Synaptic Mechanisms in the Mammalian Retina

我们的工作集中在视网膜内部的特殊突触上。 我们扩大了对视网膜内无长突细胞所建立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。我们先前发现，A17无长突细胞通过一种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突触提供快速的GABA能反馈(Chavez等人，2006年)。这种快速反馈可能对于防止杆状双极细胞突触终末易释放的小泡迅速耗尽至关重要(Singer和Diamond，2006)。我们最新的工作表明，来自A17的相互反馈扩大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对突触增益有微弱的调节作用，但不会改变作用范围。 我们对双极细胞中的带状突触生理学的了解仅限于杆状双极细胞。我们对锥体双极细胞的突触传递知之甚少，因为很难获得突触耦合的锥体双极-神经节细胞对。我们已经用基因编码的标记杂交了小鼠品系，以识别非常可能相连的特定类型的双极细胞和神经节细胞。特别是，我们利用了可以显示二型锥体双极细胞(CBC2)的小鼠品系。我们发现CBC2与所有的无长突细胞都有相互的突触联系。我们正在研究这两种细胞之间的双向通讯，我们也在研究视杆双极细胞信号在传递到CBC2之前是如何由AII无长突细胞塑造的。我们的初步发现表明，AII优先向CBCs传递关于对比度的信息，而不是亮度信息。此外，AIIs将具有不同时间动力学的信号传递给开和关锥体双极细胞，这可能反映了电和化学突触之间的根本差异。我们正在研究这些转化背后的各种细胞和突触过程，一份手稿正在准备中。 我们与Donald Zack(Johns Hopkins)合作，研究干细胞来源的视网膜神经节细胞的生理特性(Sluch等人，Sci。代表5,16595)。我们发现，这些细胞在发育的不同阶段发展出不同的放电特性，这与不同的神经节细胞亚型一致。我们还发现，胚胎干细胞来源的神经节细胞表达功能性谷氨酸受体。修改后的手稿已提交出版。 我们还与Wai Wong(NEI)合作研究了小胶质细胞在保存回路和突触形态中的作用(Wang等人，2016 J.Neurosci)。在小胶质细胞耗竭的小鼠模型中，视网膜回路和光感受器形态的许多一般特征保持完好，但双极细胞反应(由视网膜电波b波表示)受损。虽然在光免疫荧光水平上，光感受器突触看起来是正常的，但我们用电子显微镜(EM)发现，小胶质细胞依赖的视网膜的光感受器突触是营养不良的，有模糊的囊泡和不规则的突触带。 最后，我们与Richard Leapman(NIBIB)合作，扩展了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，EM断层扫描使我们能够检测到将突触小泡拴在突触带和突触前膜上的蛋白质细丝。我们发现，小泡在突触带和突触前膜上采用不同的连接关系，我们认为这可能反映了停靠和启动的突触小泡之间的形态差异。我们使用高压冷冻和EM断层扫描技术，在不同的生理条件下获得了视杆双极细胞带突触的高分辨率三维图像，这些条件应该会产生不同比例的停靠/启动的小泡。我们目前正在分析这些不同生理状态之间的形态差异，目的是确定这些突触处小泡启动的形态底物，并检测胞吐的空间模式。

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.