Confocal laser scan system to image parallel processing of the brain neurons in behaving animals

我们开发了两个共聚焦激光系统来成像行为动物的神经元活动。第一个系统由一个建立在直立显微镜上的高速扫描仪组成。它可以每秒扫描500帧，但信噪比低，无法检测荧光信号的微弱变化。第二个系统是基于传统的共焦激光系统，但扫描速度提高到每秒收集10个全帧到500个线帧。该共焦系统具有较高的信噪比，以检测与突触输入相关的荧光变化，而无需对它们进行平均。利用该系统观察了斑马鱼幼鱼后脑网状脊髓神经元的活动。由于斑马鱼幼鱼的体和脑是透明的，因此可以通过完整脑内神经元的钙离子成像来观察活体神经元的活动。特别是我们成功地在体内成像的功能抑制网络的Mauthner细胞，这是已知的启动快速逃避反应行为的鱼。这是第一次报告的成像抑制性突触行动在完整的动物。最后，我们研制了一套用于观察行为动物神经元活动的光学系统。为此，在激光扫描系统的基础上增加了高速摄像系统，以同时测量斑马鱼神经元的荧光反应和行为反应。与运动检测器相关联的高速共聚焦激光扫描系统将帮助我们了解哪些中枢神经元以及如何控制动物的简单行为。

We developed two confocal laser systems to image neuronal activities of behaving animals. The first system consists of a high-speed scanner built on an upright microscope. It can scan 500 frames per second but has a low signal-to-noise ratio to detect faint change in fluorescence signals. The second system is based on a conventional confocal laser system, but the scanning speed was improved to collect 10 full-frames to 500 line-frames every second. This confocal system has a higher signal-to-noise ratio to detect fluorescence change associated with synaptic inputs without averaging them. Using the system we observed the activities of reticulospinal neurons in the hindbrain of zebrafish larvae. Since the body and brain of larval zebrafish are transparent, the neuronal activities in vivo can be observed by calcium imaging of the neurons in the intact brain. Especially we succeeded in in vivo imaging of functional inhibitory networks on the Mauthner cell which is known to initiate fast escape response behavior of fish. It is the first report of imaging inhibitory synaptic action in intact animals. Finally we developed the optical system to observe neuronal activities of behaving animals. For this purpose, the high-speed video camera system was added to the laser-scan system to measure the fluorescence responses of neurons and behavioral responses of zebrafish simultaneously. The high-speed confocal laser scanning system associated with motion detector will help us to understand which and how central neurons contribute to controlling the simple behavior of animals.