Optical studies of the cone photoreceptor synapse

描述(申请人提供)：视锥感光细胞在黑暗中释放谷氨酸。光线使视锥细胞超极化，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，对于了解视觉信息是如何传递到双极细胞和水平细胞至关重要。以前的研究通过测量锥体细胞的突触后反应来间接推断这些细胞的突触前释放。然而，许多根本性的问题仍然存在，因为到目前为止，还没有关于锥形发射机释放的直接测量。其中的关键问题是：1)突触小泡在锥体末端的内吞和胞吐之间的时空路径是什么？2)突触前电压、钙离子浓度和胞吐作用之间的定量关系？3)视锥细胞在明暗两种情况下的释放速度是多少？4)视网膜外部的反馈突触和调制递质是如何调节释放的？5)视网膜锥体末端阵列上的侧抑制对比信号的大小和程度是什么？我们的目标是通过直接测量锥体突触前释放来回答这些问题。我们将使用包括FM1-43在内的荧光亲脂染料的活性依赖的摄取和释放作为内吞和胞吐的指标。初步结果表明，染料在锥体末端的吸收和释放是依赖于钙离子和去极化的。电子显微镜研究表明，“光转化”染料定位于突触小泡。FM1-43的释放在黑暗中迅速发生，受到光的抑制，并受到来自HCS的突触反馈的影响。我们监测了FM1-43从单独分离的视锥细胞、视网膜切片中的视锥细胞组和完整视网膜平面支架中的视锥细胞终末的二维阵列中的释放。这些准备工作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.