Effect of methadone on the developmental properties of human brain organoids

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ABSTRACT The opioid crisis has become a national epidemic and the statistics are startling. In the past decade there has been a sharp increase in heroin use, opiate prescriptions and fentanyl abuse. Overdose deaths have doubled nationally since 2000 and in 2015, and more than 33,000 deaths were attributable to overdose from opioids. In addition, there has been a major increase between 1998 and 2011 in the number of opioid-dependent pregnant women, such as with methadone dependency. Although opioids have been well studied in general, the epidemic of opioid abuse, especially in pregnant women, has unmasked how little we know about the effect of methadone on fetal brain development. In the past several years, we have taken advantage of a newer technology, the 3D- brain organoids, that facilitated enormously the investigation of early human brain development. This has provided us with an unprecedented opportunity to investigate the cellular and molecular mechanisms underlying the effect of opioids on early brain development. Using such methods, we have been able to produce exciting preliminary data showing that methadone decreases synaptic transmission and possibly affects synaptic plasticity. Based on our recent results, we have posed the following overall hypothesis: Opioid exposure leads to abnormal synaptogenesis and impaired synaptic transmission during fetal brain development. In order to address this hypothesis, we have formulated the following Specific Aims: Specific Aim 1: To determine the effect of methadone on neural network activity during development in human cortical organoids. We will use multi- electrode array recordings to explore how methadone modifies the neural network activity. Specific Aim 2: To determine the effect of methadone on cellular electrophysiological properties and synaptic function and structure during development in human cortical organoids. We will investigate AP firing properties, synaptic currents, and Na+ and K+ currents in neurons and dissect the pre- and postsynaptic mechanisms using patch-clamp, molecular and imaging techniques. Specific Aim 3: To dissect the mechanisms of the methadone-induced changes in synaptogenesis and synaptic transmission in human cortical organoids. As thrombospondins 1,2 (TSP1,2), astrocyte-secreted glycoproteins, play a role in neurite outgrowth, dendritic spine and synapse formation, we will study the effect of methadone on TSPs to obtain an understanding of the molecular pathobiology of methadone’s effect on the human fetal brain. Our studies in this application are novel and unique and address the important problem of human brain maldevelopment under the influence of methadone in pregnant women. With an understanding of the mechanisms involved in methadone effect, we believe that we can develop novel therapeutic targets to mitigate the effect of methadone on brain development in early life.