Specifying Human iPSC-derived Medium spiny Neurons for Cocaine Abuse Research

摘要 在美国，可卡因滥用仍然是一个主要的公共卫生问题。可卡因成瘾是一种慢性复发 与严重的医学和心理社会并发症相关的神经障碍。它的作用机制 人们对可卡因成瘾知之甚少，目前也没有有效的治疗方法。更好的 了解成瘾的发展对于创造有效的可卡因成瘾治疗方法是至关重要的。 我们关于可卡因成瘾的知识主要来自对动物模型的研究， 通过分析正电子发射计算机断层扫描(Positron)获得的人类神经元病理信息的有限贡献 终末期可卡因成瘾者的图像和死后脑组织。这是很困难的 获得可卡因成瘾者分化的神经元，用于分子分析。患者派生的诱导 多能干细胞(IPSCs)为研究可卡因成瘾机制提供了一个很好的平台。 本方案的目的是利用IPSC来源的人纹状体中棘神经元(MSN) 包括以伏隔核为模型研究可卡因成瘾的机制。纹状体MSN 在可卡因成瘾中起关键作用，它们从前额叶皮质接受谷氨酸能输入。谷氨酸能 在体外纹状体培养中，纹状体MSN的突触发生需要皮质神经元的输入。 目标1是从这两个来源产生IPSC来源的纹状体MSN和额叶皮质(FC)谷氨酸能神经元 可卡因依赖患者(CD)和未受影响的对照组(UC)。IPSC来源的皮质神经元将是 设计成稳定表达GFP，这样它们就可以在与纹状体MSN共培养时被识别出来，而 IPSC来源的MSN将通过抗体特异性DARPP32，GABA和 多巴胺受体(D1R和D2R)。目标2是比较形态、神经化学和 CD组和UC组之间IPSC来源的纹状体MSN的电生理特性。本研究 将为可卡因成瘾研究提供重要工具，并可能揭示关键的形态， 慢性萎缩性结肠炎和溃疡性结肠炎的神经化学和电生理差异。因此，它可以作为 为阐明可卡因成瘾的分子和细胞机制奠定基础。

Abstract Cocaine abuse remains a major public health problem in the US. Cocaine addiction is a chronic relapsing neurological disorder associated with severe medical and psychosocial complications. The mechanisms of cocaine addiction are poorly understood, and no effective treatment is currently available. A better understanding of the development of addiction is essential for creating effective therapy for cocaine addiction. Our knowledge about cocaine addiction has been generated mostly from studies with animal models, with limited contributions from information about human neuronal pathology obtained by analyzing PET (Positron emission tomography) images and postmortem brain tissues of end-stage cocaine addicts. It has been difficult to obtain differentiated neurons from cocaine addicts for molecular analysis. Patient-derived induced pluripotent stem cells (iPSCs) provide an excellent platform for exploring the mechanisms of cocaine addiction. The purpose of this proposal is to use iPSC-derived the medium spiny neurons (MSNs) in human striatum including nucleus accumbens as a model to investigate the mechanisms of cocaine addiction. Striatal MSNs play key roles in cocaine addiction, and they receive glutamatergic input from prefrontal cortex. Glutamatergic inputs from cortical neurons are required for the synaptogenesis on the striatal MSNs in striatal culture in vitro. Aim 1 is to generate iPSC-derived striatal MSNs and frontal cortical (FC) glutamatergic neurons from both cocaine-dependent patients (CD) and unaffected controls (UC). The iPSC-derived cortical neurons will be engineered to stably express GFP so that they can be identified in co-culture with striatal MSNs, while the iPSC-derived MSNs will be identified by immunostaining with antibodies specific DARPP32, GABA and dopamine receptors (D1R and D2R). Aim 2 is to compare morphological, neurochemical and electrophysiological properties of the iPSC-derived striatal MSNs between the CD and UC groups. This study will generate important tools for cocaine addiction research, and may uncover key morphological, neurochemical and electrophysiological differences between CD and UC. Therefore, it may serve as the foundation for elucidating the molecular and cellular mechanisms of cocaine addiction.