Genetic Targeting Core

项目摘要-基因定位核心 物质使用障碍(SUD)的特点是大脑回路调节失调，包括 大脑奖赏回路的活动，应激回路的反应性增加和大脑功能受损 执行大脑皮层回路。神经改变见于基底神经节、杏仁核和延髓。 前额叶皮质区域，包含广泛的内源性神经递质，包括多巴胺， 阿片肽、内源性大麻素、促肾上腺皮质激素释放因子、强啡肽、谷氨酸等。它 提示这些适应不良可能是慢性阻塞性肺疾病患者行为改变的原因 吸毒者，例如习惯养成和强迫吸毒，会进一步恶化神经元和 性欲减退的行为症状。因此，恢复大脑回路的正常功能是一个可取的目标 苏德疗法。然而，慢性药物使用对成瘾大脑回路的确切影响以及如何 操纵这些回路是否可以预防或治疗物质使用障碍仍有待阐明。 作为成瘾研究卓越中心的一部分，基因靶向核心(GT Core)将帮助用户 在获得成瘾神经机制的初步数据方面，这些数据将成为未来NIDA拨款的基础 申请。GT Core将提供针对和操作特定细胞中的成瘾电路的服务 和电路的特定方式使用最先进的遗传技术，包括光遗传学、化学遗传学 和CRISPR/Cas9基因编辑。光遗传策略使用基于微生物的光激活离子通道 (视黄素)，允许神经元快速激活(通道视紫红质)或抑制(卤视紫质， 古视紫质)，而化学发生方法使用工程离子通道(PSAM)或G蛋白- 偶联受体(DREADD)，可在药理上激活。CRISPR/Cas9基因编辑允许直接 在啮齿动物体内操纵基因组。功能性转基因的引入，如opsins和opsins DREADD或编辑本地基因需要在体内传递和在所需细胞中表达遗传物质。 这是通过病毒或非病毒载体系统实现的。细胞和电路的特异性可以通过以下方式进一步增强 使用特定的CRE驱动程序鼠标行。 GT Core将为其用户提供与三个具体目标相对应的三项主要服务：1)协助 随着基因打靶方法的选择，包括转基因小鼠品系的选择和转基因小鼠的类型 病毒载体；2)使用高精度立体定位仪器优化和实施基因传递；以及3)验证 使用荧光显微镜或蛋白质和基因定量技术进行基因打靶的有效性。这个 GT核心将与行政、行为和神经分析核心协调工作，以开发有效的 涉及多个核心的实验策略。我们的核心之间的密切合作确保了 在成瘾研究的所有领域都有专门知识，并将允许建立标准化的结果衡量标准 强调科学的严谨性和重复性。

Project Summary – Genetic Targeting Core Substance use disorders (SUD) are characterized by dysregulation of brain circuitry that involves diminished activity of the brain reward circuits, increased responsiveness of the stress circuits and impaired functioning of the executive cortical circuits. Neural changes are observed in the basal ganglia, extended amygdala and prefrontal cortical regions and encompass a wide range of endogenous neurotransmitters including dopamine, opioid peptides, endocannabinoids, corticotropin releasing factor (CRF), dynorphin, glutamate and others. It implies that these maladaptations may be causally responsible for behavioral changes often observed in chronic drug users, for example habit formation and compulsive drug taking, that further worsen the neuronal and behavioral signs of SUD. Restoration of normal functioning of the brain circuits is therefore a desirable goal of SUD therapies. However, the exact influence of chronic drug use on the addiction brain circuits and how manipulation of these circuits could prevent or treat substance use disorders remains to be elucidated. As part of the Center of Excellence in Addiction Studies, the Genetic Targeting Core (GT Core) will assist users in obtaining preliminary data on neural mechanisms of addiction that will form basis for future NIDA grant applications. The GT Core will provide services to target and manipulate the addiction circuits in a cell specific and circuits specific manner using state-of-the-art genetic techniques, including optogenetics, chemogenetics and CRISPR/Cas9 gene editing. Optogenetic strategies use microbial-based light activated ion channels (opsins) that allow fast neuronal activation (channelrhodopsins) or inhibition (halorhodopsins, archaerhodopsins), while chemogenetic approaches use engineered ion channels (PSAMs) or G protein- coupled receptors (DREADDs) that can be activated pharmacologicaly. CRISPR/Cas9 gene editing allows direct in vivo manipulation of the genome in rodents. The introduction of functional transgenes, such as opsins and DREADDs or editing of native genes requires in vivo delivery and expression of genetic material in desired cells. This is accomplished with viral or nonviral vector systems. Cell and circuit specificity can be further enhanced by the use of a specific Cre-driver mouse line. The GT Core will provide three main services to its users that correspond with the three Specific Aims: 1) assist with the selection of gene targeting methods, including the choice of the transgenic mouse line, and the type of viral vector; 2) optimize and implement gene delivery using high precision stereotaxic instruments; and 3) verify the efficacy of gene targeting using fluorescent microscopy or protein and gene quantification techniques. The GT Core will coordinate work with the Administrative, Behavioral and Neuroanalytical Cores to develop effective experimental strategies that involve several Cores. The close collaboration between our Cores ensures high expertise in all areas of the addiction research and will permit establishment of standardized outcome measures emphasizing scientific rigor and reproducibility.