of OUD in humans.