DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该项目的目标是直接测量已识别的颜色对抗神经节细胞树突状树上的突触相互作用，并测试颜色对抗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设。为了实现这一目标，我们于2001年获得了美国国立卫生研究院共享仪器拨款，以获得飞秒，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，这个设备是根据Denk和Detwiler在海德堡的马克斯普朗克研究所最初开发的系统定制的，现在已经在完整的老鼠和蝾螈视网膜上进行了广泛的测试，最近在猕猴视网膜上进行了广泛的测试。在与Peter Detwiler和他的学生的合作下，我们通过对神经节细胞体进行成像，并在机械去除内限制膜后进行全细胞记录，成功地针对了猕猴视网膜中的小细胞、阳伞细胞和其他神经节细胞进行全细胞记录。在这种记录结构中，小细胞和伞细胞表现出特有的、持久的光响应。然后通过记录移液管将细胞体和树突状树在细胞内装载钙绿，树突状树枝被定位成像钙信号以响应漫射光脉冲。正如之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轻步反应中显示出清晰而快速的钙浓度变化。最后，我们结合了一种新的视觉刺激，使用数字光投影仪，这将允许在树突记录期间应用特定于锥体的视觉刺激。

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.