Genetic targeting of cortical pyramidal neuron subtypes

描述(申请人提供)：小鼠皮质锥体神经元亚型的遗传靶向摘要研究大脑皮层神经回路的发育、组织和功能的一个关键障碍是神经元类型的惊人多样性和对其基本生物学知识的缺乏。大脑皮层神经元多样性和同一性的问题是根本性的，超越了发育和系统神经科学，是定义认知和精神障碍生物学的核心。 谷氨酸能锥体神经元约占皮质神经元的80%，具有很大的信息编码、存储、可塑性和携带皮质计算输出的能力。根据其特定的椎板位置、轴突投射模式和基因表达谱，PYN由不同的亚型组成。PYN的子集形成了多个分层的信息处理子网络，具有通往皮质和皮质下目标的不同输出通道，这些目标服从于感觉、运动、认知和情绪功能。重要的是，在各种神经精神和神经退行性疾病中，PYN亚型受到不同程度的影响。然而，严重缺乏专门和有效的遗传工具来研究PYN，阻碍了对皮质环路的理解。我们计划通过一个由冷泉港实验室的Josh Huang博士和哈佛大学的Paola Arlotta博士领导的联合项目，以及艾伦脑科学研究所的曾宏奎博士的关键合作，为小鼠主要的PYN亚型建立一个全面的遗传工具集。我们已经发现了一套区分主要PYNS亚型的特异和组合标记。我们将使用交集、减法和归纳策略来瞄准PYN亚型。AIM 1将产生~25个敲打CRE和FLP驱动系，以锥体神经元的主要亚类和谱系为靶点。AIM 2将产生多个交叉和差减报告，广泛用于标记不同的神经元亚型。目标3将描述司机的特殊性，并在公共数据库中组织和展示数据和资源。我们将结合解剖示踪、原位信使核糖核酸和免疫细胞化学来确定基因靶向的特异性。我们将使用艾伦研究所的高通量和高分辨率流水线来表征PYN亚型标记和轴突投影。我们将把所有工具和试剂存放在可供公众访问的主要存储库(JAX)(Allen Brain Atlas网站)。通过整合各种现代技术，基因靶向将为大脑皮质回路提供切入点，并促进对PYN的系统和全面分析，从细胞命运指定到电路集成、连通性，以及在皮质处理和行为中的功能。这些基因工具将进一步为自闭症、精神分裂症、躁郁症和ALS、癫痫、阿尔茨海默氏症、痴呆症等脑部疾病模型的致病机制提供敏感的探针，并可能产生治疗干预的细胞和分子靶点。虽然这项资助不像传统的R01那样是由假设驱动的，但对于许多未来的R01来说，它肯定是“使能假设”，以阐明大脑皮层在中枢神经系统架构和功能的更大框架中的基础作用。1

DESCRIPTION (provided by applicant): Genetic targeting of cortical pyramidal neuron subtypes in the mouse Abstract A key obstacle to studying the development, organization, and function of neural circuits in the cerebral cortex is the stunning diversity of neuron types and a lack of comprehensive knowledge about their basic biology. The problem of neuronal diversity and identity in the cortex is fundamental, transcending developmental and systems neuroscience, and lies at the heart of defining the biology of cognition and psychiatric disorders. Glutamatergic pyramidal neurons (PyNs) constitute ~80% of cortical neurons, are endowed with large capacity for information coding, storage, plasticity, and carry the output of cortical computation. PyNs consist of diverse subtypes based on their specific laminar locations, axonal projection patterns, and gene expression profiles. Subsets of PyNs form multiple and hierarchical subnetworks of information processing, with distinct output channels to cortical and subcortical targets that subserve sensory, motor, cognitive and emotional functions. Importantly, PyN subtypes are differentially affected in various neuropsychiatric and neurodegenerative disorders. However, the severe lack of specific and effective genetic tools for studying PyNs has hampered progress in understanding cortical circuits. We propose to build a comprehensive genetic tool set for major PyN subtypes in the mouse through a joint project led by Dr. Josh Huang at Cold Spring Harbor Laboratory and Dr. Paola Arlotta at Harvard University with key collaboration from Dr. Hongkui Zeng at the Allen Institute for Brain Science. We have discovered a set of specific and combinatorial markers that distinguish major PyNs subtypes. We will use intersection, subtraction, and inducible strategies to target PyN subtypes. Aim 1 will generate ~25 knockin Cre and Flp driver lines that target major subclasses and lineages of pyramidal neurons. Aim 2 will generate multiple intersectional and subtractive reporters that are broadly useful for labeling distinct neuronal subtypes. Aim 3 will characterize the specificity of drivers, and organize and display data and resource in public databases. We will combine anatomical tracing with mRNA in situ and immunocytochemistry to determine the specificity of genetic targeting. We will use high- throughput and high- resolution pipeline at the Allen Institut to characterize PyN subtype labeling and axon projections. We will deposit all tools and reagents in major repositories (JAX) that are publically accessible (Allen Brain Atlas website). Genetic targeting will provide entry points to cortical circuits by integrating the full range of modern technologies and facilitate a systematic and comprehensive analysis of PyNs, from cell fate specification to circuit integration, connectivity, and function in cortical processing and behavior. These genetic tools will further provide sensitive probes to pathogenic mechanisms in models of brain disorders including autism, schizophrenia, bipolar and ALS, epilepsy, Alzheimer's, dementia, and may yield cell and molecular targets for therapeutic intervention. Although this grant is not hypothesis-driven as a traditional R01, it is certainly "hypothesis-enabling" for many future R01s to elucidate the fundamental role of the cerebral cortex in the larger framework of CNS architecture and function. 1