Revealing neural circuits underlying zebrafish behavior using mesoscopic light field microscopy

摘要 了解神经回路如何驱动行为的关键一步是能够记录所有神经回路的活动。 当生物体以不受约束的方式与其环境相互作用时，神经元在生物体中的活动。一个有前途 在这方面，脊椎动物模型系统是斑马鱼幼虫，其执行复杂的视觉驱动行为 例如从早期开始狩猎，由于其透明性，可以在单一时间进行大规模神经成像， 神经元分辨率然而，到目前为止，大多数对斑马鱼狩猎行为的神经基础的研究都 使用拴着的鱼，无法自由移动。虽然一些新的分析方法最近开始克服 这一限制，这些仍然显着扰乱鱼类的自然狩猎行为，需要一个非常 浅水深度，并迅速取消横向运动，以保持鱼的领域内， 风景在这里，我们将开发一种新的“MesoLFM”成像技术，克服这两个限制， 允许真正不受约束的狩猎行为的神经基础进行调查的第一次， 斑马鱼我们将通过结合光场显微镜（LFM）、电可调透镜（ETL） 和MesoLens，一个巨大的显微镜物镜与4x/0.47 NA物镜，10毫米的工作距离和 5毫米视野。LFM允许3D体积的单次拍摄捕获，从而能够在许多Hz下成像。所述ETL 不需要在z方向上快速移动载物台或物镜，从而能够在相对较深的水中成像。的 MesoLens允许在单个视场内捕获长序列的运动， 而不需要快速的XY运动消除。首先，我们将进行光学模拟， MesoLFM，以便确定诸如微透镜阵列等组件的最佳参数。第二、 我们将根据这些规范构建MesoLFM，并使用以下测量值对其进行校准 荧光珠和标准分辨率靶。第三，我们将直接演示MesoLFM成像， 斑马鱼的大脑这项工作将建立一个新的平台，用于研究神经基础， 无拘无束的行为在未来的R 01应用程序中，我们将使用MesoLFM来揭示神经回路如何 推动自然的目标导向行为和决策。

ABSTRACT A critical step towards understanding how neural circuits drive behavior is the ability to record the activity of all neurons in an organism while it interacts with its environment in an unconstrained manner. A promising vertebrate model system in this regard is the zebrafish larva, which performs complex visually-driven behaviors such as hunting from an early age, and due to its transparency allows large-scale neural imaging at single- neuron resolution. However so far most investigations into the neural basis of zebrafish hunting behavior have used tethered fish which are unable to move freely. While some new assays have recently begun to overcome this limitation, these still significantly perturb the fish’s natural hunting behavior by requiring both an extremely shallow water depth, and rapid cancellation of lateral movement in order to maintain the fish within the field of view. Here we will develop a new `MesoLFM’ imaging technology overcoming both of these limitations, thus permitting the neural basis of truly unconstrained hunting behavior to be investigated for the first time in zebrafish. We will achieve this by combining light field microscopy (LFM), an electrically tunable lens (ETL), and the MesoLens, a giant microscope objective with a 4x/0.47 NA objective, a 10 mm working distance and a 5 mm field of view. LFM allows single-shot capture of 3D volumes, enabling imaging at many Hz. The ETL removes the need for rapid stage or objective movements in z, enabling imaging in relatively deep water. The MesoLens allows long sequences of movements to be captured within a single field of view, enabling imaging of hunting without the need for rapid xy motion cancellation. First, we will perform optical simulations of MesoLFM in order to determine the optimal parameters for components such as the microlens array. Second, we will construct the MesoLFM according to these specifications, and calibrate it using measurements of fluorescent beads and a standard resolution target. Third, we will directly demonstrate MesoLFM imaging of the zebrafish brain. Together this work will establish a new platform for investigating the neural bases of unconstrained behavior. In a future R01 application we will use the MesoLFM to reveal how neural circuits drive natural goal-directed behavior and decision-making.