MOLECULAR MECHANISMS OF SIGNAL TRANSDUCTION IN RETINA

谷氨酸是大脑中主要的兴奋性神经递质， 视网膜。由于它们在视觉处理中的重要性，正确的 谷氨酸受体(GluRs)的功能和定位是 幻象。此外，在大脑中已经表明，一种不适当的 过度刺激GluRs会导致神经细胞死亡，这是一个已知的过程 作为兴奋性毒物。尽管这种现象已经被证明发生在 视网膜，它的重要性被低估了。的多样性 GluRs是广泛的和亚型特异性的激动剂和拮抗剂。 受体已被证明可以减少或增强兴奋性毒性细胞的损伤。 该实验室的目标是更好地了解 视网膜细胞类型的功能和形态的复杂性以及 谷氨酸受体的多样性。具体而言，将开展三个项目： 1)视网膜中的代谢性谷氨酸受体。至少有六个代谢性 谷氨酸受体，即激活第二信使通路的受体 在谷氨酸刺激下，它们在视网膜中表达。使用子类型- 特异性抗体，我们将确定这些受体的定位 在视网膜上。原位杂交和免疫组织化学研究将 来分析这些受体在体内的表达模式 发育和视网膜退化，这两者都可以通过 代谢性受体。最后，可能的电压调制-和 通过代谢性受体的配基门控通道将在 分离培养的视网膜神经元。 2)双极细胞的信号转导途径。因为光感受器 细胞对光刺激有超极化反应，而双极细胞超极化 去极化时，ON双极细胞的反应是符号反转。这 这种反应可能是由一种代谢性谷氨酸受体mGluR6介导的。 然而，人们对与此相关的第二信使途径知之甚少。 受体。使用细胞分选程序，我们已经从 丰富的双极细胞种群。编码一种新的 假定的cGMP门控通道w从该材料中确定，并且它将 在功能上具有特征性。将使用相同的过程进行克隆 这一途径的其他组成部分，如G蛋白亚基。 3)视网膜中的海藻酸型谷氨酸受体。这三种电致电离性 谷氨酸受体类型、NMDA、AMPA和海人藻酸受体的功能 其中最不为人所知的是。我们已经证明了 海人藻酸受体亚单位基因的表达受 RD小鼠的视网膜变性。我们将提出特定于子单元的 抗这些受体亚基的抗体并分析这些变化 受体蛋白水平。通过原位杂交，我们也发现 GluR7在发育过程中有一个独特的表达模式，因为它 在光感受器中瞬时表达。利用最近的 构建了GlluR7基因失活的转基因小鼠，我们 将分析这个受体亚单位在视网膜形态中的作用。

Glutamate is the major excitatory neurotransmitter in the brain and the retina. Because of their importance in vision processing, the correct function and localization of glutamate receptors (GluRs) is essential for vision. In addition it has been shown in the brain, that an inappropriate over-stimulation of GluRs leads to neuronal cell death, in a process known as excitatoxicity. Even though this phenomenon has been shown to occur in the retina, its importance has been understimulated. The diversity of GluRs is extensive and subtype specific agonists and antagonists of these receptors have been shown to reduce or enhance excitotoxic cell damage. The goal of the laboratory is to better understand the relationship between the functional and morphological complexity of retinal cell classes and the diversity of GluRs. Specifically, three projects will be pursued: 1) Metabotropic glutamate receptors in retina. At least six metabotropic glutamate receptors, i.e. receptors that activate second messenger pathways upon glutamate stimulation, are expressed in the retina. Using subtype- specific antibodies, we will determine the localization of these receptors in the retina. In situ hybridization and immunohistochemical studies will be done to analyze the pattern of expression of these receptors during development and retinal degeneration, both of which could be regulated by metabotropic receptors. Finally, the possible modulation of voltage- and ligand-gated channels by metabotropic receptors will be analyzed in dissociated retinal neurons in culture. 2) Signal transduction pathway in ON bipolar cells. Since photoreceptor cells hyperpolarize in response to light stimuli, whereas ON bipolar cells depolarize, the response of ON bipolar cells is sign-inverting. This response is probably mediated by a metabotropic glutamate receptor, mGluR6. However, little is known about the second messenger pathway coupled to this receptor. Using a cell sorting procedure, we have prepared RNA from enriched populations of bipolar cells. A cDNA clone encoding a novel putative cGMP-gated channel w as identified from this material and it will be functionally characterized. The same procedure will be used to clone additional components of this pathway, such as G protein subunits. 3) Kainate-type glutamate receptors in retina. Of the three ionotropic glutamate receptor classes, NMDA, AMPA and kainate receptors, the functions of the last are the most poorly understood. We have shown that the expression of several kainate receptor subunit genes is affected by retinal degeneration in the rd mouse. We will raise subunit-specific antibodies against these receptor subunits and analyze these changes at the receptor protein level. By in situ hybridization we have also found that GluR7 has a distinctive pattern of expression during development, since it is transiently expressed in photoreceptors. Making use of very recently constructed transgenic mice,in which the GlluR7 gene is inactivated, we will analyze the role of this receptor subunit in retinal morphology.