DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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，我们在2001年获得了NIH共享仪器拨款，获得了一台飞秒、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。在过去的两年里，这个设施是定制的，仿照海德堡Max Planck的Denk和Detwiler最初开发的系统，现在已经在完整的小鼠和火蜥蜴视网膜上进行了广泛的测试，最近在猕猴视网膜上进行了测试。我们与Peter Detwiler和他的学生合作，通过对神经节细胞体成像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。在这种记录结构中，侏儒和遮阳伞细胞表现出特征和持久的光反应。然后，通过记录吸管在细胞体和树突树内装载钙绿，并以树突分支为目标，对漫反射光脉冲做出反应，对钙信号进行成像。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 were awarded an NIH shared instrumentation grant in 2001 to acquire a femtosecond, 2-photon scanning laser microscope for measuring light evoked calcium signals in the dendrites of retinal neurons. Over the last two years this facility was custom built, 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 and most recently on 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 showed characteristic and long lasting light responses in this recording configuration. The cell bodies and dendritic trees were then intracellularly loaded with Calcium Green via the recording pipette and dendritic branches were 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.