Ryan J McLaughlin - US grants

Project Summary Cannabis is the most commonly abused illicit drug in the United States. With several states recently passing legislation permitting the use of cannabis for recreational purposes, there is valid concern that this could dramatically increase the availability and use of cannabis in sensitive developmental populations. Adolescence is a period of dynamic cortical development that facilitates proper behavioral and cognitive maturation, and as such, many are worried that chronic cannabis use during this period may interfere with these important developmental processes, thereby contributing to the emergence of cognitive and/or motivational deficits in adulthood. Clinical studies have lent credence to this notion; however, the long-term causal effects of adolescent cannabis use have been difficult to elucidate. Preclinical animal models are advantageous in this respect, yet current models of cannabis exposure have been plagued by methodological concerns that limit the translatability of these data to human populations. Our laboratory has generated important new data using a novel, translationally relevant model of cannabis vapor self-administration in rats. This new method uses custom-designed equipment to deliver discrete ?puffs? of vaporized cannabis extracts in a response-contingent manner. This approach is unique in that it uses natural cannabis extracts (rather than synthetic cannabinoid receptor agonists or isolated cannabis constituents) that are delivered via the pulmonary route of administration that is most common in human users. We will use this innovative approach to determine the extent to which self-administration of vaporized cannabis extracts that are high in ?9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD) elicits long-term alterations in cortical development, cognitive flexibility, and effort-based decision making. Moreover, we will examine whether such alterations occur in an age-, sex-, and/or drug-dependent manner. For Aim 1, an automated set-shifting task will be used to assess deficits in cognitive flexibility, and an effort-discounting task will be used to assess deficits in effort- based decision-making. For Aim 2, brains will be harvested from rats tested in Aim 1 to determine whether potential treatment effects in task performance are associated with individual differences in white matter development and spine density in the prefrontal cortex (PFC). Our overarching hypothesis is that intrapulmonary self-administration of an extract high in THC will produce long-term impairments in attentional set shifting and reduce the preference for larger, more effortful rewards, especially following adolescent-onset self-administration. We further predict that these cognitive impairments will be associated with aberrations in myelination and synaptic pruning in the PFC, and that these long-term cognitive and structural impairments will be attenuated in cohorts receiving an equal ratio of the CBD-rich extract. The results are expected to positively impact the field by providing the proof-of-concept for a novel, translationally relevant model of pulmonary cannabis self-administration and delineating its long-term structural and functional consequences. Additionally, this work will provide the foundation for future studies that will dissect the mechanisms by which cannabis exposure impacts cognition, motivation, and vulnerability for addiction.

PROJECT SUMMARY The most commonly cited reason for habitual cannabis use is to cope with stress. Although this may provide acute beneficial effects, the long-term ramifications of chronic cannabis use, particularly under drug-free conditions, remain unknown. We have demonstrated that sober chronic cannabis users display blunted psychological and physiological responses to an acute laboratory stressor relative to non-users. However, this cross-sectional approach precludes the ability to establish causal relationships between cannabis use and the stress response. Preclinical animal models are particularly advantageous in this respect, yet current models of cannabis use have been plagued by methodological concerns that limit the translatability of these data to human populations. To address these limitations, our laboratory has generated important new data using a novel, translationally relevant model of cannabis vapor self-administration that uses response-contingent delivery of vaporized cannabis extracts containing high concentrations of ?9 tetrahydrocannabinol. This approach is unique in that it uses volitional exposure to natural cannabis extracts (rather than forced delivery of synthetic cannabinoid receptor agonists or isolated cannabis constituents) that are delivered via the pulmonary route of administration that is most common in human users. We will use this approach in the current proposal to determine whether cannabis vapor self-administration causes alterations in basal and stress-induced activation of the hypothalamic- pituitary-adrenal (HPA) stress axis in male and female rats. In Aim 1, we will use immunohistochemistry to quantify activation of the immediate early gene c-fos in different cell types within the paraventricular nucleus (PVN) of the hypothalamus of male and female rats trained to self-administer varying concentrations of cannabis or vehicle vapor. In Aim 2, we will perform radioimmunoassays on plasma harvested from cannabis-exposed and non-exposed rats in stress and no-stress conditions to determine the extent to which chronic volitional cannabis exposure alters basal and stress-induced recruitment of adrenocorticotropic hormone (ACTH) and corticosterone (CORT). Additionally, brains will be extracted to assess corticotropin-releasing hormone (CRH) peptide content in the central nucleus of the amygdala of cannabis-exposed and non-exposed rats under stress or non-stress conditions. We predict that rats trained to self-administer cannabis vapor will exhibit dose- dependent elevations in basal CORT, attenuated stress-induced activation of CRH-positive PVN neurons, increased reliance on passive coping strategies, and dampened recruitment of ACTH, CORT and CRH content relative to vehicle-exposed rats. Results from these studies will provide a foundation for understanding long-term consequences of chronic cannabis use and allow us to identify causal relationships between cannabis use and stress reactivity. Moreover, this work will support the feasibility of future studies that will investigate the mechanisms underlying these effects and identify whether alterations in the stress response are a risk factor for developing problematic cannabis use.

PROJECT SUMMARY Stress is a pervasive aspect of daily life and a significant risk factor for a host of mental illnesses, including major depression. In the brain, chronic stress causes adaptations in the mesolimbic dopamine system that increase vulnerability for developing depression and depression-related behaviors in clinical populations and preclinical animal models, respectively. One area of the brain that has gained attention as of late is the lateral habenula (LHb), in part because of its ability to tightly constrain dopamine activity. Notably, the LHb is hyperactive in individuals suffering from major depression, while restoring normal activity in this area has emerged as a viable therapeutic strategy in treatment-resistant patients. Although we still do not know how chronic stress leads to LHb dysfunction, one intriguing possibility is through stress-induced alterations in the endogenous cannabinoid (ECB) system. The primary role of the ECB system in the brain is to provide activity- dependent, on-demand negative feedback, which helps to maintain synaptic homeostasis. Our data indicate that chronic stress augments ECB signaling in the LHb, while local activation of this system elicits a passive- despair-like coping strategy, impairs behavioral flexibility in an attentional set-shifting task, and decreases the firing rate of dopamine neurons located in the ventral tegmental area. However, the precise role of the ECB system in the LHb and the mechanisms by which this system modulates stress-related behaviors has yet to be formally evaluated. In the current proposal, we will fill this important gap in knowledge by systematically examining how the ECB system modulates LHb function and identifying whether chronic stress-induced alterations in this system are necessary to produce deficits in dopamine cell firing and the expression of depression-related behaviors. In Aim 1, we will perform site-specific pharmacological manipulations of the ECB system in tandem with in vivo electrophysiology recordings of dopamine cell activity in freely behaving rats to uncover how stress-induced alterations in LHb ECB signaling may contribute to deficits in behavioral flexibility. In Aim 2, we will use ex vivo electrophysiology combined with retrograde labeling of LHb projections to identify the role of the ECB system in modulating excitatory and inhibitory LHb inputs and examine how chronic stress alters ECB control of synaptic strength at projectionally defined LHb synapses. In Aim 3, we will use a combinatorial viral approach to determine effects of acute, circuit-specific activation of LHb neurons on stress coping and behavioral flexibility, and test whether chronic, long-term LHb activation recapitulates the behavioral effects of chronic stress in an ECB-dependent manner. Broadly stated, the proposed research will fill a significant gap in the field by identifying how the ECB system regulates the activity of a key circuit that has been implicated in various domains of mental health, and the neurophysiological and behavioral consequences of stress-induced alterations in this system. Moreover, this work will pave the way for future studies exploring the involvement of this system in brain function and disease.

PROJECT SUMMARY The recent wave of recreational cannabis legalization in the US has left many concerned that rates of cannabis dependence and cannabis use disorder (CUD) will rise dramatically in the coming years. Approximately 9% of first-time users will become dependent on cannabis, yet there are no FDA-approved pharmacotherapies for managing CUD. This is in part due to flawed diagnostic nosology resulting in a lack of understanding of the mechanisms that give rise to CUD, as well as a conspicuous lack of translationally relevant animal models of cannabis use. To address these gaps in knowledge, we have developed and validated a novel model of cannabis self-administration that delivers vaporized cannabis extracts in a response-contingent manner via the pulmonary route of administration that is most common among human users. Our data indicate that rats exhibit stable rates of responding for cannabis vapor that produce dose-dependent elevations in plasma ?9-tetrahydocannabinol (THC) concentrations and metabolic alterations that are consistent with observations in human cannabis users. We will use this model in the proposed studies to identify behavioral and biological factors that predict high vs. low rates cannabis-seeking behavior and subsequently determine region-specific alterations in the endocannabinoid (ECB) system following vapor self-administration. To accomplish this goal, we will conduct a battery of behavioral assays in male and female rats prior to vapor self-administration training. In Aim 1, we will rigorously characterize the phenotype of rats using the National Institute of Mental Health Research Domain Criteria (RDoC) and then determine which behavioral dimensions are most strongly associated with high vs. low responding for cannabis vapor in our model. Given that the endocannabinoid (ECB) system is the primary target for cannabis and is fundamentally involved in the reinforcing effects of THC, in Aim 2 we will test whether circulating endocannabinoid (ECB) tone is a biomarker of a high-responding phenotype. Finally, given that alterations in ECB degradation have been associated with an exaggerated subjective response to the acute effects of cannabis and increased problematic drug use, we will next examine whether cannabis self- administration causes alterations in ECB hydrolysis and CB1 receptor binding in reward-relevant brain regions. We predict that measures of positive valence and arousal/regulatory systems will be the best predictors of individual rates of cannabis self-administration, and that circulating anandamide concentration will be positively associated with cannabis-seeking behavior. We further predict that anandamide degradation and CB1 receptor binding will be decreased in the mesolimbic pathway of high-responding rats following cannabis self- administration. Together, these studies will establish behavioral and biological predictors of problematic cannabis use, which can be leveraged to improve early diagnosis of CUD. These data will also help to identify more efficacious pharmacotherapeutic interventions that are grounded in the neurobiological underpinnings of the disorder, with direct application to human populations afflicted with CUD.