Dynamic modulation of retinal ribbon-type synapses

项目总结 双极细胞和无长突细胞突触是脊椎动物视网膜回路的关键组成部分，具有多个 专门化职能。控制来自带状结构的发射器输出的底层细胞机制 在截然不同的情况下，双极细胞的突触和无长突细胞的传统抑制性突触 在哺乳动物的视网膜中，环境光条件仍然知之甚少。在这里，我们建议做一系列 小鼠视网膜单个无长突细胞膜片钳电生理和钙成像实验。 这使我们能够测量突触前钙电流和诱发的膜电容变化 实时检测活细胞中突触小泡的胞吐作用。我们将应用钙成像和膜 对AII无长突细胞的电容技术，AII无长突细胞是哺乳动物视网膜中释放的主要中间神经元 甘氨酸位于包含一大簇突触小泡的常规活动区。我们将确定 单个AII无长突细胞的总体胞吐能力。我们将检验所有无长突细胞 含有三个不同的、容易释放的囊泡池，这些囊泡具有不同的大小和胞吐动力学。 维持和调节AII无长突细胞甘氨酸释放和短期可塑性的机制 都是未知的。我们将检验cAMP水平调节甘氨酸释放动力学的假设，即大小 容易释放的囊泡池，以及所有无长突细胞突触的短期可塑性。我们还将 在出生后早期发育的所有无长突细胞上进行这些测量。我们将测试 小泡与胞吐作用的钙感受器之间的耦合在早期变得更紧密的假设 随着钙电流的增大以及随着钙通道与对接的钙通道变得更加共存而发生的发展 所有无长突细胞活动区的小泡。最后，使用电压钳位和电流的组合- 我们将测量单个AII无长突细胞的胞吐程度，它是由 生理刺激，即不同强度的光刺激。总而言之，这项拨款提案的结果 将提供一个更好的理解AII无长突细胞如何通过 动态和可塑性抑制性突触。

PROJECT SUMMARY Bipolar cell and amacrine cell synapses are key components of the vertebrate retinal circuitry with multiple specialized functions. The underlying cellular mechanisms that control transmitter output from the ribbon-type synapses of bipolar cells and the conventional inhibitory synapses of amacrine cells, under vastly different ambient light conditions, are still poorly understood in the mammalian retina. Here we propose to do a series of patch-clamp electrophysiology and calcium imaging experiments on single amacrine cells of the mouse retina. This allows us to measure both presynaptic Ca2+ currents and evoked changes in membrane capacitance that assay synaptic vesicle exocytosis in real time from a living cell. We will apply calcium imaging and membrane capacitance techniques to the AII amacrine cell, a major interneuron in the mammalian retina that releases glycine at conventional active zones that contain a large cluster of synaptic vesicles. We will determine the overall capacity for exocytosis of a single AII amacrine cell. We will test the hypothesis that AII amacrine cells contain three distinct readily releasable pools of vesicles that have different sizes and kinetics of exocytosis. The mechanisms that maintain and modulate glycine release and short-term plasticity from AII amacrine cells are not known. We will test the hypothesis that cAMP levels modulate the kinetics of glycine release, the size of the readily releasable vesicle pool, and the short-term plasticity at AII amacrine cell synapses. We will also perform these measurements on AII amacrine cells during early postnatal development. We will test the hypothesis that the coupling between vesicles and the Ca2+ sensor for exocytosis becomes tighter during early development as Ca2+ currents increase in size and as Ca2+ channels become more colocalized with docked vesicles at the active zones of AII amacrine cells. Finally, using a combination of voltage-clamp and current- clamp recordings we will measure the degree of exocytosis from single AII amacrine cells that is evoked by physiological stimuli, namely, light stimuli of different intensities. In summary, the results of this grant proposal will provide a better understanding of how AII amacrine cells modulate bipolar cell terminal release via dynamic and plastic inhibitory synapses.