Synaptic Mechanisms in the Mammalian Retina

我们的工作集中在视网膜内部的几种不同的突触和细胞类型上。 在整合来自网络的突触输入并将视觉信号传递到大脑其他部分的视网膜神经节细胞(RGC)中，我们发现某些类型的NMDA型谷氨酸受体(NMDAR)在特定条件下被特异性地定位以限制它们的激活。实验室的解剖学研究表明，NMDAR主要定位于突触上的突触周围，而它们定位于突触外的突触后密度(Zhang和Diamond，2006)。此外，包含NR2B亚单位的NMDAR更有可能成为突触外靶点和突触上的靶点，而含有NR2A的NMDAR主要是突触和突触外的。这种截然不同的突触下分布与已知对谷氨酸受体靶向重要的支架蛋白的表达模式有关(手稿正在准备中)。我们发现PSD-95与含有NR2A的受体共定位，而SAP102的表达与含有NR2B的受体更一致。有趣的是，我们还发现NR1亚基的特定剪接变体与特定NR2亚基共定位。我们的生理学结果支持这些解剖学发现，因为突触诱发的ON反应是由含有NR2B的受体比非突触介导的(手稿正在准备中)。我们还发现，不同亚基的分数贡献可以通过突触释放NMDAR共激动剂甘氨酸或丝氨酸来调节。这些结果表明，NMDAR可能在视觉世界中对光的递增和递减的突触处理中起着不同的作用。我们正在通过测量神经节细胞树突中NMDAR介导的突触钙信号来更详细地检验这一想法。我们的初步结果表明，突触的NMDAR介导的钙瞬变表现出与通过电压门控钙通道内流不同的动力学。我们正在测试细胞内钙储存和挤出机制在树突状钙动力学中的相对贡献。 我们还扩大了对视网膜内无长突细胞进行的抑制性突触连接的研究，以了解前馈和反馈抑制如何在这个网络中对信号处理做出贡献。我们发现，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视杆双极细胞终末提供快速的GABA能反馈(Chavez等人，2006年)。这种快速反馈是由A17无长突细胞中钙离子通透性AMPA受体的激活驱动的，这可能是防止杆状双极细胞突触终末的易释放小泡迅速耗尽所必需的(Singer和Diamond，2006)。最近的工作(两篇论文即将提交)表明，对双极细胞的另外两种反馈抑制表现出不同的特征和调制机制。除了提供有关反馈的有价值的信息外，这项工作还使我们能够开始了解哺乳动物视网膜中大量的无长突细胞(&gt；24种细胞类型)的功能。最后，在另一个项目中，我们正在结合电生理和成像技术来探索A17无长突细胞中信号整合的机制。我们发现，活跃的钙依赖的电导可能是划分A17树突内信号处理的必要因素，形成哺乳动物视网膜暗视(弱光)基础的杆状通路的关键组成部分。

Our work focuses on several different synapses and cell types in the inner retina. In retinal ganglion cells (RGCs), which integrate synaptic input from the network and relay the visual signal to the rest of the brain, we find that certain types of NMDA-type glutamate receptors (NMDARs) are localized specifically to limit their activation under certain conditions. Anatomical work in the lab shows that NMDARs are localized primarily perisynaptically at ON synapses, while they are localized in the postsynaptic density at OFF synapses (Zhang and Diamond, 2006). Moreover, NMDARs containing the NR2B subunit are more likely to be perisynaptically targeted and at ON synapses, while NR2A-containing NMDARs are primarily synaptic and at OFF synapses. This contrasting subsynaptic distribution correlates with the expression pattern of scaffolding proteins known to be important for glutamate receptor targeting (manuscript in preparation). We find that PSD-95 is co-localized with NR2A-containing receptors, while the expression of SAP102 is more aligned with NR2B containing receptors. Interestingly, we also find that specific splice variants of the NR1 subunit co-localize with specific NR2 subunits. Our physiological results support these anatomical findings, as synaptically-evoked ON responses are mediated by a greater fraction of NR2B-containing receptors than OFF synapses (manuscript in preparation). We also find that the fractional contribution of different subunits can be modulated by synaptic release of NMDAR coagonist, either glycine or serine. These results indicate that NMDARs may play distinct roles in synaptic processing of light increments and decrements in the visual world. We are examining this idea in greater detail by measuring synaptic Ca signals mediated by NMDARs in ganglion cell dendrites. Our initial results indicate that synaptic, NMDAR-mediated Ca transients exhibit different dynamics than those due to influx through voltage-gated calcium channels. We are in the process of testing the relative contribution of intracellular calcium stores and extrusion mechanisms in the dynamics of dendritic calcium. We also 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 find that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell terminals via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback, driven by activation of calcium-permeable AMPA receptors in the A17 amacrine cell, may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). More recent work (two papers nearing submission) indicate that two other types of feedback inhibition onto bipolar cells exhibit different characteristics and mechanisms of modulation. In addition to providing valuable information about feedback, this work is enabling us to begin to make functional sense of the vast array of amacrine cells (> two dozen cell types) in the mammalian retina. Finally, in another project we are combining electrophysiological and imaging techniques to explore the mechanisms of signal integration in A17 amacrine cells. We find that active, calcium-dependent conductances may be essential to compartmentalize signal processing within A17 dendrites, forming a crucial component of the rod pathway that underlies scotopic (low light) vision in the mammalian retina.