光遗传学解析视网膜内层抑制性环路

Within the inner retina, inhibitory neural circuits, composed of a wide variety of amacrince cells and ganglion cells via highly complicated spatial connections and neural transmitter systems, play an important role in the precise neural computation of visual system. The project proposed here tries to efficiently dissect the inhibitory circuits in the inner retina by using optogenetic techniques in combination with patch clamping and multielectrode array recording. We will focus on two types of ganglion cells, direction-selective ganglion cell and intrinsically photosensitive ganglion cell, which are respectively representative neurons for image-forming and non-image-forming visual functions. Experiments will be conducted in retinas from genetic mice, in which the light-gated ion channel Channelrhodopsin-2 are specifically expressed in inhibitory interneurons. We will optogenetically manipulate populational activities of amacrine cells in a specific manner, and/or influence responses of these cells individually using high-spatial resolution light stimulation, and make a precise analysis of dynamic changes in postsynaptic ganglion cell activities, with an aim of understanding how the inhibitory circuits are constructed for a specific functional type of ganglion cells and their neighboring amacrine cells, through distinct anatomical connections, various cell types, and different neurotransmitters. This study will provide insights into the roles of inhibitory circuits in mediating the functional activities of ganglion cells performing image-forming and non-image-forming visual tasks, respectively.

在内层视网膜，各种无长突细胞和神经节细胞通过复杂多样的空间联系和递质系统构成的抑制性环路，在视觉系统实施精确神经计算的过程中起重要作用。本项目应用光遗传学技术，与膜片钳及多通道微电极阵列记录技术相结合，高效地解析视网膜内层的抑制性环路。研究将特别关注负责成像视觉和非成像视觉的各一类代表性神经元——方向选择性神经节细胞和自感光神经节细胞。我们将以抑制性中间神经元特异表达光通道Channelrhodopsin-2的遗传学小鼠视网膜为标本，通过光照特异地操控无长突细胞的群体活动，和/或以高空间分辨率的方式作单细胞光调控，同时精确分析其突触后神经节细胞电活动的动态变化，目的是回答以下重要问题：对于特定功能的神经节细胞，其与无长突细胞组成的各种抑制性环路是通过怎样的解剖学联系、细胞学成分以及神经递质基础构筑的？本研究将为理解各类抑制性环路如何介导成像视觉和非成像视觉相关的神经节细胞的功能提供启示。

围绕无长突细胞（AC）构筑的视网膜内层突触环路结构、功能、递质复杂多样，是视网膜神经计算的重要基础。视网膜是中枢神经系统的重要组分，深入研究上述环路有助于理解大脑工作原理。本项目通过多学科技术，鉴定了两个特定类型AC被荧光分子标记，且表达光敏离子通道ChR2的遗传学小鼠品系，并以之为工具，通过光遗传学刺激和膜片钳记录相结合的策略，研究基于AC所构筑的视网膜微环路。ChAT-ChR2-EYFP小鼠视网膜中，免疫双标实验表明，ChR2-EYFP除少量表达于M1型自感光神经节细胞外，大量存在于一种新型的AC——非GABA能非甘氨酸能AC（nGnG AC）。膜片钳记录结合药理学实验显示，这些被特异标记的nGnG AC对全野光刺激呈现ON-OFF型的超级化膜电位反应。在加入谷氨酸能突触阻断剂后，蓝光刺激依然诱发膜电位去极化，表明ChR2表达具有功能性。其后进行的光遗传学操纵实验揭示，nGnG AC向多种节细胞提供不同强度的抑制性输入，且其中确实含有非GABA能非甘氨酸能的递质成分。在另一品系——VGAT--ChR2-EYFP小鼠视网膜中则发现，ChR2-EYFP特异表达于绝大多数种类的AC和水平细胞。在谷氨酸能及胆碱能突触阻断的条件下，全野蓝光刺激导致这些抑制性中间神经元的群体性的强烈激活，并在突触后节细胞上诱发兴奋性电流。在ON型、OFF型、ON-OFF型节细胞上均能记录到这一电流，且其不能被缝隙连接阻断剂阻断，但可被钴离子完全消除，这表明多种节细胞上存在源自AC的、非谷氨酸能非胆碱能的兴奋性化学突触输入。上述结果为探索内层视网膜神经环路及中枢神经系统信号加工机制提供了全新信息。