Optical studies of the cone photoreceptor synapse

描述（由申请人提供）：视锥光感受器在黑暗中强直性释放谷氨酸。光使视锥细胞发生超极化，导致谷氨酸释放逐渐减少。了解视锥细胞如何调节递质释放对于理解视觉信息如何传递到双极细胞和水平细胞至关重要。先前的研究通过测量这些细胞的突触后反应间接推断了锥细胞的突触前释放。然而，许多基本问题仍然存在，因为到目前为止，还没有直接测量锥体发射器释放的方法。关键问题包括：1)突触囊泡在锥体末端的胞吞作用和胞吐作用之间的时空路径是什么？2)突触前电压、Ca2+浓度和胞吐之间的定量关系是什么？3)明暗环境下视锥细胞的释放速率是多少？4)外视网膜的反馈突触和调节递质如何调节释放？5)视网膜锥体末端阵列侧抑制对比信号的大小和范围是什么？我们的目标是通过直接测量视锥细胞的突触前释放来回答这些问题。我们将使用荧光亲脂性染料（包括FM1-43）的活性依赖性摄取和释放作为内吞和胞吐的指标。初步结果表明，染料在锥体末端的摄取和释放依赖于Ca2+和去极化。电镜研究显示“光转化”染料定位于突触囊泡。FM1-43的释放在黑暗中迅速发生，受到光的抑制，并受到hc突触反馈的影响。我们监测了单独分离的视锥细胞、视网膜切片中的视锥细胞群和完整视网膜平面支架中视锥终端的二维阵列中fm1 -43的释放。这些准备使我们能够回答有关锥体神经递质释放调节的亚细胞到系统水平的问题。这些结果将提供有关正常视力机制的基本信息，并将为视网膜疾病中发生的突触功能障碍提供见解。

DESCRIPTION (provided by applicant): Cone photoreceptors tonically release glutamate in the dark. Light hyperpolarizes cones and causes a graded reduction in glutamate release. Understanding how cones regulate transmitter release is crucial for understanding how visual information is transmitted to bipolar and horizontal cells. Previous studies have indirectly inferred presynaptic release from cones by measuring postsynaptic responses from these cells. However, many fundamental questions remain because, until now, there has been no direct measure of cone transmitter release. Among the key questions are: 1) What is the spatio-temporal pathway taken by synaptic vesicles as they journey between endocytosis and exocytosis in the cone terminal? 2) What is the quantitative relationship between presynaptic voltage, Ca2+ concentration, and exocytosis? 3) What is the rate of release from cones in the light and dark? 4) How is release modulated by feedback synapses and modulatory transmitters in the outer retina? 5) What is the size and extent of the lateral-inhibition contrast signal on arrays of cone terminals in the retina? Our goal is to answer these questions by directly measuring presynaptic release from cones. We will use the activity-dependent uptake and release of fluorescent lipophilic dyes, including FM1-43, as indicators of endocytosis and exocytosis. Preliminary results show that uptake and release of the dyes into cone terminals are Ca2+ and depolarization-dependent. Electron microscopic studies show localization of "photoconverted" dye to synaptic vesicles. FM1-43 release occurs rapidly in the dark, is suppressed by light, and is affected by synaptic feedback from HCs. We have monitored FM 1-43 release from individually dissociated cones, groups of cones in retinal slices, and 2-dimensional arrays of cone terminals in intact retinal flat mounts. These preparations allow us to answer subcellular-to systems-level questions about the regulation of cone neurotransmitter release. These results will provide fundamental information about the mechanisms of normal vision and will provide insights about synaptic dysfunction that occur in retinal diseases.