A cAMP-Related Gene Network in Microglia Is Inversely Regulated by Morphine Tolerance and Withdrawal
Kevin R Coffey1, Atom J Lesiak2, Russell G Marx1
1Puget Sound VA Health Care System and Department of Psychiatry & Behavioral Sciences, University of Washington School of Medicine, Seattle, WA, 98105, USA.
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.
Insights
Microglia play a role in opioid withdrawal. Opioid tolerance downregulates cAMP-associated genes in microglia, while withdrawal rapidly upregulates them, suggesting a mitigating function.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Microglia, the brain's immune cells, are increasingly recognized for their involvement in opioid dependence and withdrawal.
- Mu Opioid Receptors (MOR) are present on microglia, influencing their function through direct and indirect (neuron-mediated) pathways.
- Understanding microglial roles is crucial for developing novel therapeutic strategies for opioid use disorder.
Purpose of the Study:
- To investigate the direct effects of opioids on microglial gene expression and function during tolerance and withdrawal.
- To elucidate the specific molecular pathways and cellular mechanisms underlying microglial responses to opioids.
Main Methods:
- Ribosome-associated RNA sequencing (RiboTag-Seq) was employed on striatal microglia following morphine tolerance and naloxone-precipitated withdrawal.
- RNA-sequencing data were validated using fluorescent in-situ hybridization and immunohistochemistry.
- Chemogenetic activation of Gi/o-coupled DREADD receptors in CX3CR1-expressing cells was performed during morphine withdrawal.
Main Results:
- Significant inverse changes in RNA translation were observed during opioid tolerance and withdrawal.
- Weighted Gene Co-expression Network Analysis (WGCNA) identified a cAMP-associated gene network, crucial for microglial motility and morphology, downregulated during tolerance and upregulated during withdrawal.
- Activation of Gi/o-coupled DREADD receptors in microglia exacerbated opioid withdrawal symptoms.
Conclusions:
- Microglial Gi-signaling and cAMP-associated gene networks exhibit inverse regulation during opioid tolerance and early withdrawal.
- These findings suggest a potential role for microglia in mitigating the adverse consequences of opioid exposure.
- Targeting microglial pathways may offer new avenues for treating opioid dependence and withdrawal.
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