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Research Component 5:  Probing Cortical-Nigral Dynorphinergic Circuits in Alcohol Abuse

Primary Investigator: Dr. Thomas Kash

Co-Investigator: Dr. Monserrat Navarro

Excessive drinking cost the United States $249 billion in 2010 with 77% attributed to lost productivity, health care costs, and accidents as a result of binge drinking (Sacks et al. 2015). Despite this high cost, the precise mechanisms that underlie escalated drinking remain elusive. The intermittent access (IA) paradigm is a rodent schedule of alcohol drinking that reliably leads to voluntary escalated alcohol intake and preference as well as dysregulated emotional behavior. While numerous neurochemical systems have been identified as playing a role in alcohol-related behavioral pathology, one of the most promising leads for treatment is the Kappa Opioid Receptor (KOR) and its endogenous ligand Dynorphin (Dyn). Consistent with the important role of this system in excessive alcohol drinking, we found that pharmacological blockade of KOR leads to a suppression of IA drinking. Using a reporter mouse, we found that Dyn-containing neurons in the Insular Cortex (ICDyn) that projected to the substantia nigra (SN) exhibited increased fos labeling following 8 weeks of IA. Interestingly, fellow ARC PI, Charlotte Boettiger has found that the insular cortex (IC)®SN pathway shows increased functional connectivity in a population of human binge drinkers, and that the strength of this connectivity correlates with depression, anxiety and distress. Taken together, these preliminary data suggest that the IC®SN circuit and KOR signaling play a key role in alcohol abuse, potentially via regulation of dysphoric behavioral states. In this proposal we will use a multidisciplinary approach to test the central hypothesis: Intermittent access to alcohol causes dysregulation of Dyn / KOR systems in the insular cortex to substantia nigra circuit that drives negative behavioral states leading to increased ethanol consumption.

 AIM 1: Investigate physiological adaptations in the IC®SN circuit following IA

 Our preliminary results using a Dyn reporter mouse line demonstrate that the anterior insula is enriched in Dyn neurons that exhibit increased fos labeling in mice drinking high levels of alcohol.  Further, a subset of these ICDyn neurons project to the SN. These data suggest that the Ins®SN circuit is strengthened following alcohol exposure. Using a combination of slice electrophysiology, optogenetics, and anatomical techniques, we will probe the impact of both 1 week and 8 weeks IA exposure on the function of this circuit. In addition, as we have found that KOR antagonism can alter both alcohol consumption and alcohol-induced anxiety, we will determine functional adaptations in the KOR system within the IC and SN. Taken together, these experiments are a rigorous physiological analysis that will demonstrate how alcohol drinking impacts the IC®SN circuit.

 AIM 2: Investigate the role of the IC®SN circuit in IA-induced anxiety-like behavior and escalated alcohol consumption

 Our preliminary results suggest that the IC®SN circuit is recruited following 8 weeks of IA. In addition, our preliminary results, as well as published studies have found that IA can lead to increases in anxiety-like behavior. In this aim we will use multiplexed chemogenetic approaches to test the hypothesis that heightened activity in the IC®SN circuit can drive altered anxiety-like behavior and escalated alcohol consumption. Additionally, in collaboration with the Imaging Core and using an identical chemogenetic approach, we will test the role of the IC®SN in driving aberrant resting state functional connectivity MRI (rs-fcMRI) following IA. These studies will expand our understanding of the causal role of the IC®SN circuit in driving both escalated alcohol consumption and alcohol induced anxiety-like behaviors.

AIM 3: Investigate the role of IC®SN Dynorphin signaling on escalated alcohol consumption

 Our preliminary results demonstrated that KOR signaling plays a critical role in escalated alcohol consumption and that ICDyn neurons exhibit increased activity following IA. This suggests that dynorphin released from IC neurons is a molecular signal driving changes in behavior. While in Aim 2, we focus on the circuit, this aim will use converging genetic approaches to probe the role of ICDyn neurons and dynorphin in the IC in both escalated alcohol drinking and anxiety like behavior. Specifically, we will chemogenetically manipulate ICDyn neurons to determine their role in both escalated alcohol drinking and IA-induced anxiety-like behavior. We will then examine the function of locally produced ICDyn in escalated alcohol drinking and IA-induced anxiety-like behavior using a viral-based Dynoprhin shRNA knockdown approach. Together, these studies will provide a rigorous evaluation of how Dynorphin in the IC®SN circuit regulates alcohol drinking.

Public Health Significance

Through this work the field will obtain an understanding of both how the insular cortex is altered following repeated cycles of excessive alcohol consumption and how it contributes to escalated alcohol intake and alcohol induced anxiety. This understanding will be vital to interventions aimed at reversing the neuroadaptations that take place as a consequence of repeated heavy drinking.