Cell Communication in Retina

描述（由申请人提供）：本研究的总体目标是了解通过哺乳动物视网膜的内丛状层（IPL）处理信息的神经元电路。由于我们对锥体双极细胞连接的了解不够，对兔神经节细胞突触学的了解也不够，所以实验设计的重点是特定的锥体双极细胞回路，它们从锥体蒂接收的兴奋输入，以及它们向神经节细胞树突施加的兴奋输入。因此，我们的网络分析涉及利用视网膜的细胞结构组织。这是了解视网膜功能的关键，通过检查识别的锥体双极细胞轴突终端的连接，激发开-关定向选择（DS）和关闭α神经节细胞。我们描述了一个横向前馈抑制微电路涉及两个不同的双极细胞；一个双极细胞的二极体直接刺激DS细胞，而另一个双极体的二极体则刺激一个星爆无突细胞，该细胞前馈与DS细胞发生突触。免疫标记和细胞内染色结合共聚焦显微镜，用于鉴定与DS细胞突触的双极细胞，以便聚焦电镜（EM）分析。双极细胞鉴定后，这些细胞内的轴突终末！用电镜连续重建染色细胞，观察其突触结构；两个对比的电子密度标记，前嵌入金取代银强化过氧化物酶和DAB/过氧化物酶，用于建立两个细胞之间的突触前和突触后相互作用。结合微波和化学固定和/或速冻，结合各种包埋前和包埋后的EM免疫标记技术，包括DAB/过氧化物酶和免疫金，鉴定GABA和胆碱能突触，并确定这些受体如何被星爆腺分泌细胞用于与DS细胞和彼此之间的通信。全细胞记录用于表征off -双极细胞树突上与off - α神经节细胞突触的嗜离子性谷氨酸受体，并确定双极细胞是否主要使用相同的谷氨酸受体与off - α神经节细胞突触。一旦功能性谷氨酸受体被确定，双重免疫标记被用来确认它们在神经节细胞树突上的存在，神经节细胞在那里接受来自双突触的兴奋。

DESCRIPTION (provided by applicant): The overall objective of this research is to understand the neuronal circuitry that processes information through the inner plexiform layer (IPL) of the mammalian retina. Because our knowledge of cone bipolar cell connectivity is insufficient, as is the knowledge of rabbit ganglion cell synaptology, the experimental design is focused on specific cone bipolar cell circuits, the excitatory input they receive from cone pedicles and the excitatory input they exert onto ganglion cell dendrites Therefore, our network analysis involves exploiting the cytoarchitectural organization of the retina, which is the keystone to understanding retinal function by examining the connectivity of identified cone bipolar cell axon terminals that excite ON-OFF directional selective (DS) and OFF-alpha ganglion cells. We have described a lateral feed-forward inhibitory microcircuit involving two different bipolar cells; the dyad of one bipolar cell directly excites the DS cell, while the dyad of the other excites a starburst amacrine cell which feeds forward to synapse with the DS cell. lmmunolabeling and intracellular staining, in combination with confocal microscopy, are used to identify bipolar cells synapsing with the DS cell in order to focus the electron microscopy (EM) analysis. After the bipolar cells are identified, the axon terminals of these intracellular!y stained cells are serially reconstructed with EM to examine their synaptology; two constrasting electron dense markers, pro embedding gold-substituted silver intensified peroxidase and DAB/peroxidase, are used to establish pro- and postsynaptic interactions between two cells. Combined microwave and chemical fixation and/or rapid-freezing are used with various pro- and postembedding EM immunolabeling techniques involving DAB/peroxidase and immunogold to identify the GABA and cholinergic synapses and determine how these receptors are used by the starburst amacrine cell to communicate with the DS cell and each other. Whole cell recordings are used to characterize the ionotropic glutamate receptors on the dendrites of OFF-bipolar cell(s) synapsing with OFF-alpha ganglion cells and to determine whether the bipolar cell(s) primarily uses the same glutamate receptor to synapse with the OFF-ganglion cell. Once the functional glutamate receptors are characterized, double immunolabeling is used to confirm their presence on the dendrites of the ganglion cell where it receives excitation from dyad synapses.