Nguyen Jeffrey P, Shipley Frederick B, Linder Ashley N, Plummer George S, Liu Mochi, Setru Sagar U, Shaevitz Joshua W, Leifer Andrew M
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; Department of Physics, Princeton University, Princeton, NJ 08544;
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544;
Proc Natl Acad Sci U S A. 2016 Feb 23;113(8):E1074-81. doi: 10.1073/pnas.1507110112. Epub 2015 Dec 28.
The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal's position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal's position and orientation. Custom software tracks the 3D position of the animal's head in real time and two feedback loops adjust a motorized stage and objective to keep the animal's head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal's behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.
为了深入了解神经元群体如何产生动物行为，需要具备在清醒且不受束缚的动物身上获取大规模神经元活动记录的能力。我们展示了一种仪器，它能够以细胞分辨率记录自由活动的秀丽隐杆线虫头部大多数神经元的细胞内钙瞬变，同时记录动物的位置、姿势和运动。该仪器在不受束缚且活动的动物身上提供具有细胞分辨率的全脑成像。我们使用转盘共聚焦显微镜以每秒6个头部体积的速度捕获表达钙指示剂GCaMP6s的神经元的3D体积荧光图像。一套三个摄像头监测神经元荧光以及动物的位置和方向。定制软件实时跟踪动物头部的3D位置，两个反馈回路调整电动载物台和物镜，以便在动物自由活动时将其头部保持在视野范围内。我们观察到多达77个神经元的钙瞬变超过4分钟，并将这种活动与动物的行为相关联。我们对由于动物运动导致的系统噪声进行了表征，并表明在不同线虫中，多个神经元与对应于向前、向后和转弯运动的行为模式存在显著相关性。
