Repeated fentanyl abstinence intensifies opioid withdrawal and induces a proinflammatory state in striatal microglia

Opioid withdrawal is a serious obstacle to self-initiated abstinence, and previous experiences of opioid withdrawal may exacerbate the severity of subsequent incidences. To study the impact of repeated opioid withdrawal episodes, we compared male and female mice after one or five cycles of fentanyl exposure and withdrawal. We selectively expressed hemagglutinin-tagged ribosomes (RiboTag) in microglia of transgenic mice to immunoprecipitate and sequence RNA actively undergoing translation (the "translatome") from striatal microglia during fentanyl withdrawal. Key changes were confirmed by RTqPCR of RiboTag RNA. Repeated bouts of fentanyl treatment and withdrawal impacted striatal microglia much more than a single cycle of fentanyl followed by withdrawal. Multiple withdrawal cycles reduced ramification of microglial processes, suggesting a more reactive cell state, and induced more severe behavioral withdrawal signs in mice. Five cycles of fentanyl exposure and withdrawal increased the expression of gene networks associated with innate immunity signaling. Indeed, 100% of the genes associated with the "microglia core sensome", were upregulated after five cycles of withdrawal. Together these results suggest that mouse striatal microglia initiate a proinflammatory response following five, but not one, opioid exposure and withdrawal experiences and suggest that drug therapies targeting microglial innate immune responses may mitigate the severe withdrawal associated with repeated opioid tolerance and withdrawal. Significance statementRepeated cycles of fentanyl administration and withdrawal caused worsened behavioral signs of withdrawal in mice. This is the first such study to directly examine the effects of repeated opioid withdrawal on mouse behavior. We also found that repeated opioid withdrawal increased the expression of RNAs related to the proinflammatory "microglia core sensome". Furthermore, microglia in the mouse striatum were present at a higher density with reduced ramification, which suggests that multiple opioid withdrawal experiences cause a significant change to microglial signaling-state.