Extracellular vesicles from morphine-exposed prefrontal cortex carry transcriptomic and proteomic signatures of synaptic dysfunction

Extracellular vesicles (EVs) released by neurons and glial cells mediate intercellular communication in the brain and regulate synaptic function, neuronal survival, and neuropathological processes. Although chronic opioid exposure induces widespread neuroadaptations, the role of brain-derived EVs (BDEVs) in these processes remains poorly understood. We isolated BDEVs from the prefrontal cortex of rats subjected to chronic morphine exposure and performed integrative analyses of their RNA and protein cargo. Total RNA sequencing was combined with unbiased proteomics to define morphine-induced alterations. Functional validation was conducted by exposing primary cortical neurons to BDEVs from morphine- or vehicle-treated animals, followed by transcriptional profiling. Morphine significantly reprogrammed the transcriptome and proteome of BDEVs, with enrichment of pathways related to synaptic plasticity, endoplasmic reticulum stress, mitochondrial dysfunction, and neurodegeneration. Among the most robust changes, the synaptic regulator ARC and the ER stress marker Hspa5 were consistently modulated at both mRNA and protein levels. In functional assays, morphine-derived BDEVs altered neuronal expression of genes associated with synaptic remodeling and excitability, including Ncam1 and Kcnn1 . These findings provide the first evidence that chronic opioid exposure reprograms BDEV cargo in a brain region critical for addiction, and that these vesicles are sufficient to induce transcriptional changes in recipient neurons. BDEVs emerge as active mediators of morphine-induced neuroadaptations and hold promise as biomarkers and therapeutic targets in opioid-related neuropathologies.