Administrative Supplements for Generating Multiple Circuit And Neuron Type Specific AAV Vectors With Cross-Species Applicability

 描述(由申请人提供)：检查特定脑核和特定神经元类型的连接性和功能的能力对于理解大脑在微电路和中尺度水平上的工作方式至关重要。因此，能够以神经系统内选定类型的神经元和/或神经元组为目标的工具，特别是易于应用并可在许多不同物种(模式生物)中使用的工具，对神经科学界非常重要。在小鼠中，已经产生了少量的转基因株，它们在特定的神经元类型或脑区域表达GFP或Cre重组酶，而这样的工具在其他生物中是必不可少的，但在其他生物如大鼠和非人灵长类动物中是不可用的。在初步研究中，我们发现，在几个超保守的增强子元件的控制下，非手术的携带GFP或Cre的AAV载体在外周和中枢神经系统中产生了不同的、特异的和可重复的表达模式。在这里，我们建议基于保守的增强子来生成区域、神经元类型特定和活动相关的AAV载体。脑室注射的简便性和AAV载体的灵活性将使其能够在不需要产生多个转基因株系的情况下获得广泛的应用，并且增强子的保守性使得这些载体很容易适用于多种物种。具体地说，我们将产生一套在不同增强子控制下表达GFP、Cre或其他转基因的AAV载体，包括(1)通过比较基因组研究确定的脊椎动物超保守增强子，(2)驱动抑制性中间神经元表达的增强子，以及(3)保守的神经元活性依赖增强子。这些载体将首先通过脑室注射在小鼠身上进行筛选。具有所需表达模式的AAV载体的子集将使用相同的脑室内注射在大鼠和绒猴身上进行测试。我们预计在三年的资助期内，将产生多个AAV载体，使特定电路和神经元类型的研究成为可能，并适用于多种物种。

 DESCRIPTION (provided by applicant): The ability to examine the connectivity and functions of specific brain nuclei and specific neuron types is critical for understanding how the brain works at both microcircuit and mesoscale levels. Therefore tools that enable targeting of selected types of neurons and/or groups of neurons within the neural system, especially tools that are easily applicable and can be used in many different species (model organisms) are important for the neuroscience community. In mouse, a small number of transgenic lines have been generated that express GFP or Cre recombinase in certain selected neuron types or brain areas, whereas such tools are essential but not available in other organism such as rat and non-human primates. In preliminary studies, we found that non- surgical intra-ventricular injection of AAV vectors carrying GFP or Cre under the control of several ultra conserved enhancer elements gave rise to a different yet specific and reproducible pattern of expression in the peripheral and central nervous system. Here we propose to generate region-, neuron type-specific, and activity-dependent AAV vectors based on conserved enhancers. The ease of intra-ventricular injection and the flexibility of AAV vectors will enable a wide range of applications without the need of generating multiple transgenic lines, and the conserved nature of enhancers allow these vectors to be readily applicable in multiple species. Specifically, we will generate a suite of AAV vectors expressing GFP, Cre, or other transgenes under the control of different enhancers, including (1) vertebrate ultra conserved enhancers identified through comparative genomic studies, (2) enhancers to drive expression in inhibitory interneurons, and (3) conserved neuronal activity-dependent enhancers. These vectors will first be screened in mouse using intra-ventricular injections. Subset of AAV vectors with desired expression patterns will be tested in rat and marmoset using the same intra-ventricular injections. We expect in the three-year funding period to generate multiple AAV vectors that enable circuit- and neuron-type-specific investigations and are applicable in multiple species.