Cell Type Specific Tracing of Neocortical Circuits Using Viral Vectors

描述(由申请人提供)：新皮质由密集的兴奋性神经元组成，这些神经元被不同的和更稀疏分布的抑制性神经元阵列所平衡。因此，皮质抑制神经元提供了一种在多种条件下工作的增益控制。例如，在初级视觉皮质(V1)，抑制对于单个神经元的几个基本功能特征是必不可少的，包括对刺激对比度、大小和方向的偏好。虽然抑制性神经元用于控制兴奋，但尚不清楚抑制性和兴奋性细胞类型是否通过同一套皮质环路调节，也不知道这些环路的功能选择性是否不同。确定这两种主要细胞类型的神经回路的起源将为皮质处理的基本机制提供巨大的洞察力。然而，为了做到这一点，有必要开发一种针对细胞类型的特定神经解剖示踪技术。这里的建议是通过利用重组病毒载体(腺相关病毒和慢病毒)的神经趋向性和细胞类型特异性启动子(GAD-67和β-CaMKII)的选择性来传递和允许两个关键基因(TVA和RabG)的表达来开发这种技术。因此，这些基因将选择性地在抑制性或兴奋性新皮质细胞中表达，但不会 两者都有。在这些特定细胞类型中表达的TVA和RabG蛋白将允许靶向病毒感染和作为单突触逆行示踪剂的新型转基因和假型狂犬病病毒(ENVA-RabG)的反式互补(Wickersham，Lyon等人，2007，Neuron)。这些基因的细胞类型特异性表达和ENVA-RabG狂犬病病毒的靶向选择性的高度创新组合，将使独立追踪局部抑制或兴奋细胞簇的连接成为可能。此外，一旦开发出来，它将被用来追踪抑制性和兴奋性神经元的远程连接 在CATV1中，相对于方位图；将通过本征信号光学成像得到的图。通过这种方式，可以第一次确定输入对新大脑皮层抑制神经元的功能偏好，并将其与输入对兴奋细胞群体的偏好进行比较。最后，虽然这项提议的目标是最终实施这项新技术来理解CAT V1的内在电路，但这项技术将适用于任何哺乳动物物种和新皮质的任何区域。 与公共卫生相关：这里建议开发的方法将允许对哺乳动物大脑中两种主要类型的神经元的输入进行选择性的神经解剖学追踪，即抑制性和兴奋性，而不是像目前的技术那样，不加区别地同时追踪这两种类型。该方法涉及许多高度创新的工具的开发和组合，包括重组病毒、细胞型特异性启动子、转基因和假型狂犬病病毒，以及内在信号光学成像。一旦开发出来，这项技术就可以用于新皮质的任何区域，首次确定抑制性和兴奋性神经元的神经回路是如何比较的，然后将这些回路与细胞类型功能的差异联系起来。

DESCRIPTION (provided by applicant): The neocortex is comprised of a dense population of excitatory neurons that are balanced by a diverse and more sparsely distributed array of inhibitory neurons. As such, cortical inhibitory neurons provide a gain control that works under a myriad of conditions. In primary visual cortex (V1), for example, inhibition is essential for severl basic functional characteristics of individual neurons, including preferences for stimulus contrast, size, and orientation. While inhibitory neurons serve to control excitation it is not knon whether inhibitory and excitatory cell-types are mediated through the same set of cortical circuits, nor whether the functional selectivity of these circuits differ. Determining the origins f the neural circuits to these two major cell-types will provide tremendous insight into the basic mechanisms of cortical processing. However, in order to do so it is necessary to develop a technique for cell-type specific neuroanatomical tracing. The proposal here is to develop such a technique by taking advantage of the neurotropism of recombinant viral vectors (adeno associated virus and lentivirus) and selectivity of cell-type specific promoters (GAD-67 and ¿-CamKII) to deliver and allow for expression of two key genes (TVA and RabG). These genes will thereby be selectively expressed in either inhibitory or excitatory neocortical cells, but not both. The TVA and RabG proteins expressed in these specific cell types will allow for targeted viral infection and trans-complementation of a novel genetically modified and pseudo-typed rabies virus (EnvA-¿RabG) that acts as a monosynaptic retrograde tracer (Wickersham, Lyon et al., 2007, Neuron). This highly innovative combination of cell-type specific expression of these genes and the targeted selectivity of the EnvA-¿RabG rabies virus will enable independent tracing of the connections of local clusters of inhibitory or excitatory cells. Moreover, once developed it will be used to trace the long-range connectivity of inhibitory and excitatory neurons in cat V1 with respect to the orientation map; maps that will be derived through intrinsic signal optical imaging. In this way, the functional preference of inputs to inhibitory neurons in neocortex can be determined for the first time and compared to the preference of inputs to excitatory cell populations. Finally, while the goal of this proposal is to ultimately implement th new technique to understand intrinsic circuits in cat V1, this technique will be available to other for use in any mammalian species and any region of neocortex. PUBLIC HEALTH RELEVANCE: The method proposed to be developed here will allow for selective neuroanatomical tracing of inputs to the two major classes of neurons in the mammalian brain, inhibitory and excitatory, rather than indiscriminant tracing of both types together, as current techniques do. This method involves the development and combination of a number of highly innovative tools including recombinant viruses, cell- type specific promoters, a genetically modified and pseudo-typed rabies virus, and intrinsic signal optical imaging. Once developed the technique can be used in any region of neocortex to determine, for the first time, how neural circuits of inhibitory and excitatory neurons compare and then relate these circuits to differences in cell type function.