Effect of methadone on the developmental properties of human brain organoids

摘要 阿片类药物危机已经成为全国性的流行病，统计数据令人震惊。在过去的十年里 海洛因的使用、鸦片类药物处方和芬太尼的滥用急剧增加。服药过量死亡人数翻了一番 自2000年以来和2015年，在全国范围内，有33,000多人死于阿片类药物过量。在……里面 此外，1998年至2011年期间，阿片类药物依赖孕妇的数量大幅增加 女性，如对美沙酮有依赖。尽管阿片类药物总体上已经得到了很好的研究，但这种流行病 阿片类药物滥用，特别是在孕妇中，揭示了我们对美沙酮的影响知之甚少 关于胎儿大脑发育的研究。在过去的几年里，我们利用了一种较新的技术，3D- 脑有机化合物，这极大地促进了对人类早期大脑发育的研究。这为我们提供了 美国获得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。使用这些方法，我们已经能够制作出令人兴奋的初步 数据显示，美沙酮减少突触传递，并可能影响突触的可塑性。基于 根据我们最近的研究结果，我们提出了以下总体假设：阿片类药物暴露会导致 胎儿脑发育过程中突触发生异常和突触传递受损。为了 针对这一假设，我们制定了以下具体目标：具体目标1：确定效果 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。我们将使用多个 电极阵列记录，以探索美沙酮如何改变神经网络的活动。具体目标2： 测定美沙酮对细胞电生理特性及突触功能和结构的影响 在人类皮质有机体的发育过程中。我们将研究AP的触发特性、突触电流和 神经元的Na+和K+电流，并用膜片钳分子技术剖析突触前和突触后的机制 和成像技术。具体目标3：剖析美沙酮诱导的血管紧张素转换酶变化的机制 人脑皮质类器官中的突触发生和突触传递。作为血栓蛋白1，2(TSP1，2)， 星形胶质细胞分泌的糖蛋白，在轴突生长、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生物学 对人胎脑的影响。我们对这一应用的研究是新颖和独特的，并解决了重要的 美沙酮对孕妇脑发育不良的影响带着一个 了解了美沙酮的作用机制，我们相信我们可以开发出新的治疗方法 目标是减轻美沙酮对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.