Development of an imaging system for in vivo real-time monitoring of neuronal activity in deep brain of free-moving rats.
Development of an imaging system for in vivo real-time monitoring of neuronal activity in deep brain of free-moving rats.
复制标题
开发用于体内实时监测自由移动大鼠脑深部神经元活动的成像系统。
DOI：
10.1007/s00418-017-1576-2
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Ozawa H.
中科院分区：
文献类型：
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作者：
Iijima N;Miyamoto S;Matsumoto K;Takumi K;Ueta Y;Ozawa H.
我们新开发了一种系统，该系统允许通过光纤监测转基因修饰以表达增强型绿色荧光蛋白（eGFP）的大鼠脑深部的荧光信号强度。光纤的一端连接到蓝色半导体激光振荡器/绿色荧光检测器。另一个末端插入到表达eGFP的神经元附近。由于光纤容易受到动物运动引起的扭曲应力的影响，我们还开发了一种笼子，其中地板会自动转动，以响应大鼠头部的转动。这减轻了光纤上的扭转应力。然后，该系统能够在数小时内实时监测清醒和不受约束的大鼠的荧光。利用该系统，我们可以连续监测eGFP在精氨酸加压素-eGFP转基因大鼠中的表达。此外，我们观察到盐负荷条件下室旁核中eGFP表达的增加。然后，我们通过由3000根细光纤组成的束对下丘脑中表达eGFP的GnRH神经元进行体内成像。通过将光纤束连接到荧光显微镜和特殊的笼系统的组合，我们能够捕获并保留自由移动大鼠的eGFP表达神经元的图像。我们相信，我们新开发的用于监测和成像脑深部神经元中eGFP表达的方法将有助于长期分析清醒和不受限制的动物的神经元功能。
We have newly developed a system that allows monitoring of the intensity of fluorescent signals from deep brains of rats transgenically modified to express enhanced green fluorescent protein (eGFP) via an optical fiber. One terminal of the optical fiber was connected to a blue semiconductor laser oscillator/green fluorescence detector. The other terminal was inserted into the vicinity of the eGFP-expressing neurons. Since the optical fiber was vulnerable to twisting stresses caused by animal movement, we also developed a cage in which the floor automatically turns, in response to the turning of the rat’s head. This relieved the twisting stress on the optical fiber. The system then enabled real-time monitoring of fluorescence in awake and unrestrained rats over many hours. Using this system, we could continuously monitor eGFP-expression in arginine vasopressin-eGFP transgenic rats. Moreover, we observed an increase of eGFP-expression in the paraventricular nucleus under salt-loading conditions. We then performed in vivo imaging of eGFP-expressing GnRH neurons in the hypothalamus, via a bundle consisting of 3000 thin optical fibers. With the combination of the optical fiber bundle connection to the fluorescence microscope, and the special cage system, we were able to capture and retain images of eGFP-expressing neurons from free-moving rats. We believe that our newly developed method for monitoring and imaging eGFP-expression in deep brain neurons will be useful for analysis of neuronal functions in awake and unrestrained animals for long durations.