Functional connectomics of the binocular optic flow processing circuit in zebrafish

神经元之间相互连接的复杂性给我们理解大脑的功能和功能障碍带来了重大挑战。使用现代电子显微镜(EM)技术的连接学方法不仅提供了解剖连接，而且结合生理或行为证据，提供了对潜在神经元计算的机械理解。斑马鱼幼体由于其脑体积小，以及光学和遗传可及性，是一种很有前途的结合基于EM的连接和功能成像的系统。我们建议利用这些优势来重建具有定义的功能和行为的视觉电路，即水平光流的处理。我们之前的成像研究表明，斑马鱼顶盖前的神经元执行光流的双目计算。值得注意的是，我们确定的功能反应类型使我们能够预测能够解释如何计算不同模式的双目光流的线路图。这个接线图预测了有待实验验证的前提电路主题。在这个提议中，我们将通过承担以下两个工作计划来直接测试我们预测的接线图。首先，我们将使用我们最近在突触分辨率下获得的斑马鱼幼体的全脑连续块面电子显微镜(SBEM)堆叠来重建光流反应神经元。重要的是，这个堆栈是从之前用双光子钙成像记录的动物身上获得的，它带有一组单眼和双眼光流刺激，包含了大约200个神经元的生理信息。通过追踪这些具有生理特征的神经元，我们将确定它们通过直接突触相互连接。其次，我们将使用一种结合钙成像和可光激活绿色荧光蛋白荧光标记的遗传工具，在光学显微镜(LM)水平上检测视流反应细胞的神经递质类型。这一工具在神经递质特异性转基因品系的驱动下，将在单细胞水平上将对视流的生理反应与兴奋性和抑制性细胞的细胞形态联系起来。通过比较光镜显示的EM重建细胞和神经递质特异性单细胞的形态特征，我们将能够将兴奋或抑制的身份分配给重建的神经元。最后，我们将整合所有的实验数据来执行双目光流处理电路的计算建模和仿真。采用两种互补的方法，我们的功能引导的连接学分析将使我们能够理解双目运动视觉计算的连接机制。我们计划将我们的SBEM数据集及其重建结果提供给研究界，希望促进神经科学不同学科之间未来的合作。

The complexity of interconnectivity between neurons presents a major challenge in our understanding of functions and dysfunctions of the brain. Connectomics approaches using modern electron microscopy (EM) techniques have provided not only anatomical connectivity, but also mechanistic understanding of underlying neuronal computation, when combined with physiological or behavioral evidence. Larval zebrafish is a promising system for combining EM-based connectomics and functional imaging, due to its small brain size, as well as its optical and genetic accessibility. We propose to capitalize on these advantages to reconstruct a visual circuit with a defined function and behavior, namely, the processing of horizontal optic flow. Our previous imaging study demonstrated that neurons in the zebrafish pretectum perform binocular computation of optic flow. Notably, functional response types we identified allowed us to predict a wiring diagram that was able to explain how different patterns of binocular optic flow are computed. This wiring diagram predicts prerequisite circuit motifs that remain to be experimentally verified.In this proposal, we will directly test our predicted wiring diagram by undertaking the following two work programmes. First, we will reconstruct the optic flow responsive neurons using a whole brain serial block-face electron microscopy (SBEM) stack of larval zebrafish that we have recently acquired at synapse resolution. Importantly, this stack was obtained from an animal previously recorded with two-photon calcium imaging with a battery of monocular and binocular optic flow stimuli and contains physiological information of about two hundred neurons. By tracing these physiologically characterized neurons, we will determine their interconnectivity via direct synapses. Secondly, we will examine the neurotransmitter type of optic flow-responsive cells at light microscopy (LM) level using a genetic tool that combines calcium imaging and fluorescent labeling by photoactivatable GFP. This tool, when driven by neurotransmitter-specific transgenic lines, will link physiological response to optic flow with cellular morphology of excitatory and inhibitory cells at the single-cell level. By comparing morphological features of the EM reconstructed cells and neurotransmitter-specific single cells revealed by the LM, we will be able to assign the excitatory or inhibitory identity to reconstructed neurons. Finally, all experimental data will be integrated to perform a computational modeling and simulation of the binocular optic flow processing circuit.Taking two complementary approaches, our function-guided connectomics analysis will allow us to understand connectivity mechanisms underlying the computation of binocular motion vision. We plan to make our SBEM dataset and its reconstruction results accessible to the research community in the hope of fostering future collaborations between different disciplines in neuroscience.