Accumulating evidence has suggested that prenatal exposure to methadone causes multiple adverse effects on human brain development. Methadone not only suppresses fetal neurobehavior and alters neural maturation, but also leads to long-term neurological impairment. Due to logistical and ethical issues of accessing human fetal tissue, the effect of methadone on brain development and its underlying mechanisms have not been investigated adequately and are therefore not fully understood. Here, we use human cortical organoids which resemble fetal brain development to examine the effect of methadone on neuronal function and maturation during early development. During development, cortical organoids that are exposed to clinically relevant concentrations of methadone exhibited suppressed maturation of neuronal function. For example, organoids developed from 12th week till 24th week have an about 7-fold increase in AP firing frequency, but only half and a third of this increase was found in organoids exposed to 1 and 10 μM methadone, respectively. We further demonstrated substantial increases in INa (4.5-fold) and IKD (10.8-fold), and continued shifts of Na+ channel activation and inactivation during normal organoid development. Methadone-induced suppression of neuronal function was attributed to the attenuated increase in the densities of INa and IKD and the reduced shift of Na+ channel gating properties. Since normal neuronal electrophysiology and ion channel function are critical for regulating brain development, we believe that the effect of prolonged methadone exposure contributes to the delayed maturation, development fetal brain and potentially for longer term neurologic deficits.

Opioid use disorder (OUD) among pregnant women has become an epidemic in the United States. Pharmacological interventions for maternal OUD most commonly involve methadone, a synthetic opioid analgesic that attenuates withdrawal symptoms and behaviors linked with drug addiction. However, evidence of methadone’s ability to readily accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on prenatal brain development. We utilized human cortical organoid (hCO) technology to probe how this drug impacts the earliest mechanisms of cortico-genesis. Bulk mRNA sequencing of 2-month-old hCOs chronically treated with a clinically relevant dose of 1 μM methadone for 50 days revealed a robust transcriptional response to methadone associated with functional components of the synapse, the underlying extracellular matrix (ECM), and cilia. Co-expression network and predictive protein-protein interaction analyses demonstrated that these changes occurred in concert, centered around a regulatory axis of growth factors, developmental signaling pathways, and matricellular proteins (MCPs). TGFβ1 was identified as an upstream regulator of this network and appeared as part of a highly interconnected cluster of MCPs, of which thrombospondin 1 (TSP1) was most prominently downregulated and exhibited dose-dependent reductions in protein levels. These results demonstrate that methadone exposure during early cortical development alters transcriptional programs associated with synaptogenesis, and that these changes arise by functionally modulating extra-synaptic molecular mechanisms in the ECM and cilia. Our findings provide novel insight into the molecular underpinnings of methadone’s putative effect on cognitive and behavioral development and a basis for improving interventions for maternal opioid addiction.

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Opioid use disorder (OUD) among pregnant women has become an epidemic in the United States. Pharmacological interventions for OUD involve methadone, a synthetic opioid analgesic that attenuates withdrawal symptoms and behaviors linked with maternal drug addiction. However, methadone's ability to readily accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on prenatal brain development. We took advantage of human cortical organoid (hCO) technology to probe how this drug impacts the earliest mechanisms giving rise to the cerebral cortex. To this end, we conducted bulk mRNA sequencing of 2-month-old hCOs derived from two cell lines that were chronically treated with a clinically relevant dose of 1μM methadone for 50 days. Differential expression and gene ontology analyses revealed a robust transcriptional response to methadone associated with functional components of the synapse, the underlying extracellular matrix (ECM), and cilia. Further unsupervised co-expression network and predictive protein-protein interaction analyses demonstrated that these changes occurred in concert, centered around a regulatory axis consisting of growth factors, developmental signaling pathways, and matricellular proteins. Our results demonstrate that exposure to methadone during early cortico-genesis fundamentally alters transcriptional programs associated with synapse formation, and that these changes arise by modulating extra-synaptic molecular mechanisms in the ECM and cilia. These findings provide novel insight into methadone's putative effect on cognitive and behavioral development and a basis for improving interventions for maternal opioid addiction.

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.