The current epidemic of opioid overdoses has been propelled by both illicit and prescribed narcotic pain medications. Extensive opioid use and repeated abstinence increases the likelihood of severe withdrawal and perpetuates the vulnerability to relapse via means of negative reinforcement. The negative emotional valence of withdrawal can last long after the initial, dramatic physical signs, involving a protracted negative emotional state, drug craving, and a high likelihood of relapse. These combined symptoms are commonly referred to as being “dope sick”. Addicted individuals often prefer to continue drugs rather than face withdrawal. Being “dope-sick” has many attributes of a severe inflammatory state and this led us to investigate the involvement of microglia, the innate immune cells that reside in the brain, in opioid tolerance and withdrawal, and this was supported by a CEBRA R21 grant (R21-DA044757). That CEBRA R21 grant resulted in our findings of dramatic changes in ribosome-bound mRNAs—the “translatome”—in microglia using RNA sequencing of RiboTag purified microglial RNAs. Those results provided us with the leads that form that basis for this proposal. Many of the changes related to cyclic AMP signaling and its downstream targets, and experimental chemogenetic stimulation of Gi/o signaling was found to actually worsen opioid withdrawal. With the understanding that glia are partners in plasticity, we suspect that the relapsing/remitting nature of opioid dependence serves to prime and condition microglia, shifting the impact from tempering withdrawal during initial stages to exacerbating withdrawal and opioid seeking after multiple cycles of tolerance and withdrawal. Thus, investigations into the role of glia in withdrawal may provide new therapeutic avenues. We propose three Aims using fentanyl and a recently developed transgenic mouse that allows conditional and microglia-specific Cre and TdTomato expression without disrupting microglia function. In Aim 1 we will analyze the trajectory of the changing microglial translatome after one vs. five cycles of opioid dependence and spontaneous withdrawal. In Aim 2 we will examine the physical and behavioral consequences of one vs. five cycles of opioid dependence and withdrawal, to explore the idea that intermittent cycles of dependence and withdrawal exacerbate the negative consequences of withdrawal. We will then investigate the hypothesis that the purinergic receptors P2Y12 and P2X7 are involved in microglial responses during initial and delayed phases of opioid withdrawal. In Aim 3 we will use an in vitro brain slice model with 2- photon confocal imaging of microglia. We will study the microstructure and motility of microglia using time- lapse microscopy. We will measure real-time changes in cyclic AMP using a FRET-based biosensor and calcium dynamics with GCaMP6. These three Aims integrate the temporal, behavioral, and molecular consequences of microglial engagement during opioid dependence ...