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，但除了少数类别外，所有这些类别对视力的贡献是未知的。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，以至于很难用单个微电极甚至微电极阵列从任何给定类别的足够多的细胞中记录以表征其功能作用。微电极技术的另一个限制是记录过程是侵入性的，需要穿透眼球，或者在准备眼杯的情况下，需要剜出眼球。这就排除了在相同细胞上重复实验的能力，并限制了在同一动物上进行的已获得电反应的行为实验。然而，报告分子的开发正在取得快速进展，这些报告分子允许用多光子荧光对单个神经元的电反应进行光学监测。此外，最近用于校正眼睛像差的自适应光学技术的发展，现在可以在活体灵长类动物的眼睛中以~ 2微米的分辨率对单个神经节细胞进行成像。我们将开发一项视网膜生理学的新技术，即活体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，以同时绘制猴子视网膜斑块中数百个神经节细胞中的每个细胞的电活动。我们将利用病毒转导将基因编码的钙指示剂（GCaMP3）插入神经节细胞，探索两种传递方法来进一步改善猕猴神经节细胞的病毒转导：与加州大学伯克利分校的John Flannery合作，在玻璃体内注射腺相关病毒（AAV）；与Salk研究所的Ed Callaway合作，将假型马贫血免疫缺陷病毒（EAIV）逆行运输注射到视网膜受体细胞核中。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。我们将在小鼠模型上进行FACILE的早期开发，并将成熟的技术应用于猴视网膜。在第4-5年，我们将通过解决长期以来关于猕猴视网膜是否包含方向选择神经元的争论来证明该方法的价值，例如那些在其他几种哺乳动物的视网膜中已被确定的神经元。

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.