Yip Mighten C, Gonzalez Mercedes M, Lewallen Colby F, Landry Corey R, Kolb Ilya, Yang Bo, Stoy William M, Fong Ming-Fai, Rowan Matthew J M, Boyden Edward S, Forest Craig R
George W Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 315 Ferst Dr, Atlanta, GA, 30363, USA.
Ocular and Stem Cell Translational Research Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
bioRxiv. 2024 Aug 15:2024.03.30.587445. doi: 10.1101/2024.03.30.587445.
Significant technical challenges exist when measuring synaptic connections between neurons in living brain tissue. The patch clamping technique, when used to probe for synaptic connections, is manually laborious and time-consuming. To improve its efficiency, we pursued another approach: instead of retracting all patch clamping electrodes after each recording attempt, we cleaned just one of them and reused it to obtain another recording while maintaining the others. With one new patch clamp recording attempt, many new connections can be probed. By placing one pipette in front of the others in this way, one can "walk" across the tissue, termed "patch-walking." We performed 136 patch clamp attempts for two pipettes, achieving 71 successful whole cell recordings (52.2%). Of these, we probed 29 pairs (i.e., 58 bidirectional probed connections) averaging 91 m intersomatic distance, finding 3 connections. Patch-walking yields 80-92% more probed connections, for experiments with 10-100 cells than the traditional synaptic connection searching method.
在测量活脑组织中神经元之间的突触连接时，存在重大的技术挑战。当使用膜片钳技术探测突触连接时，人工操作既费力又耗时。为了提高其效率，我们采用了另一种方法：在每次记录尝试后，不是将所有膜片钳电极都撤回，而是只清洁其中一个电极并重新使用它来获取另一次记录，同时保持其他电极不变。通过一次新的膜片钳记录尝试，可以探测许多新的连接。通过以这种方式将一个移液管放置在其他移液管之前，就可以“走过”组织，这被称为“膜片行走”。我们对两个移液管进行了136次膜片钳尝试，成功获得了71次全细胞记录（52.2%）。在这些记录中，我们探测了29对（即58个双向探测连接），平均体细胞间距离为91μm，发现了3个连接。对于有10 - 100个细胞的实验，与传统的突触连接搜索方法相比，膜片行走产生的探测连接多80 - 92%。
