PROJECT SUMMARY An estimated 50-70 million Americans suffer from a sleep disorder, which is commonly comorbid with many psychiatric illnesses, such as alcohol use disorder (AUD). Individuals with AUD frequently report insomnia, and sleep difficulty significantly increases the likelihood of relapse. Despite the overwhelming need to control sleep disturbances to aid in the prevention of relapse, the underlying physiology of wakefulness, particularly in the context of acute and chronic alcohol use, is not well understood. The brain’s noradrenergic system mediates many behavioral states, such as arousal, alertness, and stress. Acute and chronic alcohol exposure differentially alter the activity of noradrenergic neurons as well as the release, synthesis, and turnover of norepinephrine (NE) in the brain. Recently, our lab alongside collaborators delineated a novel arousal circuit from the noradrenergic locus coeruleus (LC) to dopaminergic neurons in the ventral periaqueductal gray (vPAGDA), where stimulation of this circuit promotes wakefulness. Acute ethanol exposure also increases excitatory drive and the activity of wake-promoting vPAGDA neurons; however, the mechanism is unknown. Specific activation of alpha1-adrenergic receptors (α1ARs) increases excitatory drive onto vPAGDA neurons and subsequently causes arousal. Interestingly, α1AR expression is particularly enriched on vPAG astrocytes, and activation of astrocytic Gq signaling is sufficient to promote wakefulness. While astrocytes are essential in mediating synaptic transmission in other brain regions through purinergic signaling, the role of neuron- astrocyte interactions in the vPAG during ethanol exposure requires further investigation. This F30 aims to test the central hypothesis that purinergic transmission from neighboring astrocytes mediates the noradrenergic modulation of vPAGDA neurons, and that ethanol potentiates this signaling. The goal is to elucidate the mechanistic details mediating the increase in excitatory drive onto the wake-promoting vPAGDA neurons by activation of α1ARs and the pathologic changes to the noradrenergic system following acute and chronic alcohol exposure. In Specific Aim 1 I will utilize pharmacological and viral genetic approaches combined with ex vivo fluorescence imaging and whole cell patch-clamp electrophysiology to examine the neuron-astrocyte interactions underlying noradrenergic modulation of vPAGDA neurons. In Specific Aim 2 I will use similar approaches in addition to a mouse model of chronic intermittent ethanol exposure to determine the changes in synaptic transmission onto vPAGDA neurons following acute and chronic ethanol exposure. The results of these studies will allow our lab, our collaborators, and others to further investigate the role of neuron- astrocyte physiology related to sleep dysfunction in alcohol use disorder. This will further the work towards identifying possible therapeutic targets for the development of novel therapies to alle...