CELL BIOLOGY OF THE RETINA

描述（申请人的描述）：我们的研究目标是 了解哺乳动物的视网膜如何分析视觉世界，并编码其 空间、时间和色彩对比度转化为动作电位信息 安全传送到大脑。在视网膜中，多巴胺是调节剂 负责许多导致神经适应光的事件。到 确定控制释放的机制和神经网络 多巴胺，我们标记多巴胺能细胞（DA细胞），通过引入到 小鼠基因组人类胎盘碱性磷酸酶的cDNA连接到一个 酪氨酸羟化酶基因的启动子序列。由于存在 我们可以研究DA的突触连接 细胞，通过单细胞RT-PCR鉴定其受体和通道， 通过膜片钳技术研究其生理学。因此，我们证明， 细胞自发地以有节奏的方式激发动作电位， 突触输入，这种活动导致多巴胺的突触外释放。 本申请具有以下目的：通过单细胞RT-PCR， 发现DA细胞含有钠通道Scn 8a的转录本， 我们正计划确定它是否对心脏起搏器负责 DA细胞在其基因无效突变纯合子小鼠中的电流。到 为了研究完整视网膜中DA细胞的光反应，我们 产生了一个转基因品系，其中这些神经元被标记为绿色 荧光蛋白（GFP）。在这些动物身上，我们将研究光的影响 刺激离体视网膜DA细胞反应。 我们将继续分析DA细胞的受体和离子通道， 识别控制多巴胺释放的机制和网络。本 目的：将单细胞RT-PCR技术与联合收割机、 电生理学和免疫细胞化学。我们已经开始研究 为了获得受体和通道的代表性cDNA探针， 鉴定视网膜神经元，并通过高密度cDNA杂交对其进行测试 微阵列希望这种方法能加快并丰富我们的功能 DA细胞和其他类型的视网膜神经元的研究。

DESCRIPTION (Applicant's Description): The objective of our research is to understand how the retina of mammals analyzes the visual world and encodes its spatial, temporal and chromatic contrast into a message of action potentials for safe sending to the brain. In the retina, dopamine is the modulator responsible for many of the events that lead to neural adaptation to light. To identify the mechanisms and neural networks that control the release of dopamine, we labeled dopaminergic cells (DA cells) by introducing into the mouse genome the cDNA of human placental alkaline phosphatase linked to a promoter sequence of the gene for tyrosine hydroxylase. Because of the presence of the reporter gene product, we could study the synaptic connections of DA cells, identify their receptors and channels by single-cell RT-PCR and investigate their physiology by patch clamping. We have thus shown that DA cells spontaneously fire action potentials in a rhythmic fashion in absence of synaptic inputs and that this activity cause extrasynaptic release of dopamine. The present application has the following aims: By single-cell RT-PCR we have found that DA cells contain the transcripts for the sodium channels Scn8a and we are planning to establish whether it is responsible for the pacemaker current of DA cells in mice homozygous for a null mutation of its gene. To investigate the light responses of DA cells in the intact retina, we have produced a transgenic line in which these neurons are labeled with the green fluorescent protein (GFP). In these animals, we will study the effects of light stimulation on DA cells responses in the isolated retina maintained in vitro. We will continue our analysis of the receptors and ion channels of DA cells to identify mechanisms and networks that control dopamine release. To this purpose, we will combine the techniques of single-cell RT-PCR, electrophysiology, and immunocytochemistry. We have begun to develop methods for obtaining representative cDNA probes of receptors and channels from single, identified, retinal neurons and test them by hybridization on high density cDNA microarrays. Hopefully, this approach will speed up and enrich our functional studies of DA cells and other types of retinal neurons.