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Research Component 2: Neuroimmune Glutamatergic Regulation of Corticolimbic Circuits in Ethanol Self-Administration

Primary Investigator: Dr. Joyce Besheer

Co-Investigator: Dr. Leon Coleman

The induction of neuroimmune Toll-like receptor (TLRs) and microglial pro-inflammatory signaling are key pathologic features of alcohol use disorder (AUD). However, the mechanistic contribution of specific TLRs to AUD-related circuits and behavioral pathologies is lacking. In the current funding cycle, we found that activation of TLR3 and TLR7 via systemically administrated agonists promotes escalations in operant self-administration of ethanol1-3. Additionally, we find that the TLR3 and TLR7 agonists and alcohol vapor exposure result in robust downstream induction of the proinflammatory transcription factor interferon regulator factor-7 (IRF7) in the anterior insular cortex (aIC). This is significant given that our work implicates projections from the aIC→nucleus accumbens core (AcbC) as critical in the maintenance of operant ethanol self-administration4,5. Surprisingly, IRF7 was primarily localized in neurons alongside pro-inflammatory activation of microglia. Microglia are key initiators of inflammatory signaling in brain and can promote neuronal and circuit adaptations to ethanol. Depletion of microglia prevents induction of most proinflammatory genes by ethanol6 and blocks escalation of drinking after chronic ethanol vapor7. Microglia release endogenous TLR ligands8,9, and microglial pro-inflammatory cytokines further alter neuronal excitatory/inhibitory (E/I) balance10 – an overall theme of the UNC Alcohol Research Center (ARC) – as well as functional connectivity11-13. Therefore, we hypothesize that pro-inflammatory microglia in aIC promote neuronal IRF7 and subsequent excitatory outflow from the aIC to AcbC to promote ethanol self-administration. The overall hypothesis of this Research Component is that neuronal IRF7 induction and pro-inflammatory microglial activation as a consequence of ethanol vapor exposure together alter E/I balance across aIC→AcbC projections to promote increases in ethanol self-administration.


Specific Aim 1: Will test the hypothesis that ethanol vapor exposure induces IRF7 signaling in aIC that promotes increases in ethanol self-administration. This Aim will assess IRF7 signaling in aIC neurons and determine if IRF7 promotes escalations in operant ethanol self-administration following chronic ethanol vapor exposure (CIE-v). Male and female rats trained on ethanol self-administration will undergo CIE-v. Experiment 1.1 will examine expression of IRF7 and downstream type I interferon genes and interferon-stimulated genes (ISGs) in the aIC using NanoStringTM. Experiment 1.2 will examine IRF7-related target proteins in aIC=→AcbC projection neurons using retrograde labeling of aIC projection neurons followed by immunohistochemistry (IHC). Experiment 1.3 will examine the functional role of IRF7 on CIE-v induced escalations in ethanol self-administration using knockdown of IRF7 in the aIC (siIRF7). We predict neuronal IRF7 and its downstream interferon targets will be increased in the aIC following CIE-v and that IRF7 knockdown will prevent the CIE-v induced increases in ethanol self-administration.


Specific Aim 2: Will test the hypothesis that proinflammatory microglia activation promotes increases in ethanol self-administration and changes in E/I balance. This Aim will examine the consequences of CIE-v on proinflammatory microglial activation, E/I balance and the functional role of aIC microglia on neuronal IRF7 expression and ethanol self-administration. Experiment 2.1 will find if proinflammatory microglial activation genes (e.g., CD11b, TNFα) in aIC increase after CIE-v using tissue from Experiment 1.1. In Experiment 2.2 we will examine cellular (vGLUT1:GAT1) and synaptic (PSD95:gephyrn) markers of E/I balance using IHC. Experiment 2.3 will test if chemogenetic inhibition of proinflammatory microglia in aIC prevents CIE-v induced induction of neuronal IRF7 and escalation in ethanol self-administration. We expect that CIE-v will increase proinflammatory microglial genes and E/I balance in the aIC, with microglial inhibition blunting CIE-v induced increases IRF7 and ethanol self-administration.


Specific Aim 3: Collaborative aim to test the hypothesis that proinflammatory signaling alters E/I balance of aIC projection neurons and alters functional connectivity. This aim leverages ARC collaborations to expand the scope of our studies. Experiment 3.1 measures E/I balance in aIC→AcbC projections after CIE-v and the role of proinflammatory signaling using electrophysiology with Component 1. We expect CIE-v will shift the E/I balance of aIC→AcbC projection neurons by increasing excitatory drive of these cells which will be prevented by blocking proinflammatory signaling. Experiment 3.2 will assess the contribution of proinflammatory signaling to ethanol exposure-induced changes in functional connectivity using resting state fMRI in collaboration with Components 3 and 4 and the Scientific Resource Core. We predict that chronic binge ethanol causes induction of proinflammatory signaling and loss of functional connectivity in cortico-striatal networks. Together, these studies will inform the role of proinflammatory signaling on ethanol-induced changes in E/I balance (functional and molecular) as well as functional connectivity across key circuits involved in AUD pathology.


The UNC-ARC seeks to increase understanding of molecular and cellular pathogenesis in AUD. Using ARC resources and collaborations, the studies in this Research Component will determine the molecular consequences of high-dose ethanol exposure on IRF7 signaling and microglial activation and how these changes promote subsequent escalations in ethanol self-administration. This work has potential to broaden our understanding of the role of neuroinflammatory signaling in drinking which can inform therapeutic development.