UC San Diego

Methadone is a synthetic opioid analgesic and a common pharmacological treatment for opioid use disorder (OUD) in pregnant women. Its clinical application blocks the effects of other opioids and attenuates withdrawal symptoms or behaviors leading to relapse or overdose. However, methadone’s ability to readily enter fetal circulation, accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on fetal brain development.

In the present research, human induced pluripotent stem cell-derived cortical organoid (hCO) technology was leveraged to probe the etiology of these deficits by understanding how methadone impacts the earliest mechanisms of cortical development.

Bulk mRNA sequencing of 2-month-old hCOs chronically treated with a clinically relevant dose of 1μM methadone revealed a robust transcriptional response to methadone associated with functional components of the synapse, including pre-synaptic vesicular trafficking/release and post-synaptic signal reception machinery, as well as 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). Of the central MCPs, thrombospondin 1 (TSP1), which is necessary for synapse formation and maturation, was most prominently downregulated, with dose-dependent reductions in its protein levels. These results indicated that methadone exposure during cortical development alters transcriptional programs associated with synapse biology by modulating ECM and ciliary molecular mechanisms.

To further dissect methadone’s impact at the synapse, ultrastructural imaging was performed to visualize and quantify changes in synaptic features. Improving upon the limitations of prior ultrastructural studies of synapses in brain organoids, culture, imaging, and segmentation methodologies were modified to optimize the volumetric capture of synaptic features via electron microscopy. These techniques revealed that 10μM methadone reduces synaptic vesicle volumes as well as the range of vesicle morphologies in 3-month-old hCOs, which may have implications for the reductions in neurotransmission observed following methadone exposure.

Cumulatively, the findings presented in this dissertation provide novel insight into the molecular underpinnings of methadone’s effect on neurocognitive development, novel methods of studying these effects in brain organoids, and a basis for improving interventions for maternal OUD.