Synaptic Mechanisms in the Mammalian Retina

我们的工作集中在视网膜内层的特殊突触上。 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，来自A17的相互反馈扩展了这些突触编码亮度和计算对比度的范围（Oesch和Diamond，提交）。 来自其他无长突细胞的反馈微弱地调节突触增益，但不改变操作范围。 我们对双极细胞中带状突触生理学的理解仅限于杆状双极细胞。我们对来自锥双极细胞的突触传递了解不多，因为很难获得突触耦合的锥双极-神经节细胞对。我们已经将小鼠品系与遗传编码标记进行了杂交，这些标记识别了极有可能连接的特定类型的双极细胞和神经节细胞。特别地，我们已经利用了小鼠品系，其中可以可视化2型锥双极细胞（CBC 2）。我们发现CBC 2与AII无长突细胞形成相互的突触连接。我们正在研究这两种细胞类型之间的双向通讯，我们也在研究视杆双极细胞信号在传递到CBC 2之前是如何被AII无长突细胞塑造的。我们的初步研究结果表明，AIIs优先传输信息的对比度，而不是亮度，CBCs。此外，AIIs将具有不同时间动态的信号传递到ON和OFF锥双极细胞，这可能反映了电突触和化学突触之间的根本差异。我们正在研究这些转变背后的各种细胞和突触过程，手稿正在准备中。 我们已经与Donald Zack（Johns霍普金斯）合作来检查源自干细胞的视网膜神经节细胞的生理特征（Sluch等人，Sci. Rep.5，16595）。 我们发现，这些细胞开发不同的放电特性，这是符合不同的神经节细胞亚型在不同的发展阶段。我们还发现，ESC衍生的神经节细胞表达功能性谷氨酸受体。修订稿已提交出版。 我们还与阿威Wong（NEI）合作研究了小胶质细胞在保护回路和突触形态中的作用（Wang，et al.，2016 J. Neurosci.）。在小胶质细胞耗竭的小鼠模型中，视网膜回路和感光细胞形态的许多一般特征保持完整，但双极细胞反应（由视网膜电图b波表示）受损。虽然光感受器突触在光免疫荧光水平上表现正常，但我们使用电子显微镜（EM）发现，小胶质细胞depelted视网膜中的光感受器突触是营养不良的，具有不清楚的囊泡和不规则的突触带。 最后，我们已经扩展了我们的电子显微镜研究，与理查德Leapman（NIBIB）合作，探索在光感受器和杆双极细胞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。 我们发现，囊泡采用不同的拴系关系相对于丝带和突触前膜，我们相信这可能反映了停靠和引发的突触囊泡之间的形态差异。我们已经获得了高分辨率的三维图像的杆双极细胞带状突触，使用高压冷冻和EM断层扫描技术，在不同的生理条件下，应该引起不同的分数停靠/引发囊泡。我们目前正在分析这些不同的生理状态之间的形态差异，以确定在这些突触囊泡启动的形态基板，并检测从丝带胞吐的空间模式的目标。

Our work focuses on specialized synapses in the inner retina. 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 most recent work indicates that reciprocal feedback from A17s extends the range over which these synapses encode luminance and compute contrast (Oesch and Diamond, submitted). Feedback from other amacrine cells weakly modulates the synaptic gain but does not change the operating range. Our understanding of ribbon synaptic physiology in bipolar cells is limited to rod bipolar cells. We don't know as much about synaptic transmission from cone bipolar cells, because it is very difficult to obtain synaptically coupled cone bipolar - ganglion cell pairs. We have crossed mouse lines with genetically encoded markers identifying specific types of bipolar and ganglion cells that are very likely to be connected. In particular, we have utilized mouse lines in which type two cone bipolar cells (CBC2) can be visualized. We find that CBC2s make reciprocal synaptic connections with AII amacrine cells. We are studying bidirectional communication between these two cell types, and we are also studying how the rod bipolar cell signal is shaped by the AII amacrine cell before it is passed to the CBC2. Our preliminary findings suggest that AIIs preferentially transmit information about contrast, but not luminance, to CBCs. In addition, AIIs pass signals with different temporal dynamics to ON and OFF cone bipolar cells, likely reflecting fundamental differences between electrical and chemical synapses. We are examining various cellular and synaptic processes that underlie these transformations, and a manuscript is in preparation. We have collaborated with Donald Zack (Johns Hopkins) to examine the physiological characteristics of retinal ganglion cells derived from stem cells (Sluch, et al., Sci. Rep. 5, 16595). We find that these cells develop diverse firing properties that are consistent with different ganglion cell subtypes at different stages of development. We also found that ESC-derived ganglion cells express functional glutamate receptors. A revised manuscript has been submitted for publication. We also have collaborated with Wai Wong (NEI) to examine roles for microglia in preserving circuit and synaptic morphology (Wang, et al., 2016 J. Neurosci.). In a mouse model of microglial depletion, many general features of retinal circuitry and photoreceptor morphology remained intact, yet bipolar cell responses (indicated by the electroretinogram b-wave) were impaired. Although photoreceptor synapses appeared normal at the light immunofluorescence level, we found using electron microscopy (EM) that photoreceptor synapses in microglia-depeleted retinas were dystrophic, with indistinct vesicles and irregular synaptic ribbons. Finally, we have extended our electron microscopy studies, in collaboration with Richard Leapman (NIBIB), 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. We find that vesicles adopt different tethering relationships with respect to the ribbon and the presynaptic membrane, and we believe this may reflect morphological differences between docked and primed synaptic vesicles. We have obtained high-resolution three-dimensional images of rod bipolar cell ribbon synapses, using high-pressure freezing and EM tomographic techniques, under different physiological conditions that should give rise to different fractions of docked/primed vesicles. We are currently analyzing morphological differences between these different physiological states with the goal of determining the morphological substrate for vesicle priming at these synapses and to detect spatial patterns of exocytosis from the ribbon.