Abstract This grant application proposes to understand the role of astrocytes in mediating cocaine-induced circuit alterations that drive cocaine seeking and relapse. Outcomes of the first funding period for this R01 project demonstrate that cocaine self-administration (SA) activates an astrocyte-mediated synaptogenic mechanism to generate AMPA receptor (AMPAR)-silent synapses in principal medium spiny neurons (MSNs) in nucleus accumbens shell (NAcSh). During withdrawal from cocaine SA, a subset of these cocaine-generated NAcSh synapses mature and strengthen by recruiting AMPARs. Experimentally converting and locking cocaine- generated NAcSh synapses within their silent state during drug withdrawal substantially decreases cue- induced cocaine seeking. By measuring intracellular Ca2+ activities, preliminary studies reveal that NAcSh astrocyte activities are upregulated by cocaine administration, and that this upregulation is increased after 5-d cocaine SA, indicating a ‘sensitization’ process in astrocytes. Furthermore, after cocaine SA, NAcSh astrocytes acquire the ability to respond to cocaine-associated cues by increasing their activities, and experimentally increasing astrocyte activities re-silences cocaine-generated NAcSh synapses. These and other preliminary results lead to the current hypothesis: NAcSh astrocytes gain unique properties through cocaine experience to regulate cocaine-generated synapses and formulate specific neuronal ensembles that drive cue-induced cocaine seeking after drug withdrawal. This hypothesis will be tested by three lines of experimentation. First, using astrocyte-specific molecular tools, proposed experiments will test the specific hypothesis that astrocytic levels of mGluR5 are upregulated by cocaine SA, which, in turn, mediates sensitized in vivo responses of NAcSh astrocytes to cocaine and cocaine-associated cues after cocaine withdrawal. In parallel, selective RNA- seq of NAc astrocytes will reveal novel molecular substrates for cocaine action in this cell type. Second, using in vivo two-photon microscopy combined with slice electrophysiology, proposed experiments will test the specific hypothesis that increased activities of NAcSh astrocytes is both sufficient and necessary for cue re- exposure-induced re-silencing of cocaine-generated NAcSh synapses, and thus can be used to reduce cue- induced cocaine seeking after drug withdrawal. Third, using GCaMP-mediated in vivo Ca2+ imaging, proposed experiments will test the specific hypothesis that the neuronal ensembles are formed, in part, by astrocyte- mediated synaptogenesis in response to cocaine, and then drive cue-induced cocaine seeking after drug withdrawal. These proposed experiments will characterize several novel astrocyte-associated substrates through which addiction-related memories can be manipulated for therapeutic benefits.