NEUROCHEMICAL PATHWAYS IN THE INNER RETINA

视网膜内层的神经化学通路

基本信息

批准号：
6559176
负责人：
NICHOLAS C. BRECHA
金额：
$ 5.27万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1981
资助国家：
美国
起止时间：
1981-07-06 至 2004-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from the applicant's abstract): The overall goal of this program is to better understand visual image processing in the mammalian inner retina by defining and characterizing its transmitter systems, cell morphology and circuitry. The focus of these studies is on somatostatin (SRIF), a potent inhibitory transmitter localized to wide-field amacrine cells and its five G protein-coupled receptors, SSTR1-5. The proposed studies are focused on determining the presence of the SSTRs, their cellular expression patterns and relationship to SRIF-containing amacrine cell processes to address the hypothesis that SRIF has a paracrine mode of action and a modulatory influence at both the cellular and circuitry levels by acting at distinct SSTRs expressed by multiple cell populations. Investigations will also study the inhibitory actions of SRIF on intracellular Ca2+ levels in acutely isolated rodent rod bipolar cells and salamander photoreceptors, which strongly express SSTR2, to better define the modulatory action of SRIF at the cellular level. Specific aim 1 will determine and characterize SSTR1-SSTR5 mRNA tissue and cellular distribution using RT-PCR, RNA blot hybridization and in situ hybridization immunohistochemistry using the rodent retina. Specific aim 2 will investigate the sites of action of SRIF by defining the expression of SSTR1-SSTR5 and their relationship to SRIF fibers using immunohistochemistry in the rodent and rabbit retina. Specific aim 3 will investigate the inhibitory action of SRIF at the cellular level using acutely isolated rodent rod bipolar cells and salamander photoreceptors with fluorometric Ca2+ imaging techniques. Studies will characterize SRIF inhibition of a K+ stimulated intracellular Ca2+ increase in these cells, and SRIF's modulation of the Go/Gi G proteins that have been implicated in the cellular action of this peptide. Finally, studies will evaluate SRIF action utilizing SRRF-1 and SRRF-2 receptor agonists. These studies will use rod bipolar cells from normal and Go- or Gi-deficient mice with a gene disruption of the alpha-o, alpha-i1, alpha-i2 or alpha-i3 subunits, and from SSTR-2-deficient mice with a gene disruption of SSTR-2. Experiments will utilize sequence-specific RNA probes, antibodies, SRIF-1 and -2 receptor agonists, and a highly specific SSTR2 antagonist to accomplish the experimental objectives of this application. Proposed studies will provide new information about the role of peptides in the modulation of retinal cells and circuitry in the inner retina, thus providing the basis for a better understanding of visual image processing in the retina. These objectives are consistent with the health-related goals of the National Eye Institute to develop more effective treatments and ultimately to prevent retinal diseases.

描述（改编自申请人的摘要）：本项目的总体目标程序是为了更好地理解哺乳动物内部的视觉图像处理通过定义和表征视网膜的发射系统、细胞形态和电路。这些研究的重点是生长抑素 (SRIF)，这是一种有效的抑制性递质定位于广域无长突细胞及其 5G 蛋白质偶联受体，SSTR1-5。拟议的研究重点是确定 SSTR 的存在、它们的细胞表达模式以及与含有 SRIF 的无长突细胞过程的关系，以解决假设 SRIF 具有旁分泌作用模式和调节影响通过作用于表达的不同 SSTR，在细胞和电路水平上发挥作用由多个细胞群。调查还将研究抑制作用 SRIF 对急性离体啮齿动物杆细胞内 Ca2+ 水平的作用双极细胞和蝾螈光感受器强烈表达 SSTR2，更好地定义 SRIF 在细胞水平上的调节作用。具体目标 1 将确定和表征 SSTR1-SSTR5 mRNA 组织和使用 RT-PCR、RNA 印迹杂交和原位进行细胞分布使用啮齿动物视网膜进行杂交免疫组织化学。具体目标2将通过定义 SRIF 的表达来研究 SRIF 的作用位点使用免疫组织化学方法研究 SSTR1-SSTR5 及其与 SRIF 纤维的关系啮齿动物和兔子的视网膜。具体目标 3 将研究抑制作用使用急性离体啮齿动物杆双极观察 SRIF 在细胞水平上的作用使用荧光 Ca2+ 成像技术对细胞和蝾螈光感受器进行观察。研究将表征 SRIF 对 K+ 刺激的细胞内 Ca2+ 的抑制作用这些细胞的增加，以及 SRIF 对 Go/Gi G 蛋白的调节与该肽的细胞作用有关。最后，学习将利用 SRRF-1 和 SRRF-2 受体激动剂评估 SRIF 作用。这些研究将使用来自正常小鼠和 Go 或 Gi 缺陷小鼠的视杆双极细胞具有α-O、α-i1、α-i2或α-i3亚基的基因破坏，以及来自 SSTR-2 基因破坏的 SSTR-2 缺陷小鼠。实验将利用序列特异性 RNA 探针、抗体、SRIF-1 和 -2 受体激动剂和高度特异性的 SSTR2 拮抗剂来完成实验此应用程序的目标。拟议的研究将提供新信息关于肽在调节视网膜细胞和电路中的作用内视网膜，从而为更好地理解视觉提供基础视网膜中的图像处理。这些目标与国家眼科研究所制定更有效的健康相关目标治疗并最终预防视网膜疾病。