Project summary/Abstract With the current opioid epidemic at a national crisis level, understanding the neural mechanisms governing heroin use and relapse to drug seeking is key to discovering new treatment strategies. Exposure to stimuli previously paired with the euphoric effects of heroin are thought to maintain habitual heroin use and seeking behaviors. These heroin-conditioned effects therefore extend the negative consequences of drug use well past cessation. Dysregulation of the dorsal medial prefrontal cortex (dmPFC) is a driving factor in habitual heroin use and cue-induced relapse, yet how discrete neural network components orchestrate heroin-seeking behavior remains less clear. Using a novel head-fixed self-administration (SA) mouse behavioral paradigm and simultaneous two-photon calcium imaging, recent data from our laboratory indicates unique excitatory neuronal ensembles emerge and encode information related to heroin-conditioned cues and heroin-seeking behaviors. Astrocytes are key regulatory elements in neuronal circuits and glial calcium events are necessary for modulating neuronal network activity. However, how dmPFC astrocytes contribute to neuronal encoding of heroin-related cues and heroin-seeking behaviors remains unknown. The goal of the current project is to investigate that dmPFC astrocytes (1) are engaged throughout heroin use and (2) causally influence surrounding neuronal ensemble dynamics and relapse behaviors. To this end, we will use a combination of contemporary strategies, including in vivo two-photon imaging and chemogenetic tools, to measure and manipulate dmPFC cellular activity during heroin use and relapse. The current proposal will be the first to measure astrocytic calcium dynamics throughout habitual heroin use and relapse and determine the causal influence of astrocytic activity on neuronal encoding and heroin-seeking behavior. I propose to use a multi-virus approach and in vivo two-photon microscopy to visualize and track individual astrocytic calcium events throughout heroin-SA, extinction, and reinstatement in head-fixed mice (Aim 1). Subsequently, I will employ two-photon imaging coupled with head-fixed heroin-SA to measure and track neuronal activity with single-cell resolution. I will then stimulate an astrocyte-specific hM4D(Gi) designer receptor during relapse and assess the consequence for neuronal ensemble activity and heroin-seeking behavior (Aim 2). Based on my preliminary data and published work of my mentorship team, I hypothesize that dmPFC astrocytes display biased activation during the onset of heroin use and reinstatement of heroin seeking (Aim 1) and that astrocytic calcium events directly coordinates surrounding neuronal network dynamics and relapse behavior (Aim 2). These data would provide a wholistic view of how dmPFC network activity orchestrates habitual heroin use and seeking behavior. The current proposal is the first to track astrocytic activity throughout drug use and causally ...