Barry Setlow, PhD - US grants

The amygdala and nucleus accumbens have long been recognized as structures important for the control over behavior by reinforcing and motivational stimuli. More recent work suggests that the basolateral complex of the amygdala (BLC) and the accumbens, to which it projects strongly and unidirectionally, function as a system involved in the processing of acquired stimulus value information. However, several questions remain regarding the functions of this system, including whether it is involved in learning processes or simply behavioral expression of learned information, and whether it is equally important for processing of appetitive and aversive stimulus value information. The proposed experiments will investigate the nature of the involvement of the BLC and accumbens in these functions. Specific Aim 1 will use an appetitive second-order conditioning task (which allows assessment of the degree to which a stimulus has acquired motivational value), to determine whether the BLC and accumbens are involved in learning or performance of appetitive stimulus value information. Specific Aim 2 will use an aversively motivated second-order conditioning task to determine whether the accumbens is involved in processing of aversive stimulus value, and if so, in what capacity. Specific Aim 3 will use in vivo electrophysiology to investigate how appetitive and aversive stimulus value are encoded in accumbens neuronal responses, and the degree to which this encoding is dependent on the BLC. It is hoped that these complementary approaches will enable a more complete understanding of how these structures function together to influence motivated behavior in both normal and disease states.

DESCRIPTION (provided by applicant): Research in drug addicts shows that cocaine use is associated with alterations in cognition and motivation. Cognitive alterations associated with drug addiction include impairments in decision-making and choosing appropriate responses based on the value of likely outcomes. Motivational alterations include enhancements in the control over behavior by rewards and cues predictive of reward. Animal research has begun to establish causal links between cocaine exposure and such alterations, and it has been proposed that alterations in cognition and motivation resulting from drug exposure may act synergistically to promote drug addiction. However, the relationship between cocaine's effects on these two functions is unclear. This proposal is designed to investigate the relationship between cocaine's impairing effects on responding based on the value of likely outcomes and its enhancing effects on control of behavior by reward-related cues. Specifically, rats given various cocaine exposure conditions will be tested on a behavioral task that assesses both of these functions. The results of this experiment will determine a) whether both impaired responding based on outcome value and enhanced behavioral control by reward-related cues are observed across a wide range of cocaine exposure conditions, and b) whether cocaine's effects on these functions are correlated. These findings will be used to guide future research investigating the neural systems underlying interactions between cognitive and motivational consequences of cocaine exposure.

DESCRIPTION (provided by applicant): Drug addiction is a serious public health problem. Approximately 22 million Americans (9% of the population over 12 years of age) abuse or are addicted to alcohol and illicit drugs. Although the causes of drug addiction are multifaceted, it is becoming increasingly clear that cognitive deficits associated with drug use may contribute to the addiction process. In particular, drug addicts demonstrate poor decision-making and an abnormally increased preference for immediate over delayed gratification (i.e. - "impulsive choice"). This increased impulsive choice may contribute to the addiction process by rendering addicts more likely to choose the short-term rewards of further drug use over the delayed but ultimately more beneficial rewards (such as health, employment, and family) associated with abstinence. Because increased impulsive choice appears to persist long after cessation of drug use, this cognitive alteration may account in part for the high relapse rates that follow even the best currently available addiction therapies. Using animal models, in which clear cause-and-effect relationships can be determined, it has been established that chronic cocaine self-administration causes increased impulsive choice, just as is observed in human addicts, that can persist as long as 3 months after cocaine cessation. Cocaine self-administration also causes long-lasting neuroadaptations in a network of brain systems, including alterations in dopaminergic and glutamatergic signaling in the nucleus accumbens, a structure implicated in regulation of impulsive choice in drug-naove subjects. However, the role of these nucleus accumbens alterations in cocaine-induced increases in impulsive choice is unknown. The experiments in this proposal will test the hypothesis that alterations in dopaminergic and glutamatergic neurotransmission in the nucleus accumbens are involved in the long-lasting increases in impulsive choice caused by cocaine self-administration. Specifically, we will determine in a rat model: i) the procedural parameters under which cocaine self-administration increases impulsive choice, ii) the roles of nucleus accumbens dopaminergic and glutamatergic signaling in normal impulsive choice in drug-naove subjects, and iii) whether cocaine-induced increases in impulsive choice can be reversed by pharmacological targeting of alterations in nucleus accumbens dopaminergic and glutamatergic signaling known to result from cocaine self- administration. Knowledge gained from these experiments of the neural mechanisms by which cocaine self-administration increases impulsive choice is crucial for the development of novel effective treatments for addiction. Such treatments would have the potential to reduce the incidence of relapse and to enhance productivity and quality of life for addicted individuals. Cocaine use is associated with long-lasting increases in impulsive choice, (reflected in a preference for immediate over delayed rewards), which may contribute to addiction by rendering users more likely to choose the immediate rewards of further drug use (e.g., short-term euphoria, relief from withdrawal symptoms) over the delayed but ultimately more beneficial rewards of abstinence (better health, employment, family relationships). The experiments in this proposal will use a rat model to investigate brain mechanisms by which cocaine use causes persistent increases in impulsive choice. Knowledge gained from these experiments will be critical for development of novel treatments to reduce the likelihood of relapse and to enhance quality of life for addicted individuals.

? DESCRIPTION (provided by applicant):: Cannabis is the most widely-used illicit drug in the U.S., and over 1 million people are treated for cannabis dependence annually. Given the potential for dependence as well as deleterious effects of chronic use, there is an urgent need to better understand the mechanisms supporting cannabis use, in order to develop therapies to reduce such use. There are several animal models in which reliable intravenous self-administration of cannabinoids such as ?9THC (THC) has been demonstrated. Most human use, however, occurs through inhalation of cannabis smoke, which contains numerous cannabinoids aside from THC, some of which is psychoactive and may interact with THC to alter its reinforcing effects. Hence, an animal model that employs cannabis smoke self-administration would more closely mimic the conditions of actual human use, and would open new directions of research on cannabis use and abuse. The long-term goal of this research program is to use rodent models to investigate the reinforcing effects of smoked cannabis, with the ultimate goal of developing therapies for reducing cannabis use/abuse. As the first step toward this goal, the objective of this CEBRA R21 project is to develop and validate a system by which rats can self-administer cannabis smoke. To our knowledge, there are no reports of self-administration of smoked drugs of abuse in rodents; however, there have been several demonstrations in primates of reliable self-administration of smoked drugs of abuse, including cocaine and heroin. In addition, we have preliminary data showing development of dependence following passive cannabis smoke administration in rats, demonstrating the efficacy of cannabis smoke as a drug delivery vehicle in this species. On the basis of these published and preliminary data, our central hypothesis is that rats will self-administer cannabis smoke, and that smoke self-administration behavior will be similar to that reported for intravenous cannabinoids and other drugs of abuse. We have designed an apparatus that will allow precisely-calibrated, response-contingent delivery of cannabis smoke using experimental designs similar to those employed with other drugs of abuse. We will use this apparatus to determine whether rats will reliably show operant responding for cannabis smoke delivery, and whether this responding is sensitive to smoke THC content and CB1 receptor activation, as well as to cues predictive of smoke delivery. We have also developed multiple strategies to increase the likelihood of obtaining self-administration behavior. Successful development of a rodent cannabis smoke self-administration model will lay the groundwork for a larger research program on neurobehavioral mechanisms of cannabis smoking. In addition, this model could be adapted for use with other smoked drugs of abuse (e.g., tobacco).

DESCRIPTION (provided by applicant): Chronic cannabis use in humans is associated with a range of affective, cognitive, and neural alterations, including a higher incidence of depression and anxiety, deficits in memory and executive functions, and regional reductions in brain volume and white matter integrity. Emerging evidence indicates that these alterations are greater when cannabis use begins at an early age and can persist well into abstinence, indicating that adolescent cannabis exposure may have particularly deleterious effects on the developing brain. These findings are consistent with research in rodent models showing that administration of ?9THC or other cannabinoid agonists during adolescence causes cognitive deficits and increased negative affect in adulthood. Notably, however, there is almost no research on the effects of adolescent exposure to cannabis smoke on neurobehavioral outcomes. The lack of such research is striking, not only because smoking is the preferred route of human cannabis use, but because cannabis smoke contains numerous cannabinoid and other compounds aside from ?9THC, some of which are known to be psychoactive and may have lasting effects on the brain and behavior. Hence, in order to assess the causal impact of cannabis use, it is critical to employ a model that mimics the conditions of actual human exposure. The long-term goal of this research program is to determine how adolescent exposure to cannabis smoke affects adult emotional, cognitive, and brain structural/functional measures shown to be altered in human cannabis users. Important to this long-term goal, we have established a cannabis smoke exposure model in rats, which produces blood THC levels comparable to those found in humans and in which we have demonstrated cannabis dependence following chronic exposure. Building on these preliminary data, as well as published literature on cannabis users, our central hypothesis is that adolescent cannabis smoke exposure will cause deleterious effects on emotional and cognitive processing in adulthood that are accompanied by structural abnormalities in relevant brain circuitry. We will test this central hypothesis in a rat model by: ) determining whether adolescent cannabis smoke exposure influences negative affect (measures of depression- and anxiety-like behavior) during adulthood; 2) determining whether adolescent cannabis smoke exposure influences multiple measures of memory and executive function in adulthood; 3) determining whether adolescent cannabis smoke exposure affects brain volume and white matter integrity during adulthood, using neuroimaging techniques similar to those employed in assessments of human cannabis users. In addition to informing medical and public policy decision making, findings from this project will lay the groundwork for a broader research program directed toward understanding the mechanisms by which developmental cannabis smoke exposure affects the brain and behavior.

DESCRIPTION (provided by applicant): Drug addiction is associated with poor decision making and elevated risk taking in both males and females. Elevated risk taking may contribute to addiction and poor life outcomes by rendering individuals more likely to engage in drug use as well as other risky behaviors. The causal relationships between drug use and elevated risk taking, as well as the neural mechanisms which might mediate such relationships, are unclear. However, a better understanding of these mechanisms could lead to novel approaches for treating addiction. The long- term goal of this research program is to understand the behavioral and neural mechanisms underlying elevated risk taking associated with drug use in both males and females. Important to this long-term goal, preliminary work with a novel rat model of risky decision making shows that elevated risk taking is predictive of subsequent cocaine self-administration, and that chronic cocaine self-administration in turn causes lasting elevations in risk taking, indicating a bidirectional relationship between risk taking and cocaine use. Additiona preliminary data indicate that elevated risk taking in drug-na?ve rats is associated with lower levels of striatal D2 receptor expression, and that activation of these receptors can reduce risk taking. Building on these preliminary findings, the objective of this proposal is to fully characterize the relationships between elevated risk taking and cocaine self-administration, and to determine whether these relationships are mediated by alterations in D2 and D3 dopamine receptor signaling. Our central hypothesis is that elevated risk taking is mediated by low levels of striatal D2 receptors and high levels of striatal D3 receptors, either as a pre-existing conditin or resulting from chronic cocaine self-administration. We further predict that normalizing the activity or expression of these receptors will be efficacious for reducing risk taking. Using an integrative approach in which we combine behavioral assessment of risk taking with cocaine self-administration and pharmacological, molecular, and anatomical assays, we will test our central hypothesis by: 1) characterizing the bidirectional relationship between elevated risk taking and cocaine self-administration in both males and females, and determining if cocaine-induced increases in risk taking are associated with alterations in striatal D2 and D3 receptors; 2) determining if alterations in striatal D2 and D3 receptors are sufficient to cause elevated risk taking, and if acute targeting of these receptors can reverse cocaine-induced elevations in risk taking; 3) determining if chronic environmental or pharmacological normalization of striatal D2 and D3 receptors can reverse cocaine- induced elevations in risk taking. A better understanding of the role of striatal dopamine signaling in risk taking will provide foundational knowledge necessary to develop intervention strategies targeting elevated risk taking in order to reduce drug use.

PROJECT SUMMARY. The ability to make effective decisions is critical for managing finances, health care, and other activities of daily living necessary to maintain personal independence. Decision making is altered in both normal aging and in Alzheimer's disease (AD), albeit in different ways. Specifically, healthy older adults tend to make less impulsive and risky choices compared to young adults, whereas AD is associated with greater impulsivity and maladaptive risk taking relative to age-matched healthy controls. While distinct, these decision biases associated with aging and AD can both be maladaptive and have significant implications for life quality. Optimizing decision making could broadly benefit functional outcomes and promote independent living among older adults; however, development of interventions is currently hindered by a poor understanding of the neural mechanisms underlying maladaptive decision making in aging and AD. To begin to address this gap in knowledge, our labs have shown that aged rats, which lack overt AD pathology, exhibit reductions in impulsive and risky choice in a manner similar to that observed in aged humans. Moreover, preliminary data suggest that these age differences in decision making are mediated by the basolateral amygdala (BLA). The BLA is implicated in affective processing and is highly interconnected within decision-making circuits. Using in vivo optogenetic approaches in both young and aged rats, we have identified multiple, temporally distinct contributions of BLA to both impulsive and risky choice and shown these contributions of BLA to decision making change with age. The long-term goal of this research program is to determine the mechanisms by which decision making is altered in aging and AD. The immediate objective is to determine the effects of aging and early tau pathology on BLA function and its role in decision making. Our overarching hypothesis is that aging and tau pathology disrupt adaptive decision making through alterations in BLA excitatory and inhibitory dynamics. Aim 1 will determine how optogenetic activation and inhibition of BLA at discrete stages of the decision process influence aged rats' decision making. Parallel biochemical assays will evaluate the influence of age on BLA synaptic and excitatory/inhibitory signaling proteins in conjunction with decision making behavior. Aim 2 will address similar questions in conjunction with a virally-mediated approach that induces medial temporal lobe tau pathology in a manner that is anatomically relevant to early stage AD. Aim 3 will use optogenetic approaches to determine how aging alters the contributions of distinct BLA efferent circuits to decision making, and will use cellular electrophysiology to evaluate effects of aging on anatomically defined subsets of BLA efferent neurons. Completion of these experiments will reveal at the biochemical, cellular, and systems level how the BLA is influenced by aging and tau pathology, as well as how such alterations contribute to maladaptive decision making. The information will be significant because it will provide foundational knowledge that is critical for development of novel interventions to maximize decision quality in both normal aging and AD.

Project Summary Substance use disorders are a significant public health issue, costing tens of thousands of lives and hundreds of billions of dollars annually . Considerable efforts have been expended to elucidate the neural mechanisms underpinning the acute and chronic actions of drugs of abuse on the nervous system, with the hope that better understanding of these mechanisms will lead to novel therapeutics. Much of this research has targeted receptors on neurons that are localized to cell bodies, axons, or dendrites; however, neurons also contain primary cilia, which are microtubule-based organelles that project from the cell bodies of all neurons. The importance of cilia function for human health is highlighted by the number of diseases caused by cilia dysfunction, several of which are associated with cognitive and motivational deficits. Neuronal cilia express a variety of G protein-coupled receptors (GPCRs), several of which are rarely expressed outside of cilia. Notably, several of these receptors, including the receptor for melanin-concentrating hormone (MCHR1) and the orphan GPCR, GPR88, have been shown to modulate responses to drugs of abuse. Despite what we know about cilia, our understanding of how cilia regulate neuronal function and behavior is still limited, and, in particular, there has been no prior research on interactions between neuronal cilia and drugs of abuse. The long-term goal of this research is to determine how ciliary signaling contributes to integration of neuromodulatory signals that regulate short- and long-term responses to drugs of abuse. As a first step toward this goal, the objective of our R21 proposal is to determine the role of primary cilia on dopaminergic and GABAergic neurons in the VTA and nucleus accumbens, respectively, in regulation of cocaine-induced behavioral plasticity and reward. This will be accomplished through molecular-genetic approaches to target cilia loss on specific neuronal types, in combination with behavioral pharmacological approaches. The proposed experiments will allow us to test our central hypothesis that neuronal cilia within mesolimbic circuitry are critical regulators of cocaine-induced plasticity and reward. We will test this hypothesis through two Specific Aims. Experiments in Aim 1 will use novel transgenic mouse strains to determine a) how cilia loss on dopaminergic and/or GABAergic neurons affects locomotion and locomotor sensitization induced by acute and repeated cocaine, respectively; b) whether locomotor alterations can be rescued by virally- mediated restoration of cilia in targeted brain regions, and c) how repeated cocaine alters cilia morphology and MCHR1 and GPR88 expression in mesolimbic brain regions. Experiments in Aim 2 will use similar approaches to determine whether cilia on dopaminergic and/or GABAergic neurons are necessary and sufficient for the rewarding effects of cocaine using a conditioned place preference task. The proposed research is innovative, as neuronal cilia have heretofore not been assessed in the context of drugs of abuse. The proposed research is significant, as cilia represent a unique neuronal signaling environment, a better understanding of which could lead to novel targets for therapies aimed at reducing substance use.

PROJECT SUMMARY: Older adults are the fastest-growing group of cannabis users in the US. Older adults use cannabis for a variety of reasons, including pain, insomnia, anxiety, and for recreation. Cannabis can, however, also exert robust effects on cognition. Almost all research on cannabis/cannabinoids and cognition has been conducted in young adults, and largely shows that acute administration impairs mnemonic and executive functions mediated by the medial temporal lobe and prefrontal cortex (which are also those most vulnerable to decline in aging and age- related neurodegenerative disease). In contrast, a few studies suggest that cannabinoids can exert distinct effects on the aged compared to the young brain, and preliminary data from our labs show that cannabis can actually enhance cognition selectively in aged rats. Indeed, cannabinoids have been proposed as potential treatments for the age-related neurodegenerative condition Alzheimer's disease (AD), and some preclinical research shows that cannabinoids can attenuate markers linked to AD pathology (e.g., neuroinflammation). Aging studies evaluating cannabis to date, however, are very limited and have not employed either cannabis itself or routes of administration that model those used most frequently by people (smoking and oral consumption). As such, it is unclear how cannabis, as it is actually used, affects cognitive decline and the synaptic dysfunction and AD-like pathology that contribute to cognitive impairments in older subjects. The long-term goal of our program is to determine how cannabis affects cognitive decline in aging and AD, and to determine the mechanisms of such effects. The objective of the current proposal is to model the two most common routes of human cannabis use (smoking and oral consumption) in well-characterized rat models of age-related cognitive decline, and to use these models to begin to elucidate effects of cannabis on behavioral and neurobiological dysfunction associated with aging and AD. Our overarching hypothesis is that cannabis can benefit cognition in aging by attenuating age-associated synaptic dysfunction, neuroinflammation, and tau pathology. Aim 1 will determine how acute cannabis affects performance in young adult and aged rats, as well as the synaptic mechanisms supporting effects of cannabis on cognition in aged subjects. Aim 2 will assess effects of chronic cannabis on cognition in young adult and aged rats, as well as on excitatory/inhibitory signaling and inflammatory markers linked to age-related cognitive impairments. Aim 3 will assess effects of chronic cannabis on AD-like tau pathology and cognition using a novel, targeted AAV-based approach in aged rats. The proposed experiments will be significant because they will provide foundational data concerning whether and how cannabis administration relevant for human consumption yields benefits for age-related cognitive decline and neuropathology.

PROJECT SUMMARY. One in three older adults exhibits some form of cognitive deficit, with 13% of individuals over age 65 meeting the clinical diagnosis of Alzheimer's disease (AD). Even in the absence of overt pathology, age-related cognitive dysfunction can be sufficiently severe as to disrupt instrumental activities of daily living and, consequently, the ability to maintain personal independence. In aging and AD, mnemonic functions supported by the hippocampus (HPC) and executive functions supported by the prefrontal cortex (PFC) are particularly vulnerable to decline. Both HPC and PFC undergo molecular and electrophysiological alterations with age that perturb the balance between excitatory and inhibitory (E/I) signaling necessary for optimal cognition. In addition, aberrant E/I signaling in aging increases susceptibility to AD neuropathology. Moreover, age-associated increases in peripheral inflammation can dysregulate E/I signaling, exacerbate AD pathology, and impair cognition. An ideal intervention for improving cognitive outcomes in aging would thus: 1) benefit multiple aspects of cognitive function with minimal side effects, 2) act to re-establish E/I homeostasis across the aged brain, 3) attenuate the accumulation of AD pathology that can worsen cognitive dysfunction, and 4) be readily translated across species. Electrical vagus nerve stimulation (VNS) has been used safely and effectively for 30 years to treat epilepsy and depression, and published and preliminary data show that it positively influences central nervous system E/I signaling. VNS also reduces pro-inflammatory cytokines in the periphery, as well as tau levels in AD patients. Most importantly, data in both animal and human subjects show that VNS enhances multiple forms of PFC- and HPC-dependent cognition that are compromised in aging. Despite these promising findings, VNS has not been rigorously evaluated as a potential treatment for age-associated cognitive decline. The objective of this proposal is to determine if chronic VNS mitigates deleterious neurobiological and inflammatory consequences of aging and improves cognitive function in aged subjects. Our rationale is that such studies will provide a foundation for use of VNS as a treatment for cognitive impairments in aging. Our overarching hypothesis is that chronic VNS will benefit cognition in aging by restoring E/I homeostasis, reducing inflammation, and protecting against AD- associated pathology. Aim 1 will determine whether VNS normalizes molecular and electrophysiological signatures of E/I dysregulation and reduces peripheral markers of inflammation in aging. Aim 2 will determine whether VNS remediates multiple forms of age-associated cognitive impairment. Aim 3 will use a targeted AAV- based approach to determine whether VNS protects against neuropathology and cognitive decline associated with AD-like tau pathology. These experiments will be significant as they will help to determine the utility of VNS as an intervention for treating cognitive decline in aging and AD.