1 Project Summary/Abstract 2 Alcohol use disorder (AUD) is a chronic, relapsing disorder characterized by loss of control over drinking, 3 continued use of ethanol despite negative consequences, and development of tolerance and withdrawal. 4 Three medications are approved for the treatment of AUD. However, not everyone responds to these 5 treatments and AUD patients would benefit from having more treatment options. The experiments 6 outlined in this proposal aim to identify molecular mechanisms that could lead to treatments for AUD. For 7 people with AUD, withdrawal leads to a clinical syndrome characterized by neuronal hyperexcitability and a 8 negative affective state that promotes relapse. Because of its importance in driving relapse, 9 pharmacologically targeting the withdrawal state is a promising treatment strategy. Astrocytes have been 10 implicated in the withdrawal syndrome because of their importance in regulating glutamate homeostasis in 11 brain. When searching for new treatments, it makes sense to target the withdrawal state. To understand 12 mechanisms underlying withdrawal, I have developed a novel in vitro model of every-other-day ethanol 13 exposure in primary astrocyte cell culture. My central hypothesis is that astrocytes mediate molecular 14 mechanisms by which chronic ethanol exposure causes neuronal hyperexcitability during alcohol 15 withdrawal. I will test this by pursuing two specific aims: 1) Determine if astrocytic FOLH1 contributes to 16 neuronal hyperexcitability in alcohol withdrawal, and 2) Identify additional genes and pathways in astrocytes 17 that contribute to neuronal hyperexcitability associated with alcohol withdrawal. In both aims, primary 18 astrocytes will be exposed to 44mM every other day ethanol for two weeks followed by a prolonged period 19 of abstinence to mimic ethanol withdrawal. Aim 1 will identify the impact of inhibiting FOLH1 activity using 20 2-PMPA in astrocytes on neuronal hyperexcitability by performing microelectrode arrays. Aim 2 seeks to 21 validate the novel in vitro model by identifying alcohol-responsive genes and gene networks using 22 differential expression and gene co-expression network analyses and comparing the findings to those 23 identified in previous in vivo drinking models. The research proposed here is innovative and significant as 24 it provides a tool for the alcohol research community to test hypotheses by using a novel every other day 25 ethanol exposure cell culture model which in mice leads to escalation and high levels of voluntary drinking 26 in rodents. Ultimately, the research proposed here has the potential to identify unique astrocytic proteins 27 that regulate neuronal excitability and might be suitable targets for developing new treatments for AUD.