Functional Imaging of Ganglion Cells in the Living Mammalian Eye

活体哺乳动物眼中神经节细胞的功能成像

• 批准号: 8212083
• 负责人: William H Merigan
• 金额: $ 62.49万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212083
• 关键词: Address; Anemia; Animals; Behavioral; Brain; Cell Nucleus; Cells; Collaborations; Dependovirus; Development; Electrophysiology (science); Equus caballus; Eye; Eye Development; Eye Enucleation; Fluorescence; Functional Imaging; Gene Delivery; Grant; Image; Immunologic Deficiency Syndromes; In Vitro; Individual; Injection of therapeutic agent; Institutes; Life; Macaca; Mammals; Maps; Methodology; Methods; Microelectrodes; Monitor; Monkeys; Motion; Mus; Neurons; Optics; Pathway interactions; Penetration; Physiological; Physiology; Preparation; Primates; Process; Reporter; Reporting; Resolution; Retina; Retinal; Retinal Ganglion Cells; Rodent; Role; Staging; Stimulus; Technology; Testing; Viral; Virus; Vision; Visual; adaptive optics; base; calcium indicator; cell type; cellular imaging; cellular transduction; density; ganglion cell; improved; in vivo; information processing; intravitreal injection; mouse model; new technology; novel; novel strategies; public health relevance; research study; response; retrograde transport; transduction efficiency; visual information

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.

描述（由申请人提供）：灵长类动物视网膜含有超过 17 类神经节细胞，但除了其中少数几类之外，所有神经节细胞对视力的贡献尚不清楚。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，需要穿透眼球，或者在眼杯准备的情况下，需要摘除眼睛。这排除了在相同细胞上重复实验的能力，并限制了在已获得电反应的相同动物上进行行为实验。然而，报告分子的开发正在迅速取得进展，这些报告分子可以通过多光子荧光对单个神经元的电反应进行光学监测。此外，最近开发的用于校正眼睛像差的自适应光学器件现在可以以约 2 微米的分辨率对活体灵长类动物眼睛中的单个神经节细胞进行成像。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，以同时绘制贴片中数百个神经节细胞中每个神经节细胞的电活动图的猴子视网膜。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（John Flannery）合作，玻璃体内注射腺相关病毒（AAV）伯克利分校和与 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索尔克研究所。 FACILE 的开发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，并将成熟的技术部署在猴子视网膜中。在第 4-5 年，我们将通过解决关于猕猴视网膜是否包含方向选择性神经元（例如在其他几种哺乳动物的视网膜中已发现的那些）的长期争论来证明该方法的价值。 公共健康相关性：灵长类动物视网膜含有超过 17 类神经节细胞，但除了其中少数几类神经节细胞外，所有神经节细胞对视力的贡献尚不清楚，这是由于现有的生理学方法在理解新细胞类型方面的弱点所致。该项目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，以同时绘制数百个神经节细胞中每个神经节细胞的电活动图。一块猴子视网膜这种新方法将用于检查未知神经节细胞类别中是否存在定向选择性神经节细胞，就像其他哺乳动物视网膜中一样。这种方法将加速我们对灵长类视觉信息处理中从视网膜到大脑的许多通路的全面贡献的分析。