Probing functional connectivity in vivo via holographic and molecular targeting

皮层回路的功能和连通性之间的关系一直是神经科学中一个长期存在的主题。尽管几十年来，人们已经描述了初级感觉区皮层信息处理的主要途径，但对皮层层内和层间突触连接的数量和强度如何构建刺激选择性知之甚少。最近的证据表明水平兴奋连接的重量与刺激偏好之间存在相关性。插入多个微电极来记录和绘制连接的传统方法无法应用于体内数十到数百个神经元。在拟议的项目中，我们的目标是通过利用来自两个合作团队的互补专业知识，即波前整形方法（Emiliani实验室）和光遗传致动器工程（Hegemann实验室），揭示小鼠V1视觉处理背后的连接模式。在体外和体内实验中，我们的全息光图方法已经证明了其通过双光子激发对动作电位（AP）产生的毫秒级控制能力。体细胞靶向视蛋白的使用确保了单细胞的无偏激活。为了将毫秒AP诱导扩展到体内位于L2/3、L4和L5层的目标神经元，我们将为2P成像和2P刺激定制光学和分子参数，深度可达脑表面以下约1mm。为了评估最小串扰激活的视蛋白/报告蛋白组合，我们将测试定制设计的不同吸收光谱、通道动力学和膜靶向的视蛋白，这些视蛋白将与钙或电压指示器结合在光学系统中，基于我们最新的3D光成型方案。为了克服深层成像和深层刺激中组织散射造成的光强损失，我们一方面将优化物镜瞳孔下填充、时间聚焦和结构光模式的光学策略，另一方面我们将测试红移视蛋白或报告蛋白的优势。与将体内定向选择性测定与事后在体外鉴定的连通性相关联相比，在这里，我们开始用光学方法探索体内的功能连接。我们将通过全息照明将单突触连接映射到表达体细胞靶向视蛋白的突触前神经元上，同时通过贴片电极监测突触后兴奋电位（EPSP）。通过将全息点依次移动到皮质层的神经元上，我们将能够识别相对于突触后细胞的水平和垂直连接。主要优点是我们的全息方法可以在突触前神经元中毫秒级地生成单个AP，从而可以根据EPSP延迟确定单突触连接。此外，通过电压指示器读出的活动将允许全光绘制神经元连接的方向性。

The relationship between the function and connectivity of cortical circuits has been a long-standing theme in neuroscience. Although since decades the predominant pathways of cortical information processing in primary sensory area have been described, little is known about how stimulus selectivity is constructed by the number and strength of synaptic connections within and between cortical laminae. Recent evidences indicate a correlation between the weight of horizontal excitatory connections and stimulus preference. The classical approach of inserting multiple microelectrodes to record and map connectivity cannot be applied for tens to hundreds of neurons in vivo. In the proposed project, we aim at uncovering the connectivity patterns underlying visual processing in mouse V1 by harnessing the complementary expertise from the two partner teams, i.e., wavefront shaping methods (Emiliani Lab) and engineering of optogenetic actuators (Hegemann Lab).Our holographic light-patterning approach has proved its capability of millisecond control of action potential (AP) generation via two-photon (2P) excitation in vitro and in vivo. The use of soma-targeted opsin ensures unbiased single-cell activation. To extend millisecond AP induction to target neurons situated at layer L2/3, L4, and L5 in vivo, we will tailor optical and molecular parameters for 2P imaging and 2P stimulation up to ~1 mm deep below the brain surface. To assess an opsin/reporter combination of minimum crosstalk activation, we will test custom-designed opsins of different absorption spectra, channel kinetics, and membrane targeting to be combined with calcium or voltage indicators in an optical system based on our latest scheme of 3D light-shaping. To overcome the light intensity loss due to tissue scattering for deep imaging and deep stimulation, on the one hand we will optimize optical strategies of underfilling objective pupil, temporal focusing, and structured light-patterning, and on the other hand we will test the benefits of red-shifted opsins or reporters.In contrast to correlating orientation selectivity determination in vivo with connectivity identified post hoc in vitro, here we set out to probe the functional wiring all in vivo with optical approaches. We will map monosynaptic connections via holographic illumination onto a presynaptic neuron expressing soma-targeted opsin while monitoring the postsynaptic excitatory potential (EPSP) via a patch electrode. By sequentially moving the holographic spots onto neurons across cortical layers, we will be able to identify both horizontal and vertical connections relative to the postsynaptic cell. The main advantage is that our holographic method enables millisecond generation of single AP in a presynaptic neuron, thus allowing determining monosynaptic connection based on EPSP latency. Furthermore, activity readout via voltage indicator will permit all-optical mapping the directionality of neuronal connections.