DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目的目标是直接测量已识别的颜色对手神经节细胞树突触上的相互作用，并验证颜色对抗性是由锥体信号通路和神经节细胞树突之间的选择性连接决定的假说。为了实现这一目标，我们构建了一台飞秒双光子扫描激光显微镜，用于测量视网膜神经元树突中的光诱发钙信号。它是以海德堡Max Planck的Denk和Detwiler最初开发的系统为模型的，现在已经在完整的小鼠和火蜥蜴视网膜以及猕猴视网膜上进行了广泛的测试。我们与Peter Detwiler和他的学生合作，通过成像神经节细胞体并在机械去除内界膜后进行全细胞记录，成功地将侏儒、阳伞和其他神经节细胞靶向完整的猕猴视网膜进行全细胞记录。在这种记录配置中，侏儒和遮阳伞细胞表现出特征和持久的光响应。然后，通过记录吸管在细胞体和树突树内装载钙绿，并以树突分支为靶点，对漫反射光脉冲进行钙信号成像。正如以前在火蜥蜴和小鼠神经节细胞中发现的那样，猕猴神经节细胞树突在光刺激下表现出明显而快速的钙浓度变化。最后，我们加入了一种新的视觉刺激，使用了数字投影仪，这将允许在树突记录期间应用视锥特定的视觉刺激。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to directly measure synaptic interactions on the dendritic trees of identified color opponent ganglion cells and to test the hypothesis that color opponency is determined by selective connections between cone-signal pathways and ganglion-cell dendrites. To achieve this aim we constructed a femtosecond, 2-photon scanning laser microscope for measuring light evoked calcium signals in the dendrites of retinal neurons. It has been modeled after the system originally developed by Denk and Detwiler at the Max Planck in Heidelberg and has now been extensively tested on intact mouse and salamander retina as well as macaque retina. In collaboration with Peter Detwiler and his students, we have successfully targeted both midget, parasol and other ganglion cells for whole cell recording in the intact macaque retina by imaging ganglion cell bodies and making whole-cell recordings after mechanical removal of the inner limiting membrane. Midget and parasol cells show characteristic and long lasting light responses in this recording configuration. The cell bodies and dendritic trees are then intracellularly loaded with Calcium Green via the recording pipette and dendritic branches are targeted for imaging calcium signals in response to diffuse light pulses. As found previously for salamander and mouse ganglion cells, macaque ganglion cell dendrites show clear and rapid changes in calcium concentration in response to light steps. Finally, we have incorporated a new visual stimulus, using a digital light projector, which will permit the application of cone-specific visual stimuli during dendritic recording.