Development of a slice preparation of the mammalian retina

哺乳动物视网膜切片制备的开发

• 批准号: 07558294
• 负责人: KANEKO Akimichi
• 金额: $ 0.64万
• 依托单位: Keio University, School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558294/
• 关键词: retina; slice preparation; patch clamp; goldfish; mouse; シナプス

The vertebrate retina is a thin nervous tissue with a thickness of approximately 200 mum consisting of 5 types of neurons. To understand the mechanism of visual information processing in the retina, it is important to know the characters of membrane ionic channels, transmitter receptors and the input-output relation in all types of neurons composing the retina. Patch clamp studies on dissociated and cultured retinal neurons provided a vast amount of knowledge on the elementary processes. However, the isolated cell preparation is not suitable for the study of the dynamics of the synapse. The classical intracellular recording technique is used to record light-induced voltage responses of each neuron, but the obtained data is insufficient for quantitative analysis. The slice preparation has a powerful advantage in that the neural circuit is preserved and the cells composing a synapse are accessible under the direct visual control. The present study was made to develop a technique with which slice preparations can be made of the mammalian retina and the cells in the slice preparation can be recorded by a patch clamp technique.With collaboration of the machine shop of our University, we designed and constructed a slicer suitable for the retina. The isolated retina is mounted on a Millipore filter, with the receptor side up, and the retina-filter paper is sectioned vertically by a razor blade attached to the arm of the slicer. Once sectioned, the preparation is advanced by a micrometer for 100-200mum. By repeating this process, we obtained retinal slices of the thickness we wanted. The slice is immediately moved to a neighboring recording chamber that can be mounted on the stage of the microscope for recording and examining the effects of various pharmacological agents by bath application. We began experiments on goldfish retina, and are now studying the retina from the mouse.

脊椎动物视网膜是一种薄的神经组织，厚度约为200毫米，由5种神经元组成。为了了解视网膜视觉信息加工的机制，了解构成视网膜的各类神经元的膜离子通道、递质受体和输入输出关系的特性是非常重要的。膜片钳对分离和培养的视网膜神经元的研究提供了大量关于基本过程的知识。然而，分离细胞的制备并不适合于突触动力学的研究。经典的细胞内记录技术用于记录每个神经元的光致电压响应，但所获得的数据不足以进行定量分析。切片制备的优势在于保留了神经回路，并且构成突触的细胞在直接的视觉控制下是可接近的。本研究旨在建立一种哺乳动物视网膜切片制备技术，并利用膜片钳技术记录切片制备中的细胞。我们与学校的机械车间合作，设计并制作了适合视网膜的切片机。分离的视网膜被安装在一个Millipore过滤器上，受体的一面朝上，视网膜滤纸被连接在切片机手臂上的刀片垂直切割。一旦切片，该制剂将在100-200mum的范围内推进1微米。通过重复这个过程，我们得到了我们想要的厚度的视网膜切片。切片立即移动到相邻的记录室，该记录室可以安装在显微镜的舞台上，用于记录和通过沐浴应用检查各种药理学试剂的效果。我们开始在金鱼的视网膜上做实验，现在正在研究老鼠的视网膜。

项目成果

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Aoki K：“小鼠胚胎嗅觉受体神经元初始寻路过程中 N-CAM、波形蛋白和 MAP1B 的差异表达。”

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Takahashi,K.-I.,Miyoshi,S.& Kaneko,A.: "GABA-induced chloride current in catfish horizontal cells mediated by non-GABA_A receptor channels." Jph.J.Physiol.45. 437-456 (1995)

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