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。 为了扩大我们对带状突触生理学的理解，我们正在记录我们认为可能与突触相关的基因标记细胞。最近对小鼠视网膜的连接分析预测了特定的锥体双极细胞亚型和三级神经元(无长突细胞和神经节细胞)之间的联系。我们正在努力利用这一新信息来发现可靠连接的细胞类型，这将使我们能够检查锥体上和锥体外双极细胞突触传递的动力学。 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杆状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. 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. To expand our understanding of ribbon synapse physiology, we are recording from genetically labeled cells that we think are likely to be synaptically connected. Recent connectomic analyses of the mouse retina predict connectivity between specific cone bipolar cell subtypes and third-order neurons (amacrine and ganglion cells). We are working to take advantage of this new information to discover reliably connected cell types that will allow us to examine the dynamics of synaptic transmission from ON and OFF cone bipolar cells. 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 obtainedhigh-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.