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

我们开发了两种共聚焦激光系统来成像行为动物的神经元活动。第一个系统由建立在直立显微镜上的高速扫描仪组成。它可以扫描500帧每秒，但具有较低的信噪比，以检测荧光信号的微弱变化。第二种系统基于传统的共聚焦激光系统，但扫描速度提高到每秒收集10个全帧到500个线帧。该共聚焦系统具有较高的信噪比，可以检测与突触输入相关的荧光变化，而无需平均它们。利用该系统对斑马鱼幼体后脑网状脊髓神经元的活动进行了观察。由于斑马鱼幼虫的身体和大脑是透明的，因此可以通过对完整大脑神经元的钙成像来观察神经元在体内的活动。特别是我们成功地在毛特纳细胞上的功能抑制网络的体内成像，这是已知的启动鱼类快速逃逸反应行为的细胞。这是首次报道在完整动物中成像抑制突触作用。最后，我们开发了光学系统来观察行为动物的神经元活动。为此，在激光扫描系统中加入高速摄像机系统，同时测量斑马鱼神经元的荧光反应和行为反应。与运动检测器相结合的高速共聚焦激光扫描系统将帮助我们了解中枢神经元是如何控制动物的简单行为的。

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.