Probing functional connectivity in vivo via holographic and molecular targeting

大脑皮层环路的功能和连通性之间的关系一直是神经科学中的一个长期主题。虽然几十年来，初级感觉区皮层信息处理的主要途径已经被描述，但关于刺激选择性是如何由皮质板层内和皮质板层之间突触连接的数量和强度构成的，人们知之甚少。最近的证据表明，水平兴奋性连接的重量与刺激偏好之间存在相关性。插入多个微电极以记录和映射连接的经典方法不能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两个合作团队的互补专业知识，即波前整形方法(Emily Iani Lab)和光遗传致动器工程(Hegemann Lab)，揭示小鼠V1视觉处理的连接模式。我们的全息光图案化方法已经证明了其通过双光子(2P)激发在体外和体内毫秒控制动作电位(AP)产生的能力。胞体靶向视蛋白的使用确保了单细胞的无偏向激活。为了将毫秒AP诱导扩展到体内位于L2/3、L4和L5层的靶神经元，我们将定制2P成像和2P刺激的光学和分子参数，最深可达~1 mm深的脑表面。为了评估最小串扰激活的OPTIN/报告器组合，我们将基于我们最新的3D光整形方案，测试定制设计的不同吸收光谱、通道动力学和膜靶向的OPTIN与钙或电压指示器在光学系统中的组合。为了克服组织散射对深度成像和深度刺激造成的光强损失，一方面我们将优化目标光瞳不足、时间聚焦和结构光图案化的光学策略，另一方面我们将测试红移光学元件或记者的好处。相对于体内定向选择性测定与体外后确定的连接性的相关性，我们试图用光学方法探索所有在体内的功能连线。我们将通过全息照明将单突触连接映射到表达SOMA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)。通过将全息斑点顺序移动到皮层上的神经元上，我们将能够识别相对于突触后细胞的水平和垂直连接。主要的优点是，我们的全息方法能够在突触前神经元中产生毫秒级的单个AP，从而允许基于EPSP潜伏期确定单突触连接。此外，通过电压指示器的活动读数将允许全光学映射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.