Brigitte L. Kieffer - US grants

The molecular genetic approach to the study of the role of opioid receptors in ethanol dependence has recently yielded fascinating results. The non-dependent mice, knockout of the mu opioid receptor blocks ethanol drinking and operant responding for ethanol. In contrast, knockout of the delta receptor produces a time-dependent increase in ethanol preference and operant responding for ethanol that correlates with anxiety-like responses of the knockout animals. The limitations of these studies are that (i) these studies are based on constitutive manipulation of opioid receptor genes, therefore compensatory mechanisms could take place during development and account for the observed phenotypes and (ii) the knockout of the opioid receptors occurs throughout the brain and body. To address these issues, novel molecular genetic approaches will be used to produce conditional knockouts of mu and delta receptors. An attempt will be made to knockout these receptors in the adult, and in specific brain regions consistent with the overall hypothesis of INIA regarding the hypothesized role of the extended amygdala in excessive alcohol consumption.

DESCRIPTION (provided by applicant): Gene targeting in mice to study the role of neuropeptide receptors in ethanol dependence has recently yielded fascinating results. Knockout of the mu opioid receptor (MOP) gene blocks ethanol drinking and operant responding for ethanol. Knockout of the Corticotropin Releasing Factor receptor 1 (CRF1) gene reduces levels of anxiety under basal and alcohol withdrawal conditions. Altogether data demonstrate that blockade of these receptor systems reduce alcohol intake. Limitations of these "conventional" gene targeting studies are that (i) gene knockout occurs early, therefore compensatory mechanisms could take place during development, and (ii) knockout of the receptors occurs throughout the entire animal, therefore no information on the recruited neurocircuitry is provided. To address these issues, we will induce the knockout of MOP and CRF1 receptor genes specifically in the extended amygdala (EA) of adult animals, based on the overall hypothesis of INIA (Integrative Neuroscience Initiative on Alcoholism) regarding the role of the EA in excessive alcohol consumption. First, we will take advantage of two existing mutant mouse lines, one with a floxed MOP receptor gene (recently created in our laboratory), and another with a floxed CRF1 receptor gene (collaboration). Second, we will develop a novel transgenic mouse line expressing Cre recombinase in the EA. To do this, we will use a BAG promoter for the WFS1 (Wolfram syndrom 1) gene, that we have recently identified as an EA marker gene (Specific Aim 1). Third, we will breed floxed mice with the WFS1-Cre mouse to produce the conditional knockout of MOP (Specific Aim 2) and CRF1 (Specific Aim 3) receptor genes in the EA of adult mice. The two conditional lines will be fully characterized for receptor expression throughout the brain, for morphine responses (MOP) and for basal behaviors (Specific Aims 2 and 3). The two conditional lines will finally be extensively studied in behavioral models of excessive alcohol drinking, including the DID and WID models, as well as for acute ethanol responses and ethanol withdrawal (Specific Aim 4). Importantly, the WFS1-Cre transgenic mice generated in Specific Aim 1 will represent a unique tool for the conditional deletion of any other gene of interest in the extended amygdala, and will be generally useful in addiction research.

ABSTRACT Addiction develops when recreational drug use switches to compulsive drug taking. While the former is predominantly motivated by reward seeking, the latter is also driven by other factors that include enhanced stress reactivity, aversive aspects of drug withdrawal and emergence of a negative affect upon protracted abstinence. In recent years the notion that reward and aversion processing engage overlapping brain circuits has been established, together with the concept of a reward/aversion network. The habenula (Hb) encodes both rewarding and aversive aspects of external stimuli, and may therefore represent a central integrator of reward/aversion circuits. Remarkably, the medial subdivision of habenula (MHb) shows highest density of mu opioid receptors (MORs) in the brain, but the role of this particular receptor population is unknown. This project will test the hypothesis that MORs expressed in the MHb regulate specific aspects of reward and aversion processes related to drug abuse. We will capitalize on tools and preliminary findings from the previous funding period. In Aim 1, we will extensively characterize MOR-expressing neurons in the medial septum-MHb-interpeduncular nucleus (MS-MHb-IPN) pathway using viral tracers combined with knock-in MOR-mcherry, Cnrb4-Cre or novel MOR-Cre mice that we currently develop. This Aim will provide circuit-level understanding that will complement Aims 2 and 3. Aim 2 will identify behaviors, and underlying circuit mechanisms, controlled by MORs in the MHb. We will examine a range of reward/aversion behaviors potentially mediated at the Hb level (reward and reward-driven decision-making, morphine and nicotine withdrawal, aversion to morphine withdrawal and abstinence) using a novel conditional Cnrb4-MOR mouse line. Reduced physical morphine withdrawal has already been detected and DREADD approaches will be used to recapitulate this behavior, and possibly other phenotypes. Aim 3 will identify the causal impact of MOR and MOR-positive neuron activities in MS-MHB-IPN networks, and their broader impact on the brain. We will use pioneering functional magnetic resonance imaging (fMRI) in live mice, and further fMRI strategies developed in the Technical Advancement Core, to map brain-wide functional connectivity, seed-based connectional patterns and inter-node directionality in Cnrb4-MOR mice at rest and after morphine treatment. Consequences of DREADD-mediated stimulation of MOR+ neurons in the MHb will also be examined by fMRI in live animals. In sum, this proposal will reveal the role(s) of the densest and less-well studied MOR population. The three aims together will determine importance of these receptors in reward/aversion-related behaviors and elucidate the underlying circuit mechanisms. The project will also provide novel genetic mouse lines for CSORDA (Cnrb4-MOR, Project III) and the neuroscience community (MOR-Cre), and cutting edge non- invasive animal imaging that will be applicable within (PTSD model, Project IV) and outside of CSORDA.

Project Summary The goal of this project is to develop GPR88 agonists to treat alcohol use disorders. Alcoholism is a heterogeneous, chronic relapsing disorder. Available medications to treat alcoholism have limited efficacy, serious side effects, and compliance issues. Therefore, new medications based on novel targets are needed. The orphan receptor GPR88 is a G-protein-coupled receptor with robust expression in the striatum throughout the dorsal and ventral areas. Multiple lines of evidence suggest that GPR88 plays an important role in the regulation of striatal functions and is implicated in alcohol-seeking behaviors. Our preliminary results in behavioral studies using GPR88 knockout mice and a selective GPR88 agonist support the hypothesis that GPR88 agonism is beneficial to treat alcohol addiction and dependence. Based on the research conducted in our probe project R21 MH103708, we have developed the first potent, selective, and brain-penetrant small molecule GPR88 agonists. In this application, we propose to refine our early lead compounds to produce GPR88 agonists for in vivo studies through three iterative specific aims. In Aim 1, we will optimize potency, receptor selectivity, and drug-like properties of GPR88 agonists using medicinal chemistry. In Aim 2, we will characterize compounds using GPR88 functional assays (cAMP and GTP?S binding assays). Potent compounds will then be characterized using a battery of ADMET and pharmacokinetic assays. In Aim 3, we will evaluate the efficacy of select compounds, developed in Aims 1 and 2, in animal models of alcohol drinking and reinforcement. Overall, completion of this project will provide in vivo probes to further characterize the GPR88 system and pharmacologically validate GPR88 as a novel target for treatment of alcoholism.