Simultaneous two-photon imaging and two-photon optogenetics of cortical circuits in three dimensions.
Simultaneous two-photon imaging and two-photon optogenetics of cortical circuits in three dimensions.
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DOI：
10.7554/elife.32671
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发表时间：
2018-02-07
期刊：
影响因子：
7.7
通讯作者：
Yuste R
中科院分区：
文献类型：
--
作者：
Yang W;Carrillo-Reid L;Bando Y;Peterka DS;Yuste R
同时成像和操纵神经活动可以使神经回路的功能解剖。在这里，我们结合了双光子光遗传学与同步体积双光子钙成像，以细胞分辨率在三维（3D）中测量和操纵小鼠新皮层中的神经活动。利用混合全息方法，我们同时光刺激小鼠视觉皮层2/3层中超过150 μ m深度的80个神经元，同时成像周围神经元的活动。我们通过在3D选定的中间神经元组中进行光激活，抑制清醒动物中附近锥体神经元对视觉刺激的反应，验证了该方法的有用性。我们的全光学方法可以用作读取和写入神经元活动的通用平台。现代显微镜为观察大脑提供了一个窗口。第一台光学显微镜只能放大组织薄片中的细胞。相比之下，今天的光学显微镜可以对活体动物大脑表面以下的细胞进行成像。即便如此，这仍然具有挑战性，原因有几个。一个是大脑是三维的。另一个原因是大脑组织会散射光线。试图观察大脑深处的神经元有点像试图通过一杯牛奶来观察它们。大多数光在其穿过组织的途中散射，结果是很少的光到达目标神经元。Yang等人现在使用一种称为全息术的技术来解决这些挑战。全息术通过分裂光束然后以特定的方式重新组合光束来产生物体的3D图像。Yang等人将这项技术应用于红外激光束，选择红外线，因为它在脑组织中的散射比可见光少得多。将每一束红外线射到不同的神经元上，可以同时产生大脑外层（皮层）内多个细胞的3D图像。全息红外显微镜可以与两种称为光遗传学和钙成像的技术一起使用，其中光敏蛋白质被插入神经元。根据引入的蛋白质，照射到神经元上的光线要么改变它们的活性，要么使它们在活跃时发出荧光。就像计算机可以“读”和“写”数据一样，全息显微镜可以读出现有的神经元活动或写入新的活动模式。通过结合这些技术，Yang等人能够同时刺激80多个神经元-同时可视化周围神经元的活动-在小鼠皮层的多个深度。这种新的显微镜技术虽然比现有方法有了明显的进步，但仍然无法对整个大脑皮层的神经元进行成像和控制。下一个目标是进一步将这种方法扩展到多个大脑区域，并随意操纵任何神经元子集的活动。神经科学家将从3D成像和改变活神经回路活动的能力中受益匪浅。在未来，临床医生可能能够使用这种技术来治疗大脑疾病，通过调整异常神经回路的活动。
The simultaneous imaging and manipulating of neural activity could enable the functional dissection of neural circuits. Here we have combined two-photon optogenetics with simultaneous volumetric two-photon calcium imaging to measure and manipulate neural activity in mouse neocortex in vivo in three-dimensions (3D) with cellular resolution. Using a hybrid holographic approach, we simultaneously photostimulate more than 80 neurons over 150 μm in depth in layer 2/3 of the mouse visual cortex, while simultaneously imaging the activity of the surrounding neurons. We validate the usefulness of the method by photoactivating in 3D selected groups of interneurons, suppressing the response of nearby pyramidal neurons to visual stimuli in awake animals. Our all-optical approach could be used as a general platform to read and write neuronal activity. Modern microscopy provides a window into the brain. The first light microscopes were able to magnify cells only in thin slices of tissue. By contrast, today’s light microscopes can image cells below the surface of the brain of a living animal. Even so, this remains challenging for several reasons. One is that the brain is three-dimensional. Another is that brain tissue scatters light. Trying to view neurons deep within the brain is a little like trying to view them through a glass of milk. Most of the light scatters on its way through the tissue with the result that little of the light reaches the target neurons. Yang et al. have now tackled these challenges using a technique called holography. Holography produces 3D images of objects by splitting a beam of light and then recombining the beams in a specific way. Yang et al. applied this technique to an infrared laser beam, opting for infrared because it scatters much less in brain tissue than visible light. Directing each of the infrared beams to a different neuron can produce 3D images of multiple cells within the brain’s outer layer, the cortex, all at the same time. The holographic infrared microscope can be used alongside two techniques called optogenetics and calcium imaging, in which light-sensitive proteins are inserted into neurons. Depending on the proteins introduced, shining light onto the neurons will either change their activity, or cause them to fluoresce whenever they are active. Just as a computer can both “read” and “write” data, the holographic microscope can thus read out existing neuronal activity or write new patterns of activity. By combining these techniques, Yang et al. were able to stimulate more than 80 neurons at the same time – and meanwhile visualize the activity of the surrounding neurons – at multiple depths within the mouse cortex. This new microscopy technique, while a clear advance over existing methods, still cannot image and control neurons throughout the entire cortex. The next goal is to further extend this method across multiple brain areas and manipulate the activity of any subset of neurons at will. Neuroscientists will greatly benefit from the ability to image and alter the activity of living neural circuits in 3D. In the future, clinicians may be able to use this technique to treat brain disorders by adjusting the activity of abnormal neural circuits.