Synaptic Inputs to Retinal Ganglion Cells

描述(申请人提供)：哺乳动物视网膜的神经节细胞大约有十几种类型，每种类型都将不同的视觉场景编码传输到大脑。这项研究的长期目标是了解特定类型的神经节细胞实现这些停滞的机制。我们开发了一种界面孵化系统，允许在一个无监督的、类似培养的系统中对成年兔视网膜的多个样本进行数天的维护。编码RNAi或标记突触蛋白的基因被生物地导入单个视网膜神经节细胞。 有两个问题涉及某些视网膜神经节细胞中潜在的方向选择性的突触事件。首先，我们建议使用RNAi来抑制单个神经节细胞中的GABA能或胆碱能反应性。记录将揭示这些直接(突触后)输入对方向选择性的贡献。其次，我们将研究星状突起的无长突细胞共同释放GABA和Ach的情况。这两种神经递质是从相同的细胞部位释放的，还是从不同的细胞部位释放的？这将通过定位它们的囊泡运输蛋白来研究。 第三个问题是一个更一般的问题。我们最近的实验表明，神经节细胞树突的兴奋性输入在空间上的分布符合均匀间隔定律：突触似乎相互排斥。此外，它们还系统地避开树枝上的分支点。我们现在打算看看这些规则是否适用于所有类型的神经节细胞；模拟它们的生理后果；以及看看同样的规则是否适用于抑制性突触。 这里介绍的方法可以用于任何物种的成年动物。它们消除了对转基因动物的需要，增加了可以同时研究的样本数量，并允许标记具有较长半衰期的蛋白质。它们可以用于正常组织或疾病模型。我们希望它们将对研究基础和临床问题的实验室有所帮助。

DESCRIPTION (provided by applicant): The ganglion cells of mammalian retinas exist in roughly a dozen types, each transmitting a different encoding of the visual scene to the brain. The long-term goal of this research is to learn the mechanism by which specific types of ganglion cells achieve these codlings. We have developed an interface incubation system that allows maintenance of multiple samples of adult rabbit retinas for several days in an unsupervised, culture-like system. Genes coding for RNAi or tagged synapse proteins are biolistically transfected into individual retinal ganglion cells. Two questions involve the synaptic events underlying directional selectivity in certain retinal ganglion cells. First, we propose to use RNAi to knock down GABAergic or cholinergic responsiveness in individual ganglion cells. Recording will reveal the contribution of these direct (postsynaptic) inputs to direction selectivity. Second, we will investigate the co-release of GABA and Ach by the starburst amacrine cells. Are the two neurotransmitters released from the same cellular sites or different ones? This will be studied by localizing their vesicular transport proteins. The third question is a more general one. Our recent experiments suggest that the excitatory inputs to ganglion cell dendrites are spatially distributed according to an even-spacing law: the synapses seem to repel each other. In addition, they systematically avoid branch points in the dendritic arbor. We now propose to see if these rules apply to all types of ganglion cells; to model their physiological consequences; and to see if the same rules apply to inhibitory synapses. The methods introduced here can be used in adult animals of any species. They obviate the need for transgenic animals, increase the number of samples that can be simultaneously studied, and allow labeling of proteins that have long half-lives. They can be used in normal tissue or in disease models. We hope that they will be useful to laboratories studying both basic and clinical problems.