A cAMP-Related Gene Network in Microglia Is Inversely Regulated by Morphine Tolerance and Withdrawal

Background: Microglia have recently been implicated in opioid dependence and withdrawal. Mu opioid receptors are expressed in microglia, and microglia form intimate connections with nearby neurons. Accordingly, opioids have both direct (mu opioid receptor mediated) and indirect (neuron interaction mediated) effects on microglia function. Methods: To investigate this directly, we used RNA sequencing of ribosome-associated RNAs from striatal microglia (RiboTag sequencing) after the induction of morphine tolerance and followed by naloxone-precipitated withdrawal (n = 16). We validated the RNA sequencing data by combining fluorescent in situ hybridization with immunohistochemistry for microglia (n = 18). Finally, we expressed and activated the Gi/o-coupled hM4Di DREADD (designer receptor exclusively activated by designer drugs) in Cx3cr1-expressing cells during morphine withdrawal (n = 18). Results: We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal. Weighted gene coexpression network analysis revealed an intriguing network of cyclic adenosine monophosphate (cAMP)-associated genes that are known to be involved in microglial motility, morphology, and interactions with neurons that were downregulated with morphine tolerance and upregulated rapidly by withdrawal. Three-dimensional histological reconstruction of microglia allowed for volumetric, visual colocalization of messenger RNA within individual microglia that validated our bioinformatics results. Direct activation of hM4Di in Cx3cr1-expressing cells exacerbated signs of opioid withdrawal rather than mimicking the effects of morphine. Conclusions: These results indicate that Gi signaling and cAMP-associated gene networks are inversely engaged during opioid tolerance and early withdrawal, perhaps revealing a role of microglia in mitigating the consequences of opioids.