DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目的目标是直接测量确定的颜色对手神经节细胞的树突树上的突触相互作用，并测试的假设，颜色的一致性是由锥信号通路和神经节细胞树突之间的选择性连接。为了实现这一目标，我们构建了一个飞秒，双光子扫描激光显微镜测量光诱发的视网膜神经元树突钙信号。它是在海德堡马克斯普朗克的Denk和Detwiler最初开发的系统之后建模的，现在已经在完整的小鼠和蝾螈视网膜以及猕猴视网膜上进行了广泛的测试。与Peter Detwiler和他的学生合作，我们成功地将侏儒，阳伞和其他神经节细胞作为完整猕猴视网膜中的全细胞记录的目标，通过对神经节细胞体进行成像，并在机械去除内界膜后进行全细胞记录。侏儒和阳伞细胞在这种记录配置中显示出特征性和持久的光响应。然后通过记录移液管将细胞体和树突树细胞内加载钙绿色，并靶向树突分支以响应漫射光脉冲成像钙信号。正如先前在蝾螈和小鼠神经节细胞中发现的那样，猕猴神经节细胞树突在响应光阶时显示出钙浓度的明显和快速变化。最后，我们已经纳入了一个新的视觉刺激，使用数字光投影仪，这将允许应用程序的视锥细胞特定的视觉刺激在树突状记录。

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.