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Methadone disrupts early synaptic development in human cortical organoids

In the last decade, the national opioid epidemic has yielded a 131% increase in pregnant women diagnosed with opioid use disorder (OUD), which is characterized by opioid abuse and dependence. Pharmacological treatments for OUD commonly involve methadone, a synthetic opioid analgesic that attenuates withdrawal symptoms. The ability to readily enter fetal circulation, accumulate in neural tissue, and cause long‐term neurocognitive sequelae, has led to concerns regarding the drug’s effect on fetal brain development. Since little is known about the impact of methadone on human fetal brain development, we took advantage of the in vitro induced pluripotent stem cell (iPSC)‐derived human cortical organoid (hCO) technology to probe its effects on the earliest developmental stages. Our lab has demonstrated that 1‐week of chronic exposure to a clinically relevant 1μM dose of methadone leads to the suppression of synaptic transmission in 8‐ to 10‐week‐old hCOs. Therefore, we hypothesized that long term chronic exposure to methadone from the earliest stages of cortical differentiation would alter the molecular mechanisms underlying synaptic development. To test this, we conducted bulk mRNA sequencing of 2‐month‐old hCOs derived from two cell lines that had been chronically treated with 1 μM methadone for 50 days. Differential expression analyses revealed a robust transcriptional response to methadone in these hCOs, with over 2000 genes that were significantly differentially expressed independently of baseline transcriptional differences between the cell lines (|FC| ≥ 1.5, p‐adj < 0.05). Gene set enrichment (GSEA) and gene ontology (GO) analyses revealed alterations in pathways associated with both structural and functional synaptic development, including the downregulation of pathways associated with primary cilia and extracellular matrix (ECM). At a molecular level, preliminary co‐expression analysis (CEMiTool) of 1063 genes associated one or more of these three cellular components yielded a primary co‐expression module of 417 genes associated with synaptic, ciliary, and extracellular matrix function that were also predicted to interact at the protein level. During brain development, primary cilia regulate the integrity and composition of the ECM, influencing synapse formation, differentiation, maturation, and refinement. These results indicate that chronic methadone changes gene expression that fundamentally alter synaptic formation and structure, potentially leading to the observed alterations in synaptic transmission.