Department of Neurobiology, Harvard Medical School Boston, MA, USA.
Front Cell Neurosci. 2010 Mar 12;4:3. doi: 10.3389/fncel.2010.00003. eCollection 2010.
Nearby neurons in mammalian neocortex demonstrate a great diversity of cell types and connectivity patterns. The importance of this diversity for computation is not understood. While extracellular recording studies in visual cortex have provided a particularly rich description of behavioral modulation of neural activity, new methods are needed to dissect the contribution of specific circuit elements in guiding visual perception. Here, we describe a method for three-dimensional cellular imaging of neural activity in the awake mouse visual cortex during active discrimination and passive viewing of visual stimuli. Head-fixed mice demonstrated robust discrimination for many hundred trials per day after initial task acquisition. To record from multiple neurons during operant behavior with single-trial resolution and minimal artifacts, we built a sensitive microscope for two-photon calcium imaging, capable of rapid tracking of neurons in three dimensions. We demonstrate stable recordings of cellular calcium activity during discrimination behavior across hours, days, and weeks, using both synthetic and genetically encoded calcium indicators. When combined with molecular and genetic technologies in mice (e.g., cell-type specific transgenic labeling), this approach allows the identification of neuronal classes in vivo. Physiological measurements from distinct classes of neighboring neurons will enrich our understanding of the coordinated roles of diverse elements of cortical microcircuits in guiding sensory perception and perceptual learning. Further, our method provides a high-throughput, chronic in vivo assay of behavioral influences on cellular activity that is applicable to a wide range of mouse models of neurologic disease.
哺乳动物新皮层中的邻近神经元表现出极大的细胞类型和连接模式多样性。这种多样性对计算的重要性尚不清楚。虽然视觉皮层的细胞外记录研究为神经活动的行为调节提供了特别丰富的描述，但需要新的方法来剖析特定电路元件在指导视觉感知中的作用。在这里，我们描述了一种在清醒小鼠视觉皮层中进行三维细胞水平神经活动成像的方法，用于主动辨别和被动观看视觉刺激。在初始任务获取后，头部固定的小鼠每天可进行数百次的稳健辨别。为了在操作行为中以单试分辨力和最小伪影记录多个神经元，我们构建了一个用于双光子钙成像的灵敏显微镜，能够快速在三维中跟踪神经元。我们使用合成和遗传编码钙指示剂，在数小时、数天和数周的辨别行为中，均实现了细胞钙活性的稳定记录。当与小鼠中的分子和遗传技术（例如，细胞类型特异性转基因标记）结合使用时，这种方法允许在体内识别神经元类群。来自相邻神经元的不同类群的生理测量结果将丰富我们对皮质微电路中不同元素在指导感觉感知和感知学习中的协调作用的理解。此外，我们的方法提供了一种高通量、慢性体内测定行为对细胞活性影响的方法，适用于广泛的神经疾病小鼠模型。
