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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. Fulton Crews

Neuroimmune signaling and ethanol drinking-induced neuroimmune activation contribute to increased ethanol drinking and possibly alcoholism. Studies have shown that ethanol preference and drinking are modulated by neuroimmune gene expression and that cycles of ethanol exposure lead to long-lasting changes in ethanol self-administration (SA) and expression of neuroimmune genes, including neuronal Toll-like receptor 3 (TLR3). We have shown that TLR3 expression in post-mortem human alcoholic brains correlates with lifetime ethanol consumption. Here, we show that in rats with a history of operant ethanol SA, treatment with the TLR3 agonist Polyinosinic:polycytidylic acid (Poly(I:C)), followed by an abstinence period, results in escalated ethanol SA relative to saline-treated controls. Moreover, we observe parallel Poly(I:C)-induced increases in TLR3 gene expression in the nucleus accumbens (Acb) and the insular cortex (IC), and increases in gene expression of Gi-coupled Group II metabotropic glutamate receptors (mGluR2 and 3) in the nucleus accumbens (Acb), specifically the Acb core (AcbC; confirmed by immunohistochemistry), consistent with altered glutamate signaling. We focus on the AcbC given its role in modulating ethanol SA and on insular cortex (IC)àAcbC projections as these glutamatergic inputs from the IC have the potential to impact the AcbC and modulate escalations in ethanol SA. Further, we find that an mGluR2/3 agonist LY379268 blocks the escalation in ethanol SA following Poly(I:C)-TLR3 activation, but does not alter SA in saline controls, providing further support for a glutamatergic disruption following TLR3 activation. Moreover, our preliminary studies show inhibition of the ICàAcbC projections by activation of Gi-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) reduces ethanol SA, implicating this circuit in modulating ethanol SA, and we propose hyperactivity/recruitment of these projections following Poly(I:C). The overall hypothesis that neuroimmune signaling (i.e., TLR3 activation) induces glutamatergic adaptations in the Acb and ICàAcb circuitry that increase ethanol SA will be tested by the following aims:

Specific Aim 1. To test the hypothesis that Poly(I:C)-TLR3 activation induces neuroadaptations in glutamatergic signaling in cortico-accumbal brain regions. The studies in this aim will examine the molecular and functional glutamatergic adaptations that occur as a consequence of TLR3 activation in rats with a history of ethanol SA and in parallel sucrose SA-trained controls. We will build upon the preliminary data showing increased TLR3 expression in Acb and IC, and increased mGluR2/3 in the AcbC. Therefore, following the Poly(I:C)-TLR3 agonist protocol discovered to increase operant ethanol SA, we will use immunohistochemistry (IHC) to examine changes in the expression of mGluR2/3, and neuroimmune signaling targets and markers of  excitatory synapses. In collaboration with Component 5, we will examine glutamatergic adaptations in the AcbC using electrophysiological analyses. In general we predict increases in the neuroimmune signaling targets in the Acb and IC and increased mGluR2/3 expression and increased glutamatergic activity in the AcbC following Poly(I:C)-TLR3 activation. These approaches will provide converging results regarding the impact of Poly(I:C) exposure on both molecular and functional changes.

Specific Aim 2. To test the hypothesis that mGluR2/3 activation/inhibition modulates ethanol self-administration following TLR3 activation. Experiments in this Aim will build on the preliminary findings that mGluR2/3 activation blocks the escalation in ethanol SA observed following Poly(I:C)-TLR3 activation. First, we will examine whether Poly(I:C) increases in drinking are sensitive to systemically administered mGluR2/3 agonist and antagonist. Next, we will assess the effects of intra-AcbC mGluR2/3 activation on ethanol SA following Poly(I:C)-TLR3 activation. Importantly, parallel sucrose trained controls will be tested to determine whether changes in mGluR2/3 activation/antagonism are specific to a history of ethanol SA. The mGluR2/3 agonist is expected to reduce relapse-like drinking following Poly(I:C), whereas the mGluR2/3 antagonist may exacerbate the relapse-like drinking following Poly(I:C).

Specific Aim 3. To test the hypothesis that ICàAcbC projections modulate the escalation in SA following TLR3 activation.  To test this hypothesis, a chemogenetic approach will be used to inhibit (Gi DREADDs) and activate (Gq DREADDs) the ICàAcbC projections and examine the impact on drinking. We predict that inhibition of these projections will block the Poly(I:C)-induced escalation in SA. Conversely, activation of the projections is predicted to increase SA that is more pronounced following Poly(I:C). We will also assess IC-Acb connectivity using MRI within the Scientific Core. We predict greatest connectivity as a consequence of TLR3 activation in ethanol-SA trained rats that will be blunted by mGluR2/3 activation.

This proposal tests novel neuroimmune signaling and insular cortex-reward circuits that integrate into, benefit from and contribute to the UNC Alcohol Research Center’s focus on neurocircuit plasticity induced by ethanol. The proposed studies investigate novel mechanisms and circuits associated with the development of AUDs that could lead to new therapies.