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 (MOR) receptors are expressed in microglia, and microglia form intimate connections with nearby neurons. Accordingly, opioids have both direct (MOR 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-Seq) after the induction of morphine tolerance and followed by naloxone precipitated withdrawal (n=16). We validated the RNA-Seq data by combining fluorescent in-situ hybridization with immunohistochemistry for microglia (n=18). Finally, we expressed and activated the Gi/o-coupled hM4Di DREADD receptor in CX3CR1-expressing cells during morphine withdrawal (n=18). Results: We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal. WGCNA analysis revealed an intriguing network of 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 mRNA within individual microglia that validated our bioinformatics results. Direct activation of Gi/o-coupled DREADD receptors 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.

Chemogenetic inhibition of lateral habenula projections to the dorsal raphe nucleus reduces passive coping and perseverative reward seeking in rats.

The lateral habenula (LHb) processes information about aversive experiences that contributes to the symptoms of stress disorders. Previously, we found that chemogenetic inhibition of rat LHb neurons reduced immobility in the forced swim test, but the downstream target of these neurons was not known. Using an intersectional viral vector strategy, we selectively transduced three different output pathways from the LHb by injecting AAV8-DIO-hM4Di into the LHb and CAV2-CRE (a retrograde viral vector) into one of the three target areas as follows: dorsal raphe nucleus (DRN), ventral tegmental area (VTA), or rostromedial tegmentum (RMTg). Using the forced swim test, we found that chemogenetic inhibition of DRN-projecting LHb neurons reduced passive coping (immobility), whereas inhibition of the other pathways did not. Chemogenetic activation of DRN-projecting neurons using hM3Dq in another cohort did not further exacerbate immobility. We next examined the impact of inhibiting DRN-projecting LHb neurons on reward sensitivity, perseverative behavior, and anxiety-like behavior using saccharin preference testing, reward-omission testing, and open-field testing, respectively. There was no effect of inhibiting any of these pathways on reward sensitivity, locomotion, or anxiety-like behavior, but inhibiting DRN-projecting LHb neurons reduced perseverative licking during reward-omission testing, whereas activating these neurons increased perseverative licking. These results support the idea that inhibiting LHb projections to the DRN provides animals with resilience during highly stressful or frustrating conditions but not under low-stress circumstances, and that inhibiting these neurons may promote persistence in active coping strategies.