NEUROSCIENCE

神经科学

• 批准号: 6692960
• 负责人: Ernest Clarence Steele
• 金额: $ 38.82万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6692960
• 关键词: Macaca mulatta; antibody; biological signal transduction; calcium channel; calcium channel blockers; calcium flux; calcium indicator; cooperative study; denervation; dihydropyridines; electrophysiology; gene expression; immunocytochemistry; laboratory rat; neural information processing; neurosciences; polymerase chain reaction; protein isoforms; retina; retinal bipolar neuron; soma; visual photoreceptor; voltage gated channel

The long-term goal of this study is to understand how photoresponses are forwarded through the vertebrate retina to higher visual centers. This proposal is aimed at determining the molecular identity of the voltagegated calcium channel pore-forming alphal subunit isoforms underlying the L-type calcium currents of photoreceptors and bipolar ceils of primate retina. Immunolabeling of different alpha-1 subunit isoforms combined with immunolabeling of cone opsins will test the hypothesis that expression of specific isoforms is restricted to spectral classes of cones. Antibody studies will be combined with isoform-specific RT-PCR amplification and sequencing of transcripts to determine molecular identity of expressed alphal subunit isoforms. Studies will be performed on whole retina and eventually on single, isolated cells to establish the diversity of expression at the single cell level. To determine the role of the different isoforms in the regulation of the intracellular calcium dynamics of the soma and synaptic terminals of these cells, electrophysiology combined with optical imaging of calcium indicators will be applied. Selective blockage of calcium entry at restricted sites, or controlled ablation of cellular compartments, will indicate the location of expression of calcium channels on cones and bipolar cells. These studies will provide the foundation for further studies aimed at understanding the mechanisms that control the regional expression of calcium channel proteins and their relationship to spatially-restricted cellular processes such as transmitter release. Another aim of this study is to identify novel candidate genes for human retinal channelopathies involving abnormal processing of photoreceptor and/or bipolar cells. In humans, mutations in the alphalF calcium channel subunit gene, CACNA1F, have been linked to one form of night blindness (CSNB2), underscoring the importance of this calcium channel subunit in normal visual processing. Newly identified calcium channel alphal subunit isoforms will become candidate genes in human visual disorders involving photoreceptor and/or bipolar cell dysfunction. This study will therefore add to our current understanding of blindness as well as our fundamental understanding of the structure and function of retinal neurons.

这项研究的长期目标是了解如何通过脊椎动物视网膜转发光响应到更高的视觉中心。该建议旨在确定电压处理的钙通道孔形成的α亚基亚基的分子身份，该亚型钙型钙电流和灵长类动物视网膜的双相CEIL的L型钙电流。与锥蛋白的免疫标记的不同α-1亚基同工型的免疫标记将检验以下假设：特定同工型的表达仅限于锥形光谱类别。抗体研究将与同工型特异性RT-PCR扩增和转录本的测序结合，以确定表达的α亚基同工型的分子身份。研究将在整个视网膜上，最终在单个分离的细胞上进行，以在单细胞水平上建立表达的多样性。确定不同同工型在调节中的作用 这些细胞的躯体和突触末端的细胞内钙动力学，将应用电生理学与钙指标的光学成像结合在一起。选择性阻塞钙在受限部位或受控隔离室的钙进入将表明钙通道在锥和双极细胞上的表达位置。这些研究将为进一步的研究提供基础，旨在了解控制钙通道蛋白区域表达的机制及其与空间限制的细胞过程（例如发射器释放）的关系。这项研究的另一个目的是确定涉及光感受器和/或双极细胞异常处理的人类视网膜通道病的新型候选基因。在人类中，Alphalf钙通道亚基基因Cacna1f中的突变已与一种夜失明相关（CSNB2），强调 该钙通道亚基在正常视觉处理中的重要性。新鉴定的钙通道α亚基同工型将成为涉及光感受器和/或双极细胞功能障碍的人类视觉疾病中的候选基因。因此，这项研究将增加我们当前对失明的理解以及我们对视网膜神经元结构和功能的基本理解。