Effect of methadone on the developmental properties of human brain organoids

摘要 阿片类药物危机已经成为一种全国性的流行病，统计数据令人震惊。在过去的十年里， 海洛因使用、阿片类药物处方和芬太尼滥用急剧增加。吸毒过量死亡人数翻了一番 自2000年和2015年以来，全国有超过33，000人死于阿片类药物过量。在 此外，1998年至2011年期间， 妇女，如美沙酮依赖。虽然阿片类药物已经得到了很好的研究， 阿片类药物滥用，特别是孕妇，揭示了我们对美沙酮的影响知之甚少 对胎儿大脑发育的影响在过去的几年里，我们利用了一种新的技术，3D- 大脑类器官，极大地促进了对早期人类大脑发育的研究。这提供 这为我们提供了一个前所未有的机会来研究这种效应背后的细胞和分子机制。 阿片类药物对早期大脑发育的影响利用这种方法，我们已经能够产生令人兴奋的初步 数据显示美沙酮降低突触传递并可能影响突触可塑性。基于 根据我们最近的研究结果，我们提出了以下总体假设：阿片类药物暴露导致 胎儿脑发育过程中异常的突触发生和受损的突触传递。为了 针对这一假设，我们制定了以下具体目标：具体目标1：确定 美沙酮对人类皮质类器官发育过程中神经网络活动的影响。我们将使用多- 电极阵列记录，以探索美沙酮如何改变神经网络活动。具体目标2： 确定美沙酮对细胞电生理特性和突触功能和结构的影响 在人类皮质类器官的发育过程中。我们将研究AP放电特性，突触电流， Na+和K+电流的神经元和解剖前和突触后机制，使用膜片钳，分子 和成像技术。具体目标3：剖析美沙酮诱导的 人类皮质类器官中的突触发生和突触传递。作为血小板反应蛋白1，2（TSP 1，2）， 星形胶质细胞分泌的糖蛋白，在神经突生长，树突棘和突触形成中发挥作用，我们将 研究美沙酮对TSP的影响，以了解美沙酮的分子病理学 对人类胎儿大脑的影响我们在这方面的应用研究是新颖和独特的，并解决了重要的 孕妇在美沙酮影响下大脑发育不良的问题。与 了解美沙酮效应的机制，我们相信我们可以开发新的治疗药物， 目标是减轻美沙酮对生命早期大脑发育的影响。

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.