Genetic targeting of cortical pyramidal neuron subtypes

描述（由申请人提供）：小鼠中皮质锥体神经元亚型的遗传靶向摘要研究大脑皮质中神经回路的发育、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缺乏关于其基础生物学的全面知识。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，是定义认知和精神疾病的生物学的核心。 谷氨酸能锥体神经元（Glutamatergicpyramidecneurons，PyNs）约占大脑皮层神经元的80%，具有信息编码、存储、可塑性和计算输出等功能。PyN由不同的亚型基于其特定的层状位置，轴突投射模式，和基因表达谱。PyN的子集形成了多个和分层的信息处理子网络，具有不同的输出通道，可以到达皮层和皮层下的目标，这些目标有助于感觉，运动，认知和情感功能。重要的是，PyN亚型在各种神经精神和神经退行性疾病中受到不同的影响。然而，严重缺乏研究PyNs的特异性和有效的遗传工具，阻碍了对皮层回路的理解。我们建议通过由冷泉港实验室的Josh Huang博士和哈佛大学的保拉Arlotta博士领导的联合项目，与艾伦脑科学研究所的Hongkui Zeng博士进行关键合作，为小鼠中的主要PyN亚型建立一个全面的遗传工具集。我们已经发现了一组特异性和组合标记，区分主要的PyNs亚型。我们将使用交叉，减法和诱导策略来靶向PyN亚型。目标1将产生靶向锥体神经元的主要亚类和谱系的~25个敲入Cre和Flp驱动线。目标2将产生多个交叉和消减报告，广泛用于标记不同的神经元亚型。目标3将描述驱动程序的特殊性，并在公共数据库中组织和显示数据和资源。我们将联合收割机解剖追踪与mRNA原位和免疫细胞化学相结合，以确定遗传靶向的特异性。我们将在艾伦研究所使用高通量和高分辨率管道来表征PyN亚型标记和轴突投射。我们将把所有工具和试剂存款在可通过计算机访问的主要存储库（JAX）中（艾伦大脑图谱网站）。遗传靶向将通过整合全方位的现代技术为皮层回路提供切入点，并促进对PyN进行系统和全面的分析，从细胞命运规范到皮层处理和行为中的回路整合，连接和功能。这些遗传工具将进一步为自闭症、精神分裂症、躁郁症和ALS、癫痫、阿尔茨海默病、痴呆症等脑部疾病模型中的致病机制提供敏感探针，并可能产生用于治疗干预的细胞和分子靶点。虽然这项资助不像传统的R 01那样是假设驱动的，但对于许多未来的R 01来说，它肯定是“假设使能”的，以阐明大脑皮层在CNS结构和功能的更大框架中的基本作用。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