Dynamic modulation of retinal ribbon-type synapses

项目概要 双极细胞和无长突细胞突触是脊椎动物视网膜电路的关键组成部分，具有多个 专门功能。控制带状发射器输出的底层蜂窝机制 双极细胞的突触和无长突细胞的常规抑制性突触，在截然不同的情况下 哺乳动物视网膜对环境光条件的了解仍然知之甚少。在这里我们建议做一系列 对小鼠视网膜单个无长突细胞进行膜片钳电生理学和钙成像实验。 这使我们能够测量突触前 Ca2 电流和引起的膜电容变化， 实时测定活细胞的突触小泡胞吐作用。我们将应用钙成像和膜 电容技术应用于 AII 无长突细胞，这是哺乳动物视网膜中的主要中间神经元，可释放 甘氨酸位于包含大量突触小泡的常规活性区。我们将确定 单个 AII 无长突细胞的总体胞吐能力。我们将检验以下假设：所有无长突细胞 包含三个不同的易于释放的囊泡池，它们具有不同的大小和胞吐作用动力学。 维持和调节所有无长突细胞甘氨酸释放和短期可塑性的机制 不知道。我们将检验以下假设：cAMP 水平调节甘氨酸释放动力学、大小 易于释放的囊泡池，以及所有无长突细胞突触的短期可塑性。我们还将 在出生后早期发育期间对所有无长突细胞进行这些测量。我们将测试 假设囊泡和胞吐作用的 Ca2 传感器之间的耦合在早期变得更紧密 随着 Ca2 电流大小的增加以及 Ca2 通道与对接变得更加共定位， 所有无长突细胞活性区的囊泡。最后，使用电压钳和电流钳的组合 钳记录，我们将测量单个 AII 无长突细胞的胞吐作用程度，这是由 生理刺激，即不同强度的光刺激。综上所述，本次拨款提案的结果 将提供对所有无长突细胞如何通过调节双极细胞终末释放的更好理解 动态和可塑性抑制突触。

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.