Synaptic Mechanisms in the Mammalian Retina

哺乳动物视网膜的突触机制

• 批准号: 8746818
• 负责人: JEFFREY S DIAMOND
• 金额: $ 214.23万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746818
• 关键词: Amacrine Cells; Auditory system; Behavior; Biological Neural Networks; Calcium; Calcium Channel; Cells; Characteristics; Circadian Rhythms; Collaborations; Coupled; Dark Adaptation; Data; Dendrites; Designer Drugs; Development; Diamond; Diffuse; Diffusion; Docking; Electron Microscopy; Electrophysiology (science); Feedback; Filament; Genetic; Goals; Hair Cells; Image; Inner Plexiform Layer; Knock-out; Manuscripts; Membrane; Microelectrodes; Mining; Molecular; Mus; N-Methyl-D-Aspartate Receptors; Neurons; Neuropil; Output; Pathway interactions; Phase; Photons; Physiological; Play; Preparation; Presynaptic Terminals; Process; Property; Proteins; Retina; Retinal; Retinal Cone; Role; Signal Transduction; Specificity; Staging; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Technology; Time; Tissues; Transgenic Mice; Vesicle; Vision; Visual; Work; absorption; cell type; computerized data processing; ganglion cell; insight; large-conductance calcium-activated potassium channels; luminance; network models; postsynaptic; presynaptic; prevent; receptor; research study; response; retinal rods; ribbon synapse; simulation; tomography; tool; two-dimensional; visual process; visual processing; voltage

Our work focuses on specialized synapses and circuitry in the inner retina. This year represents a major transition in the lab, and we are working hard to put various types of technology in place that will drive our experimental approach for years to come. We have expanded our study of inhibitory synaptic connections made by amacrine cells within the inner retina, to understand how feedforward and feedback inhibition contributes to signal processing in this network. We previously discovered that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell ribbon synapses via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). Our recent work indicates that this feedback extends the range over which these synapses encode luminance and compute contrast (manuscript in preparation). In addition, another study indicates that feedback inhibition enhances the gain of synaptic responses in the rod pathway to the absorption of single photons (manuscript submitted). In addition, we have combined electrophysiological and anatomical (EM) data with mathematical simulations to explore the role of the ribbon in regulating the delivery and release of vesicles at the presynaptic membrane. Our findings suggest that the ribbon may achieve a "conveyor belt" like function simply with passive diffusion of vesicles along the two-dimensional ribbon. These simulations also make testable predictions regarding the molecular mechanism by which the vesicles diffuse along the ribbon. A manuscript is fully developed and is nearing submission. We are combining electrophysiology, imaging approaches and cellular/network modeling to explore dendritic integration in directionally-selective ganglion cells. We are also taking advantage of numerous transgenic mouse lines to image specific cell types, which will enable us to record from synaptically coupled cone bipolar - ganglion cell pairs. This has enabled us to examine how directional selectivity is encoded at various stages of the circuit. This work also suggests that NMDA receptors play a unique role in ganglion cell dendrites to amplify the directionally selective signal. A manuscript is in preparation. We are also working to understand how NMDA receptors contribute to the developmental refinement of ganglion cell dendrites, with are precisely stratified within the inner plexiform layer, the synaptic neuropil of the inner retina. We have successfully combined multiple genetic tools to acquire a mouse in which the NMDA receptors can be knocked out in a single type of ganglion cell at any time during development. This project has taken a long time to prepare, but initial experiments with the mice are very promising and suggest that we will gain exciting new insights in the role of NMDA receptors in retinal development. Our work on feedback inhibition has led us to undertake a long-term effort to understand, in a systematic way, how amacrine cells contribute to visual signaling in the inner retina. With over 40 different kinds of amacrine cell, this prospect can be a bit overwhelming. To start, we have identified narrow- and wide-field amacrine cells that can be identified and manipulated by genetic means. We have acquired mouse lines in which CRE is expressed specifically in certain amacrine cells and then cross them with floxed lines enabling the CRE-expressing neurons to be silenced chemically (through CRE-dependent expression of designer receptors exclusively activated by designer drugs). The impact of this silencing on ganglion cell signaling will be examined with a microelectrode array. In addition, the cell-specific expression of calcium and voltage indicators will enable us to examine dendritic signaling in these cells that likely underlies visual processing in these cells (see Grimes, et al., 2010) that is not accessible with somatic electrophysiological recordings. We are also studying how the biophysical properties of synapses and neurons change with different phases of the circadian cycle or dark adaptation. Initially we have focused on changes in BK channel function in AII amacrine cells, which play distinct roles in night and daytime vision (see Oesch and Diamond, 2010). A manuscript is in preparation with the initial physiological work, and we have begun to collaborate with Dr. Kevin Briggman to mine densely reconstructed retinal tissue to identify changes in the relevant retinal circuitry. Finally, we are extending our electron microscopy studies, in collaboration with Tom Reese and Richard Leapman, to explore the detailed ultrastructure of synaptic ribbons in photoreceptors and rod bipolar cells. So far, EM tomography enables us to detect protein filaments that tether synaptic vesicles to the ribbon and the presynaptic membrane. This approach may enable us to discern morphologically, for the first time, docked and primed synaptic vesicles. We have applied a similar analysis to hair cell ribbon synapses in the auditory system and found postsynaptic specializations that optimize signal transfer at these synapses (manuscript submitted).

我们的工作重点是专门的突触和电路在内部视网膜。 今年是实验室的一个重大转变，我们正在努力将各种类型的技术投入使用，这将推动我们未来几年的实验方法。 我们已经扩大了我们的研究抑制性突触连接的无长突细胞内视网膜，了解前馈和反馈抑制如何有助于在这个网络中的信号处理。 我们先前发现，A17无长突细胞通过独立于膜去极化或电压门控钙通道的释放过程向杆状双极细胞带状突触提供快速GABA能反馈（Chavez，et al.，2006年）。 这种快速反馈对于防止杆双极细胞突触末端的易释放囊泡的快速耗尽可能是必不可少的（Singer和Diamond，2006）。 我们最近的工作表明，这种反馈扩展了这些突触编码亮度和计算对比度的范围（手稿正在准备中）。 此外，另一项研究表明，反馈抑制增强了杆通路中对单光子吸收的突触反应的增益（手稿提交）。 此外，我们结合了电生理和解剖（EM）数据与数学模拟，探索丝带在调节突触前膜囊泡的传递和释放中的作用。 我们的研究结果表明，带状物可以实现“传送带”一样的功能，只是被动扩散的囊泡沿着二维带状物。 这些模拟也使可测试的预测有关的分子机制，囊泡扩散沿着带。一份手稿已完成，即将提交。 我们正在结合电生理学，成像方法和细胞/网络建模来探索定向选择性神经节细胞中的树突整合。 我们也利用许多转基因小鼠品系来成像特定的细胞类型，这将使我们能够记录突触耦合的锥双极-神经节细胞对。 这使我们能够研究方向选择性是如何在电路的各个阶段进行编码的。 这项工作还表明，NMDA受体在神经节细胞树突中发挥独特的作用，以放大定向选择性信号。 手稿正在准备中。 我们还在努力了解NMDA受体如何有助于神经节细胞树突的发育完善，这些树突在内部网状层（内层视网膜的突触神经元）中精确分层。 我们已经成功地结合了多种遗传工具，获得了一种小鼠，其中NMDA受体可以在发育过程中的任何时候在单一类型的神经节细胞中被敲除。 这个项目花了很长时间来准备，但对小鼠的初步实验非常有希望，并表明我们将获得关于NMDA受体在视网膜发育中的作用的令人兴奋的新见解。 我们对反馈抑制的研究使我们进行了长期的努力，以系统的方式了解无长突细胞如何有助于内层视网膜的视觉信号。有超过40种不同的无长突细胞，这一前景可能有点令人不知所措。 首先，我们已经确定了窄视野和宽视野的无长突细胞，这些细胞可以通过遗传手段识别和操纵。 我们已经获得了CRE在某些无长突细胞中特异性表达的小鼠品系，然后将它们与floxed品系杂交，使得表达CRE的神经元能够被化学沉默（通过设计药物专门激活的设计受体的CRE依赖性表达）。 将用微电极阵列检查这种沉默对神经节细胞信号传导的影响。 此外，钙和电压指标的细胞特异性表达将使我们能够检查这些细胞中的树突信号传导，其可能是这些细胞中视觉处理的基础（参见Grimes，et al.，2010年），这是无法访问的躯体电生理记录。 我们还在研究突触和神经元的生物物理特性如何随着昼夜节律周期或暗适应的不同阶段而变化。 最初，我们专注于AII无长突细胞中BK通道功能的变化，AII无长突细胞在夜间和白天视觉中发挥不同的作用（参见Oesch和Diamond，2010）。 一份手稿正在准备与初步的生理工作，我们已经开始与凯文·布里格斯曼博士合作，挖掘密集重建的视网膜组织，以确定相关视网膜回路的变化。 最后，我们正在与Tom Reese和Richard Leapman合作，扩展我们的电子显微镜研究，以探索光感受器和视杆双极细胞中突触带的详细超微结构。 到目前为止，电磁断层扫描使我们能够检测到蛋白质丝，这些蛋白质丝将突触囊泡拴在丝带和突触前膜上。 这种方法可能使我们能够从形态学上第一次辨别对接和启动的突触囊泡。 我们对听觉系统中的毛细胞带状突触进行了类似的分析，发现突触后的特化可以优化这些突触的信号传递。