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紧张性释放谷氨酸。光使视锥细胞超极化并导致谷氨酸释放的分级减少。理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来间接推断这些细胞的突触前释放。然而，许多基本问题仍然存在，因为到目前为止，还没有直接测量锥发射器释放。关键问题包括：1）当突触囊泡在锥体末端内吞和胞吐之间旅行时，它们所采取的时空途径是什么？2)突触前电压、Ca 2+浓度和胞吐之间的定量关系是什么？3)光和黑暗中视锥细胞释放的速率是多少？4)视网膜外层的反馈突触和调节性递质是如何调节释放的？5)视网膜上锥状终末阵列上的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FM 1 -43）的活性依赖性摄取和释放作为内吞作用和胞吐作用的指标。初步结果表明，摄取和释放的染料到锥终端是Ca 2+和去极化依赖。电子显微镜研究表明，本地化的“光转换”染料突触囊泡。FM 1 -43的释放在黑暗中迅速发生，被光抑制，并受到HC的突触反馈的影响。我们已经监测了FM 1-43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坦支架中的视锥细胞终端的二维阵列中的释放。这些准备工作使我们能够回答关于视锥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觉机制的基本信息，并将提供有关视网膜疾病中发生的突触功能障碍的见解。

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.