Development of a slice preparation of the mammalian retina

脊椎动物视网膜是一种厚度约200微米的薄神经组织，由5种类型的神经元组成。为了了解视网膜的视觉信息处理机制，了解膜离子通道、递质受体的特性以及组成视网膜的各种神经元的输入输出关系是非常重要的。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，分离的细胞制备不适合于突触动力学的研究。经典的细胞内记录技术被用来记录每个神经元的光诱导电压响应，但所获得的数据不足以进行定量分析。切片制备的强大优势在于保留了神经回路，在直接视觉控制下可以接触到组成突触的细胞。本研究旨在开发一种可以制作哺乳动物视网膜切片的技术，并利用膜片钳技术记录切片中的细胞。我们与我校机械车间合作，设计并制造了一台适用于视网膜的切片机。分离的视网膜安装在微孔滤光片上，感受器的一面朝上，视网膜滤纸由连接到切片器手臂上的剃须刀片垂直切割。一旦切开，该制剂被推进一微米100-200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。切片被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，用于通过沐浴应用来记录和检查各种药物的效果。我们开始了金鱼视网膜的实验，现在正在研究小鼠的视网膜。

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.