Functional Imaging of Ganglion Cells in the Living Mammalian Eye

描述（由申请人提供）：灵长类动物视网膜包含超过17类神经节细胞，但除了少数几类之外，所有这些神经节细胞对视觉的贡献都是未知的。这种理解上的巨大差距是由于大多数神经节细胞类型形成如此稀疏的马赛克，以至于难以用单个微电极或甚至微电极阵列来记录任何给定类别的足够细胞以表征其功能作用。微电极技术的另一个限制是记录过程是侵入性的，需要穿透地球仪，或者在眼杯制备的情况下，需要摘除眼睛。这排除了在相同细胞上重复实验的能力，并且限制了在已经获得电响应的相同动物上的行为实验。然而，在报告分子的发展，允许光学监测的电响应的单神经元与多光子荧光的快速进展。此外，最近发展的自适应光学校正眼睛的像差，现在有可能在生活的灵长类动物的眼睛，以约2微米的分辨率成像个别神经节细胞。我们将开发一种用于视网膜生理学的新技术，即活眼功能自适应光学细胞成像（FACILE），该技术将自适应光学体内成像与光学记录相结合，以同时绘制猴视网膜中数百个神经节细胞中每个细胞的电活动。我们将使用病毒转导将遗传编码的钙指示剂（GCaMP 3）插入神经节细胞，探索两种递送方法以进一步改善猕猴神经节细胞的病毒转导：与加州大学的约翰·弗兰纳里合作，Berkeley和逆行运输假型马贫血免疫缺陷病毒（EAIV）注射到视网膜，与索尔克研究所的艾德·卡拉威合作，FACILE的发展将加速从视网膜到大脑的许多通路的完整表征，并将揭示视网膜对视觉信息处理的全部贡献。我们将在小鼠模型中进行FACILE的早期开发，并在猴子视网膜中部署成熟的技术。在第4-5年，我们将通过解决长期存在的关于猕猴视网膜是否含有方向选择神经元的争论来证明这种方法的价值，例如在其他几种哺乳动物的视网膜中发现的方向选择神经元。 公共卫生相关性：灵长类动物的视网膜包含超过17类神经节细胞，但除了少数几类之外，所有这些类别对视觉的贡献都是未知的，这是现有生理学方法用于理解新细胞类型的弱点的结果。该项目将开发一种用于视网膜生理学的新技术，即活眼中的功能性自适应光学细胞成像（FACILE），该技术将自适应光学体内成像与光学记录相结合，以同时绘制猴视网膜中数百个神经节细胞中每个细胞的电活动。新的方法将被用来检查的可能性，其中未知的神经节细胞类是定向选择性神经节细胞，在其他哺乳动物视网膜。这种方法将加速我们的分析的全部贡献的许多途径，从视网膜到大脑在灵长类动物的视觉信息处理。

DESCRIPTION (provided by applicant): The primate retina contains more than 17 classes of ganglion cells, but the contribution to vision of all but a few of these classes is unknown. This large gap in understanding is due to the fact that most ganglion cell types form such sparse mosaics that it is difficult with a single microelectrode or even an array of microelectrodes to record from enough cells of any given class to characterize its functional role. Another limitation of microelectrode technology is that the recording process is invasive, requiring penetration of the globe or, in the case of an eyecup preparation, enucleation of the eye. This precludes the ability to repeat experiments on the same cells and limits behavioral experiments on the same animals in which electrical responses have been obtained. However, rapid advances are being made in the development of reporter molecules that allow optical monitoring of the electrical responses of single neurons with multiphoton fluorescence. Moreover, the recent development of adaptive optics for correcting the eye's aberrations now makes it possible to image individual ganglion cells at ~ 2 micron resolution in the living primate eye. We will develop a new technology for retinal physiology, Functional Adaptive-optics Cellular Imaging in the Living Eye (FACILE) that combines adaptive optics in vivo imaging with optical recording to map the electrical activity of each of the several hundred ganglion cells simultaneously in a patch of monkey retina. We will use viral transduction to insert a genetically encoded calcium indicator (GCaMP3) into ganglion cells, exploring two delivery methods to further improve viral transduction of macaque ganglion cells: intravitreal injection of adeno- associated virus (AAV) in collaboration with John Flannery at UC, Berkeley and retrograde transport of pseudotyped equine anemia immunodeficiency virus (EAIV) injected into retino-recipient nuclei in collaboration with Ed Callaway at the Salk Institute. The development of FACILE will accelerate the complete characterization of the many pathways from the retina to the brain and will reveal the full contribution the retina makes to visual information processing. We will undertake early development of FACILE in a mouse model, and deploy the mature technology in monkey retina. In years 4-5, we will demonstrate the value of the approach by resolving the long-standing debate about whether the macaque retina contains direction-selective neurons, such as those that have been identified in the retinas of several other mammals. PUBLIC HEALTH RELEVANCE: The primate retina contains more than 17 classes of ganglion cells, but the contribution to vision of all but a few of these classes is unknown, a consequence of the weakness of existing physiological methodology for understanding novel cell types. This project will develop a new technology for retinal physiology, Functional Adaptive-optics Cellular Imaging in the Living Eye (FACILE) that combines adaptive optics in-vivo imaging with optical recording to map the electrical activity of each of the several hundred ganglion cells simultaneously in a patch of monkey retina. The novel approach will be used to examine the possibility that among the unknown ganglion cell classes are directionally selective ganglion cells, as in other mammalian retinas. This methodology will accelerate our analysis of the full contribution of the many pathways from retina to brain in primate visual information processing.