Jane R. Taylor - US grants

Pre-clinical studies of the neural mechanisms of motor function suggest that there may be regionally specific sites advantageous for intracerebral neural graft placement into the host. One site, the substantia nigra, ultimately might prove to be the most advantageous due tot he possible restoration of natural neural circuits. Clinical studies have begun which claim superiority of one placement site over another, particularly the putamen over the caudate, without any comparative studies in primates. This project will study the effectiveness of various graft placements in order to understand the site-dependency of functional improvements in parkinsonian behavior after grafting. Monkeys with stable MPTP-induced parkinsonism will be used exclusively in this project for functional studies. A battery of behavioral test will assess the extent and nature of functional changes. Spontaneous behavior will be used to assess the general status of the subject, and tests that examine cognitive and sensorimotor function will be used to determine the effectiveness of grafts on specific functional deficits. The specific experiments will compare placements in the caudate with placements into the putamen. Control comparisons will include cannula placement sham surgeries, cerebellar tissue grafts, and unilateral substantia nigra grafts into each structure. Since multiple small grafts might innervate the entire striatum better than a confined graft of equal volume, we will test this possibility. In addition, grafts into anatomically defined subregions of the striatum may result in selective improvements in specific behavior(s) or types of improvements measured by performance on different tasks or aspects of tasks that are dependent on striatal dopamine or the integrity of frontocaudate circuitry. Some preliminary studies have suggested simple vs complex behavior, or indeed motor vs cognitive behavior, may be affected differentially by the placements of grafts into subregions of the large and heterogenous caudate. If experiments using growth factors or co-grafts further support the possibility and demonstrate suitable methods, we will also examine whether grafts of fetal SN into the SN or bridge grafts from the SN to the striatum would produce some additional functional improvements particularly in behavior dependent on external afferent inputs impinging on the SN rather than tonic terminal increases in dopamine in the MPTP denervated striatum. The general methods, experimental design outlines and assessment measures are presented separately in detail in each of the Core sections. Transplantation methods as developed and standardized by the program will be utilized in these studies and maintained consistently to provide proper controls for the principal variable of interest -- distribution of grafts within the host. This project may provide data important for understanding how graft placement affects the various behavioral functions of the nigrostriatal dopamine system and more rational ways to ameliorate deficits in dopamine system function which may be of clinical relevance.

DESCRIPTION: (Applicant's Abstract) Drug abuse is a significant social and medical problem that directly involved over two million people in the U.S. The goal of neurobiological research is to understand the complex mechanisms underlying euphoria, drug-seeking behavior and craving so that treatment strategies can be identified to combat drug abuse. The ability of psychomotor stimulant drugs to enhance the salience and significance of reward-related stimuli may be a potent determinant of the behavioral effect of these drugs. In addition to these incentive motivational effects, stimulant drugs also produce behavioral sensitization that results in augmentation of behavior after repeated drug exposures. the ventral striatum (nucleus accumbens) has been implicated in both responding for reward-related stimuli, sensitization, and the reinforcing properties of stimulants (cocaine and amphetamine). The nucleus accumbens is a region of the forebrain that has been suggested to be a limbic-motor interface that gates or mediates motivational response output. Our previous research was the first to demonstrate that dopaminergic neurotransmission within the nucleus accumbens was a component of the neural circuitry involved in incentive motivation. The current proposal seeks to examine the role of dopamine (DA) receptor subtypes within the nucleus accumbens in enhanced responding for reward-related stimuli in sensitized rats. Direct microinfusions of neurotransmitters or drugs will be used to accomplish these goals using behavioral measures of motor activity and responding for reward-related stimuli (termed "conditioned reinforcement"). There are two major objectives of these studies: 1) To investigate the ability of cocaine and amphetamine sensitization to affect responding for conditioned reinforcement and enhanced responding after drug administration. 2) To examine the ability of intra-accumbens infusions of selective dopaminergic (D1) receptor agonists (SKF82958, SKF81297) to enhance responding for conditioned reinforcement, and determine whether selective DAantagonists can block these effects in cocaine sensitized subjects. The ventral striatum is hypothesized to be critical for reward-related processes involved in both conditioned reinforcement and in sensitization. The studies should (1) contribute to our understanding of the role of the ventral striatum in the effects of psychomotor stimulant drugs and (2) help establish the relevance of the conditioned reinforcement model to processes that may contribute to drug dependence and craving.

DESCRIPTION (provided by applicant): The objective of this proposal is to define behavioral consequences and cellular correlates of repeated drug exposure on alterations in incentive motivational processes and inhibitory control contributing to cognitive deficits in addiction. Neurobehavioral studies in humans and animals are beginning to define drug-induced alterations in cognitive-motivational processes, and brain imaging studies show altered cortico-limbic-striatal activity in drug abusers. Our studies in animals have contributed to the idea that drug-induced alterations in dopamine (DA) neurotransmission and protein kinase A (PKA) signaling may result in molecular neuroadaptations. Studies from the previous funding period are among the first to show that repeated exposure to PCP, cocaine or THC produces long-lasting cognitive impairments associated with inhibitory control in monkeys and rats, deficits characteristic of, and correlated with, DA dysfunction in regions of the frontal cortex. These cognitive impairments are concomitant with alterations in incentive motivational processes and enhanced subcortical DA function. Our new data show that these persistent drug-induced behavioral changes are also associated with, and can be mimicked by, increases in DA/PKA activity, suggesting that adaptations in DA-regulated intracellular signaling molecules may underlie alterations in motivational processes. Drug-induced increases in synaptic DA stimulate D1 receptors, which increase cAMP levels, leading to activation of PKA. PKA activation increases expression of transcription factors and phosphorylation of substrate proteins involved in neuronal excitability/plasticity such as the DA and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). The current research proposes an intensive examination of how repeated drug exposure persistently alters cognitive/inhibitory function, incentive motivation and DA/PKA signaling. In Aim 1, the effects of repeated cocaine, PCP, and THC on cognitive-inhibitory functions dependent on ventromedial vs. dorsolateral frontal cortex in monkeys and rats will be examined. In Aim 2, we will investigate the effects of drug exposure on incentive motivation using stimulus-reward learning and conditioned reinforcement, and expand these studies to monkeys and cocaine self-administering rats. An additional goal is to determine whether these drug-induced behavioral changes are associated with alterations in DA/PKA-regulated signaling pathways in cortico-limbic-striatal regions and correlate putative molecular changes with behavior. We propose to focus on the DA/PKA-regulated protein DARPP-32 because it can be used to assess both inhibition and amplification of DA/PKA signaling, and it is expressed in cortical-limbic as well as striatal regions. Aim 3 will directly test the hypothesis that reduced cortical PKA activity and enhanced limbic-striatal PKA activity can mimic the effects of prior drug exposure using local infusions of PKA inhibitors/activators and examine if drug-induced deficits can be reversed by infusions of PKA modulators.

DESCRIPTION (provided by applicant): Drug abuse and dependence define behavioral states involving increased allocation of behavior towards drug seeking and taking at the expense of more appropriate behavioral patterns. As such, addiction can be viewed as increased control of behavior by the desired drug (due to its unconditioned, rewarding properties). It is also clear that drug-associated (conditioned) stimuli acquire heightened abilities to control behavior. These phenomena have been linked with dopamine (DA) function within the ventral striatum and amygdala and have been described specifically in terms of incentive motivational processes. Our data show that persistent drug-induced enhancement in incentive motivation are associated with, and can be mimicked by, increases in limbic-striatal DA/cAMP/PKA/CREB activity, suggesting that adaptations in DA-regulated intracellular signaling molecules may underlie these behavioral changes. The current proposal will determine how PKA/CREB signaling within the amygdala contributes to associative aspects of incentive motivational processes by using Pavlovian-to-lnstrumental Transfer (PIT) and responding for Conditioned Reinforcement (CR) paradigms. This project will focus on the hypothesis that repeated cocaine exposure will result in persistent enhancements in incentive motivational processes due to alterations in the PKA/CREB signaling pathway in the amygdala. Specifically, we will investigate the following hypotheses. (1) Repeated cocaine exposure enhances incentive motivation and results in alterations of PKA/CREB FosB function in the amygdala. Behavioral and molecular alterations will be examined in rats previously-exposed to cocaine either responding in the presence of (PIT) or for (CR) food-paired conditioned stimuli. (2) Activation of PKA/CREB signaling in drug naive animals will augment incentive motivation. This will be examined using infusions of direct activators/inhibitors of PKA and viral vectors that over-express CREB or mutant CREB into the basolateral or central nucleus of the amygdala. (3) Inhibition of PKA/CREB signaling will block augmented incentive motivation in cocaine-treated animals. Correlations between behavior and CREB activity will also be examined in CRE-LacZ transgenic mice. Together these studies will determine whether cocaine-induced activation of the PKA/CREB signaling pathway in the amygdala results in augmented incentive motivation and whether these effects can be mimicked or blocked by manipulations that stimulate or inhibit PKA/CREB signaling, respectively. These data may be critical for understanding the neurobiology of motivational processes and their putative impact on drug-seeking behavior. Persistent alterations in PKA/CREB signaling in limbic-striatal regions produced by cocaine are hypothesized to contribute to compulsive reward seeking in addiction.

NIH Roadmap Initiative tag; behavioral /social science research tag; behavioral habituation /sensitization; bioimaging /biomedical imaging; corticotropin releasing factor; locus of control

[unreadable] DESCRIPTION (provided by applicant): The objective of this research is to understand the neurobiological basis for sex differences in alcoholism using an animal model designed to investigate the development of habitual responding with relevance for compulsive alcohol-seeking and taking behavioral patterns. Our proposed studies will investigate the interaction between corticostriatal circuits involved in habitual (i.e., stimulus-response) and goal-directed (i.e., response-outcome) behavior. These studies will focus on the role for corticostriatal alterations in dopamine neurotransmission that likely contribute to an intricate balance between goal-directed and habitual patterns of behavior. Previous studies suggest that women progress through the landmark stages from initial use to dependence at a faster rate than men, and the proposed series of experiments will investigate whether the faster rate of habit formation that we have observed in female rodents extends to habitual responding for alcohol. Such findings of genetic sex differences in habit formation for a natural reinforcer suggest inherent differences in corticostriatal circuitry that could underlie differences in habit formation for alcohol. Additionally, we have shown that dopamine infusions into the ventromedial prefrontal cortex can restore goal-directed responding in rats trained to express an instrumental, food-reinforced, habit. Together these studies form the basis for our hypothesis that female rats will progress toward habitual patterns of alcohol-seeking and taking more rapidly than male rats and that the development of habitual responding for alcohol will depend upon increased dopamine transmission in the dorsolateral, but not dorsomedial, striatum, and on decreased dopamine transmission in the ventromedial, but not dorsomedial, prefrontal cortex. Aim I will determine whether there is a sex difference in the rate of habit formation in rats when alcohol serves as the reinforcer. Subsequent experiments will address the neurobiological mechanisms (e.g., gonadal hormones and/or sex chromosome genes) for this potential sex difference. Aim II will use parallel procedures to examine whether dopamine transmission in the medial prefrontal cortex or dorsal striatum contributes to the performance of an alcohol habit. Subsequent experiments will address which dopamine receptors contribute to regionally specific behavioral effects. We believe that this focus provides an innovative approach because no previous research has examined sex differences in habit formation for a drug reinforcer or the role of corticostriatal dopamine neurotransmission in the goal-oriented and stimulus-bound components of alcohol responding. These data will provide the foundation for the complete analysis of genetic and hormonal contributions to sex differences in habitual responding for alcohol and cues associated with alcohol. Further, the data obtained will provide the first direct comparison of prefrontal cortical and dorsal striatal dopamine involvement in habitual responding. PUBLIC HEALTH RELEVANCE Alcoholism is characterized by the development of habitual drinking patterns that occur regardless of the societal or health consequences. Previous work has indicated that habits for alcohol consumption develop faster than for natural reinforcers, and that females may have a genetic predisposition to develop habits faster. The aim of this research is to determine if females develop alcohol habits more rapidly than males, and to determine how corticostriatal neurocircuitry regulates alcohol habit formation. The results of this research will help determine therapeutic targets to help alcoholics maintain abstinence by inhibiting the ability of strong habits to control behavior, and will determine if earlier interventions for female alcoholics can improve recovery. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Drug-associated cues elicit craving and relapse in addicts and contribute to the progression and persistence of addiction. The objective of this research is to establish novel behavioral and pharmacological methods to effectively and persistently reduce relapse by decreasing the motivational significance of drug cues, and to elucidate the corresponding neurobiological mechanisms. We have identified drug-induced alterations in amygdala DA/cAMP-regulated signaling that promote reward-related learning and memory processes, result in stronger reward-associated memories, and that enhance behavioral control by cues acting as conditioned reinforcers. While we have focused on DA/cAMP-regulated signaling cascades, recent biochemical and pharmacological studies show that cAMP activates at least two distinct intracellular signaling targets: protein kinase A (PKA) and exchange protein activated by cAMP (Epac). Epac is highly expressed in the basolateral nucleus of the amygdala (BLA) and prefrontal cortex (PFC) but its role in brain is virtually unknown. Our new data confirm regulation of BLA- and PFC-dependent behaviors by both PKA and Epac. Here, we will test the hypothesis that both disrupted reconsolidation of cue-drug memories and enhanced consolidation of extinction can reduce cue-induced relapse. To understand the role of cAMP-regulated cellular events in these processes we will use selective activation or inhibition of PKA and Epac given after non-reinforced cue exposures to independently alter reconsolidation or extinction mechanisms. Subsequent tests will examine cue-induced reinstatement and the ability of cocaine-associated cues to act as conditioned reinforcers. Aim 1 will use amygdalar manipulations to disrupt memory reconsolidation to reduce the strength of cue-drug associations. Our data demonstrate that amygdala infusions of inhibitors of cAMP signaling after reactivation of a cocaine- paired cue can reduce both cue-induced reinstatement and responding with conditioned reinforcement, consistent with reports that cocaine-seeking behavior can be reduced by disruption of cue-drug memories. Aim 2 will use PFC manipulations (infralimbic vs. prelimbic) to enhance extinction of cocaine-associated cues. These studies will also investigate the impact and persistence of selected manipulations on newly acquired or older cue memories as well as characterize resulting alterations in cAMP/PKA/Epac activity. Aim 3 will use interventions identified in Aims 1 &2 to discover if a combination of approaches that disrupt reconsolidation and enhance consolidation of extinction together can have more profound effects. Reductions in cue-induced reinstatement and conditioned reinforcement as well as altered context dependency of cue extinction will be examined. We will also determine if cocaine-induced alterations in cAMP/PKA/Epac activity induce persistent, maladaptive, drug-associated memories by biasing cue-drug memories to undergo reconsolidation, as opposed to extinction when reactivated. Together these studies should identify processes that underlie cue- induced craving and relapse in order to develop novel behavioral and pharmacological treatment strategies. The goal of the proposed research is to understand how cAMP-regulated signaling processes can be used to reduce the behavioral control of cue-cocaine memories through alterations in memory reconsolidation and extinction - opposing mnemonic processes that depended on subregions of the amygdala and prefrontal cortex, respectively. We will also test the hypothesis that cocaine-induced neuroadaptations in these regions may predispose cue memories to undergo reconsolidation, as opposed to extinction, and thereby contribute to the development and persistence of maladaptive drug-associated memories and their ability to precipitate cocaine-seeking and -taking behavior. Understanding these mechanisms can be used to identify novel behavioral and pharmacological treatment strategies to effectively and persistently reduce the ability of cocaine-associated cues to induce relapse in order to combat addiction.

DESCRIPTION (provided by applicant): The objective of this research is to define the role for PFC-dependent inhibitory control in processes that likely contribute to addiction. We have shown that repeated exposure to cocaine (COC), PCP or THC, causes aberrant cognitive-motivational function and neurobiological alterations in cortico-limbic-striatal circuits in rodents and monkeys. Short-term COC exposure was sufficient to produce persistent and selective deficits in reversal learning and inhibition of pre-potent responding, effects indicative of selective OFC dysfunction, but did not alter extinction learning, attentional selection or working memory. Diminished inhibitory control was associated with increases in the motivational significance of reward-related stimuli. These drug-induced impairments were linked to, and can be mimicked by, altered cAMP-regulated intracellular signaling within the PFC and limbic-striatal regions in rodents suggesting that such adaptations may underlie alterations in inhibitory control. Proteomics analysis of synaptoneurosomes after prior chronic COC exposure to monkeys identified alterations in proteins involved in synaptic function and activity-dependent plasticity, with distinct cortico-striatal patterns. A large number of regulated proteins were associated with cAMP-regulated signaling networks, cytoskeletal, cell adhesion, vesicle trafficking and metabolic functions. Together, these data support our hypothesis of "frontal-striatal dysfunction in addiction" yet the causal relationship between PFC dysfunction and addiction remains to be determined. Here we will investigate the interaction between COC- induced and pre-existing individual differences in OFC-mediated inhibitory control function on the development of "addictive-like" behaviors in established animal models. Aim I will determine the link between OFC-dependent inhibitory control function and compulsive drug-seeking behavior. Separate groups of rats will be examined on either cue-motivated COC-seeking measured by responding on a second-order schedule of COC self-administration or COC-seeking behavior in conflict with intermittent presentation of aversive stimuli. Another goal is to determine the neurobiological mechanisms of these interactions using biochemical analyses. Aim II will test the contribution of selected drug-induced neurobiological alterations in the OFC focusing on cAMP-regulated signaling and the transcription factor Sp1 that was identified in our proteomic analyses. Sp1 controls D2 receptor expression and has not previously been implicated in stimulant addiction. Aim III will extend these data by confirming in monkeys the interaction between individual and COC-induced differences in OFC function on inhibitory control, incentive motivation, and response conflict, and the associated neurobiological alterations using state-of-the-art proteomic techniques. Viral-mediated gene transfer will be used to manipulate Sp1 in the OFC of rats (Aim II) or monkeys (Aim III) to determine its effects on OFC-dependent inhibitory control and potential role in compulsive aspects of behavior. Together these studies should significantly advance our understanding of OFC-inhibitory control and vulnerability to addiction. PUBLIC HEALTH RELEVANCE: We have shown that prior repeated exposure to addictive drugs such as cocaine produces persistent deficits in response inhibition and incentive motivational function that are associated with neurobiological alterations in cAMP/PKA-regulated signaling in cortico-limbic-striatal brain regions in rodents and monkeys. Such changes may be directly relevant to understanding addiction as an inability to modulate changes in reward-motivated behaviors may increase the motivational significance of reward-associated cues and contribute to relapse and difficulties in inhibiting drug-associated thoughts and behaviors. We hypothesize the level of orbitofrontal cortical function prior to cocaine exposures may also predict the extent of cocaine-induced dysfunction, and we will investigate how cocaine-induced and individual differences in orbitofrontal-mediated processes can interact to affect the development of cognitive dysfunction and addictive-like behavior.

The mechanisms underiying the progression from voluntary use to the compulsive drug-seeking/taking behavior that define addicfion remain largely unknown. The ultimate goal of this translafional P20 is to take advantage of combined behavioral and neurobiological studies in rats, non-human primates (NHP), and humans to test the hypothesis that dissociable forms of impulsivity contribute to the development of compulsive drug-seeking/taking behavior. We have hypothesized that impulsivity resulting from frontostriatal dysfuncfion is central to addiction but the precise relationship between impulsivity and compulsivity has not been defined. Our data show that prior cocaine (COC) exposure can selectively disrupt inhibitory control funcfions mediated by the orbitofrontal cortex (OFC) while at the same fime altering limbic-striatal function. We have also identified persistent region-specific alterafions in the synaptic proteome after COC exposure in monkeys, including widespread changes in proteins involved in coordinating synaptic plasficity. While these COC-induced deficits are associated with altered DA-regulated signaling within cortico-limbic-striatal regions the interaction between pre-existing and COC-induced individual variability in inhibitory control processes and "addictive-like" behavior has not been elucidated. Here we will examine two forms of impulsivity: impulsive acfion and impulsive decision-making using stop signal task (SST) and inter-temporal choice task (ITCT). These two tests likely depend on the dorsomedial and ventral striatum, respectively. Individual differences in impulsivity on these tasks will be invesfigated in rats that will be divided into balanced groups based on their level of performance. Rats with "low" vs. "high" impulsive performance will be tested daily during repeated COC or SAL injecfions given 6 hours after behavioral tesfing for 30 days. Animals will then be tested on acquisifion COC selfadministrafion (SA) and COC-seeking behavior measured by cue-induced reinstatement. Assessment of level and forms of impulsivity with post-exposure performance will be determined and correlated with post-mortem biochemical measures of alterations in D1/D2/D3 receptors in cortico-striatal regions. We hypothesize that individual differences in D2/D3-regulated signaling in the ventral striatum (nucleus accumbens, NAc) will predict performance on the ITCT whereas the level of D2-regulated signaling in the dorsomedial striatum (dmS) will correlate with performance on the SST. Mechanisfic tests will utilize regional viral-vector mediated overexpression of the transcripfion factor Spl to prevent the development of impulsive behavior. Spl was identified as a COC-regulated target from our previous proteomic data and is known to regulate many of the synaptic proteins reduced by COC. Our recent studies have confirmed COC-induced alterations in Sp1 and several of its downstream targets, including D2/D3 receptors, in both cortical and striatal regions. These findings are consistent with observations made in human addicts. We hypothesize that mulfiple forms of impulsivity that involve anatomically distinct regions contribute to addiction vulnerability that, together with COC-induced dysfunction, synergize to produce compulsive drug-seeking behaviors that characterize addiction.

These studies will identify neurobehavioral mechanisms underlying vulnerability to habitual ethanol-seeking behavior and relapse. We hypothesize that individual differences in the ability of incentive stimuli to control behavior can promote compulsive alcohol drinking in the face of adverse consequences. Our studies will test whether individual variation in the ability of cues to motivate behavior measured by the Pavlovian-to-lnstrumental Transfer (PIT) procedure, will predict habitual ethanol-seeking behavior (i.e., stimulus-response) and cue-induced reinstatement behavior in mice. Animals that show high levels of sucrose PIT will display enhanced ethanol habits measured by insensitivity to outcome devaluation and increased cue-induced reinstatement. Aberrant cognitive flexibility and regulation of control over reward-motivated behavior is central to these preexisting differences in PIT that we argue depend on altered dopaminergic and glutamatergic signaling in regions of the prefrontal cortex (PFC) and striatum. Our published data show that dopaminergic activation of the ventromedial (vm) PFC can restore goal-directed responding in animals that are responding habitually for food. We propose to determine if manipulations of dopaminergic and glutamatergic signaling can reduce habitual ethanol-motivated responding and cue-induced reinstatement in mice that show high levels of sucrose PIT. The role of these signaling pathways will be examined in the vmPFC and dorsal striatum given their known roles in habit formation and in the orbito-frontal cortex and nucleus accumbens, given their role in incentive Pavlovian (stimulus-outcome) learning. Aim 1 will test the hypothesis that individual variation in sucrose PIT predicts the transition from goal-directed to habitual ethanol-seeking behavior. Alterations in protein expression and activity in cortico-limbic-striatal regions will be examined in mice showing high vs. low levels of PIT and those expressing habitual vs. goal-directed ethanol-seeking behavior. Aim 2 will examine if individual differences in sucrose PIT predict reinstatement of cue-induced ethanol seeking. We will also determine if indirect stimulation of extra-synaptic metabotropic glutamate receptors with N-acetylcysteine or direct reduction in NMDA activity with memantine can prevent the expression of ethanol habits and reduce cue-induced reinstatement of ethanol seeking. Our proposed studies will provide the first direct investigation of preexisting mechanisms of incentive motivation and their relationship to habitual ethanol-seeking behavior. These data will provide a foundation for understanding potential genetic contributions to habitual ethanol behavior and identify mechanisms of dopaminergic and glutamatergic dysfunction in corticostriatal circuits that may underlie alcoholism vulnerability and relapse.

Project 1: The Interaction of DA/Glutamate Signaling in Vulnerability to Aberrant Alcohol Seeking Jane Taylor, Ph.D. Abstract Although many people can casually consume alcohol, some people develop alcohol use disorder (AUD), especially those that are family history positive (FHP) for AUD. In AUD, drinking becomes habitual and alcohol cues become powerful triggers that invigorate alcohol seeking and ultimately trigger relapse to alcohol consumption. Unfortunately, there are few effective therapies that reduce the strength of these cues. Our goal is to use cutting edge behavioral, transgenic and viral techniques to provide a more complete understanding of the neurobiological mechanisms of habitual alcohol seeking and relapse. We will test the overarching hypothesis that habitual alcohol seeking and relapse in FHP and AUD is due to enhanced D1-R-mediated NMDA-R function and decreased D2-R-mediated NMDA-R function in the dorsal striatum. In CTNA3 we found that vulnerability to habitual ethanol (EtOH) seeking and relapse in mice was associated with two distinct endophenotypes of enhanced cue reactivity. First, mice that exhibited high Pavlovian Approach (PA) exhibited greater habitual alcohol seeking relative to mice that exhibited low PA. Second, mice that exhibited high Pavlovian to Instrumental Transfer (PIT) exhibited greater cue-induced reinstatement of EtOH seeking relative to mice that exhibited low PIT. Here, we will study the molecular basis of these predictors of compulsive EtOH seeking. Specifically, we propose to investigate the role of DA signaling cascades that underlie the transition from goal-directed to habitual alcohol seeking (Aim 1), and cue-induced relapse (Aim 2). We will use cell- specific viral knockdown of the key DA-signaling molecules that regulate NMDA-R function, the tyrosine kinase Fyn and the tyrosine phosphatase STEP, in D1-Cre and D2/A2A-Cre mice to selectively determine the roles of Fyn and STEP in D1 or D2 dorso-medial and dorso-lateral striatal neurons: The AAV-flox-Fyn-shRNA and AAV-flox-STEP-shRNA constructs were designed specifically for this project. We will then test a novel systemic Fyn inhibitor to prevent the transition to habitual EtOH-seeking. In CTNA3, we also found that cortical neural cell adhesion molecule (NCAM) expression predicted cue-reactivity and was required for cessation of EtOH-seeking behaviors. The Clinical Core identified NCAM-1 as the gene most closely associated with Fyn kinase in the AUD ?risk? gene network. We will investigate dorso-striatal NCAM signaling-dependent NMDA-R expression in the regulation of habitual alcohol seeking and relapse through enhanced polysialation: The AAV- PST virus is a novel means to enhance PSA-NCAM expression. Finally, we are uniquely positioned to study sex differences in EtOH seeking and DA/NMDA signaling. In CTNA3 we found the transition to habitual EtOH seeking is accelerated in male mice relative to female mice, and this depends on chromosomal complement rather than gonadal status: Cre-mouse lines will be used for the first time to extend these findings. Overall, this project will yield reverse translational data through parallel behavioral assessments and targeted brain-specific molecular analyses that will inform the use of Fyn-kinase inhibition as a potential pharmacotherapy for AUD.

Although many people engage in occasional drug use, few become drug-dependent. The mechanisms by which addiction vulnerability and drug-induced pathological factors interact to increase the likelihood of drug dependence remain unknown. Mounting evidence suggests that drug dependence is strongly associated with dysfunctional impulsive decision-making processes in which individuals with cocaine-use disorder (CUD) undervalue (Le., discount) future rewards in favor of smaller, immediate rewards to a greater extent than their non-addicted peers. Thus, temporal discounting of reward, as measured by various intertemporal choice tasks, has been proposed as a behavioral marker of addiction, which may be due to aberrant dopamine (DA) and serotonin (5HT) signaling within midbrain regions and corticostriatal circuits including the ventromedial prefrontal cortex (vmPFC) and striatum. Studies in CUD show elevated D3 receptor availability in the midbrain, decreased 5HT1B availability in the vmPFC and heightened levels of impulsive choice. Furthermore, our pilot rat positron emission tomography (PET) data demonstrate increased striatal l1C-PHNO binding is positively correlated with subsequent cocaine self-administration and individual differences in choice behavior, which highlights the impact of vulnerability factors on cocaine use. We have also shown that D2I3 receptors modulate flexible decision-making but it is not known how these receptors impact impulsive choice or cocaine self- administration. Here we will use a delay discounting across various randomly presented delays procedure, which yields robust individual differences in rat temporal discounting, in combination with two PET tracers (11C- PHNO for D3 and 11C-P943 for 5HT18 receptors), receptor-specific pharmacology and cocaine self- administration to investigate these proposed biobehavioral markers of addiction. In Aim 1, we will investigate the relationship between individual differences in corticostriatal D3 and 5HT1B receptors and impulsive choice and cocaine self-administration. In Aim 2, we will assess the impact of cocaine self-administration on corticostriatal D3 and 5HT18 receptors and impulsive choice using longitudinal PET/behavioral analyses and ex vivo analyses. Finally, in Aim 3, we will determine the role of midbrain D3 and vmPFC 5HT18 receptor regulated signaling in cocaine-induced enhancements in impulsive choice. In accordance with pre-clinical and clinical research, we predict that individual differences in impulsive choice are mediated by vmPFC 5HT1Band that cocaine induced-changes of midbrain D3 receptors modulates this signaling, yielding enhancements in impulsivity. Our studies in rats will complement studies in CUD individuals through the use of longitudinal neuroimaging, pharmacological manipulation of brain targets, and ex vivo validation of PET imaging results, while maintaining the common behavioral metric of impulsive choice.

Although many people use drugs of abuse occasionally, few people develop a drug addiction. The likelihood that an individual transitions from occasional to compulsive patterns of drug use may depend upon aberrant pre-existing decision-making processes. We were among the first to argue that prefrontal dysfunction mediates top-down behavioral control in addiction, a perspective that is now widely recognized, and we have provided supporting evidence that decision-making deficits, incentive motivation and habits, and neurobehavioral plasticity are impacted in animal models of addiction. Nonetheless, a more mechanistic, circuit-driven approach is needed to dissect the precise relationship between individual differences in decision-making dysfunction and vulnerability to addiction from the consequence of chronic drug exposure. We will use cutting-edge viral tools to characterize the neurobiological relationship between aberrant decision-making strategies in rats on the development and persistence of addiction-like behavior. Here we focus on the role of orbitofrontal cortex (OFC) projections to distinct subcortical targets (OFC-to-amygdala and OFC-to-nucleus accumbens) in decision- making processes. Novel tasks will be used that assess the ability of rats to adapt behavior to changing reinforcement contingencies or make choices based on abstract representations of action-reinforcement contingencies. We hypothesize that by using highly-translational behavioral tasks that are dependent upon the OFC and computational models to better characterize decision-making processes, we will isolate the OFC- dependent circuitry and mechanisms that underlie addiction vulnerability. In Aim 1, we will identify precise decision-making processes that predict vulnerability to cocaine-taking behaviors by combining retro-fitted behavioral tasks with sophisticated computational analyses. In Aim 2, we will characterize the role of specific OFC circuits in decision-making behaviors that predict vulnerability to cocaine-taking behavior using a novel circuit-specific, retroviral ablation approach to identify and remove specific top-down OFC circuits. Finally, in Aim 3, we will investigate the role of plasticity mediated by neural cell adhesion molecule (NCAM) and its proplastic modified form, polysialylated NCAM (PSA-NCAM) within OFC circuits, in decision-making processes that predict vulnerability to cocaine-taking behaviors using viral tools. Overall, results from this work should provide an innovative perspective on the role of selective loss of top-down, OFC?subcortical control on decision-making processes and vulnerability to addiction. These studies have the potential to inspire the development of novel therapeutic strategies, open new areas of investigation for biological psychiatry and neuroscience, and produce highly translational results for human addiction.

Abstract The main purpose of the Biological Sciences Training Program in the Department of Psychiatry at Yale University is to train postdoctoral fellows in biological and behavioral sciences relevant to mental illness and health. To attain this goal, the program recruits two groups of postdoctoral trainees: 1) individuals with a PhD degree in neuroscience or other basic science discipline, and 2) psychiatrists interested in basic and translational neurobiology research, generally after completing two or three years of clinical residency. To help fill a nationally recognized need, the aim of the program is to both increase the number of research psychiatrists with a grounding in basic biological sciences and to develop the interest of basic neuroscientists in clinical problems as well as in interdisciplinary aspects of their own basic research. Thus, the overall aim is to promote cross-fertilization between basic researchers and individuals who are experienced clinically. Extensive research collaborations between faculty members of the training program further contribute to the interdisciplinary experience of the trainees. It is expected that trainees coming out of this program will be able to bridge the gap between basic and clinical neuroscience and conduct translation research to elucidate the molecular and cellular basis of complex behaviors related to major psychiatric illnesses. There are 33 faculty members in the program consisting of both basic and clinical researchers. The number of postdoctoral training slots requested is five, the same number as the current grant. The program typically involves training for 2 years in one primary discipline but usually includes direct or collaborative interactions with other disciplines. Interdisciplinary laboratory training is offered in molecular and cellular neurobiology, gene expression profiling, proteomics, in vivo imaging, in vivo multi-electrode recording, optogenetics, intracellular signaling, neurochemistry, electrophysiology, neuropharmacology, and behavioral models of psychiatric illness, as well as motivation, reward, cognition, learning and memory; there is an emphasis of the interactions with strong clinical research programs in the department of Psychiatry. Clinical research training is offered in behavioral assessment, genetic analysis, biological measurements, and multiple types of neuroimaging techniques. In addition to specific research training, there are courses in Clinical Neuroscience and Neuropharmacology as well as seminars and regular Workshops in which Fellows present ongoing research to the entire group of faculty and trainees. The training proposal is directly relevant to the research objectives of NIMH by fostering the training of researchers to produce new knowledge about mental disorders, maladaptive behavior, and novel treatment strategies. Thus, basic and clinical research is carried out in areas pertaining to anxiety (e.g., panic and post- traumatic stress disorder), obsessive-compulsive disorder, schizophrenia, and mood disorders.

Drug-associated conditioned stimuli (CS), or cues, contribute to both the progression and persistence of addiction. We hypothesize, as have others, that blocking reconsolidation of cocaine-cue memories could prevent cue-induced relapse. Reconsolidation is a process whereby reactivation of a memory renders it labile and vulnerable to disruption. Our published data, and that of others, confirm that limbic-striatal dopamine (DA)- dependent and several plasticity-regulated signaling mechanisms are involved in cocaine-cue reconsolidation processes. In this new proposal we hypothesize that violation of cocaine-cue expectancy renders a memory labile by triggering memory destabilization, thereby making cocaine-cue memories more sensitive to subsequent disruption by amnestic agents. The ability to induce the destabilization of cocaine-cue memories may be a key factor in enabling the use of targeted pharmacotherapies to block cocaine-cue memory reconsolidation and treat maladaptive memories. We propose to investigate directly the role of expectancy in reconsolidation of cocaine-cue memories and their impact on subsequent relapse-like behaviors by varying the difference between what is expected and experienced during reactivation and by photo stimulation of midbrain DA-containing VTA neurons to neutralize or induce prediction error (PE)-like signals. In Aim 1 we will reactivate cocaine memories by violating expectations of a) US (i.e., cocaine) magnitude and b) CS-US temporal contiguity. These manipulations should generate PE-like signals at the time of memory retrieval. According to our hypothesis and preliminary data, both positive and negative PE-like signals should induce destabilization and make the cue memory susceptible to blockade (i.e., reconsolidation) by an amnestic agent to reduce subsequent relapse to drug-seeking behavior. Relapse-like behaviors will include cue-induced and drug-primed reinstatement, and other measures, in male and female rats subjected to weeks of long-access cocaine self-administration. Select agents that potently and selectively alter memory reconsolidation processes ? the protein-synthesis inhibitor anisomycin (ANI) and the histone acetyltransferase (HAT) inhibitor garcinol ? will be used to render the destabilized memory subject to amnestic blockade. In Aim 2 we will optogenetic-based photostimulation of midbrain VTA DA neurons (TH-Cre+ rats) during cocaine-cue memory reactivation a) to artificially produce a dip or b) a spike in DA signaling, to artificially act as a negative or positive PE-like signal, respectively, to destabilize the cue memory and render it susceptible to amnestic blockade, ultimately reducing measures of cocaine relapse-like behaviors. Together these studies will define novel behavioral conditions whereby destabilization mechanisms can be used to make memories more susceptible to amnestic agents, to block reconsolidation of cocaine-cue memories, to reduce relapse-like behaviors, and to inspire the development of innovative therapeutic strategies for maladaptive memories.

P1 SUMMARY: Dramatic rates and consequences of alcohol use disorder (AUD) continue to be a major public  health concern. Treatment approaches remain limited and translational research is imperative for the  development of more effective interventions for AUD. AUD is associated with a hyper-­glutamatergic state and  CTNA5 will focus on the mGluR5 negative allosteric modulator (NAM), mavoglurant, as a novel therapeutic for  AUD. Project 1 will investigate how AUD impacts alcohol-­seeking behaviors and decision-­making processes by  focusing on translational behavioral approaches and systems-­level techniques in rodent models. Project 1 will  serve the overall CTNA mission by testing the overarching hypothesis that mavoglurant inhibition of mGluR5  will normalize alcohol-­induced disturbances in cortico-­striatal glutamate signaling and behavioral dysfunction  Our preliminary and published data establish glutamatergic projections from the prefrontal cortex (PFC) to the  striatum (nucleus accumbens, NAc) as an essential circuit involved in alcohol-­seeking behaviors. Our data also  have established key cortico-­striatal circuits that mediate distinct aspects of reinforcement learning processes  that govern habitual (model-­free) and goal-­directed (model-­based) decision making, which are known to be  altered in addictions. Aim 1 will assess the role of mavoglurant in alcohol-­seeking behaviors. Fiber  photometry in PFCà?NAc circuits will be used to assess neural activity dynamics, and, critically, the ability of  mavoglurant to block motivation for alcohol and cue-­induced reinstatement in male and female alcohol-­ exposed mice. Aim 2 will investigate the ability of mavoglurant to alter alcohol-­induced aberrant  decision-­making. Decision-­making processes will be assessed using our novel rodent version of the multi-­ stage decision-­making (MSDM) task that has been shown to be sensitive to altered response strategies in  AUD. Decision-­making strategies in male and female alcohol-­exposed mice will be determined and the ability  of mavoglurant treatment to restore both MSDM strategies and neural activity will also be characterized. While  there are known sex differences in the neurobiology of AUD, few systematic studies have investigated the  interface between sex, alcohol-­seeking behaviors, and decision-­making processes in alcohol-­exposed mice,  and these studies will provide new insights into these interactions. To extend our studies to the cellular level,  mGluR5 signaling will be assessed ex vivo and correlated with behavior. The proposed research is highly  integrated with the planned human CTNA5 studies in Projects 2 and 3, that will use analogous laboratory  models of AUD, drinking behaviors, and decision-­making tests to examine the ability of mavoglurant to alter  behavior and neural circuits. This preclinical project complements the clinical studies by generating a level of  mechanistic insight that is not possible for studies in humans. Together our studies will enable a mechanistic  understanding of the neural circuits mediating relapse to alcohol-­seeking and compulsive decision-­making  behaviors and the putative therapeutic role of mGluR5 NAMs in these AUD-­relevant behaviors.