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用于在体全细胞记录皮层神经元和胶质细胞的内在及视觉反应的触摸与电击方法。

The Touch and Zap method for in vivo whole-cell patch recording of intrinsic and visual responses of cortical neurons and glial cells.

作者信息

Schramm Adrien E, Marinazzo Daniele, Gener Thomas, Graham Lyle J

机构信息

Neurophysiology & New Microscopies Laboratory, INSERM U603 - CNRS UMR 8154, Université Paris Descartes, Paris, France.

出版信息

PLoS One. 2014 May 29;9(5):e97310. doi: 10.1371/journal.pone.0097310. eCollection 2014.

Abstract

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe "Touch and Zap", an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the "Touch". By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or "Zap", as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique.

摘要

全细胞贴片记录是定量确定脑功能生物物理学的重要工具,尤其是在体内研究中。这种方法对于研究完整大脑中皮质神经胶质细胞的功能作用特别有意义,而细胞外记录无法评估这些作用。然而,由于稳定性、记录持续时间和电质量的限制,尤其是对于电压钳、动态钳或电导测量,要获得合理的成功率仍然是一个挑战。为了解决这个问题,我们描述了“触碰与电穿孔”(Touch and Zap),这是一种全细胞贴片钳记录的替代方法,目标是比以前的方法更简单、更快且对脑组织更温和。在使用连续一串超极化电流脉冲的电流钳模式下,一旦接触细胞就立即开始形成封接,即“触碰”。通过维持电流注入,随着封接电阻增加,在数秒内从细胞贴附配置通过自限性膜电穿孔自发实现全细胞接入,即“电穿孔”。我们展示了用这种改进方法从猫和大鼠体内皮质获得的神经元和假定神经胶质细胞的内在反应和视觉反应的实例。用“触碰与电穿孔”方法获得的记录参数和生物物理特性与用传统盲目贴片方法获得的相比具有优势,表明改进方法不会损害所记录的细胞。我们发现该方法特别适合于体内皮质神经胶质细胞的全细胞贴片记录,与标准方法相比能针对更广泛的这种细胞类型群体,且接入电阻更好。总体而言,更温和的“触碰与电穿孔”方法有望在对细胞内在特性和局部网络扰动最小的情况下研究完整大脑中的定量功能特性。由于“触碰与电穿孔”方法是半自动执行的,这种方法更具可重复性且对实验者技术的依赖性更小。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1379/4038476/f6f8054f23d5/pone.0097310.g001.jpg

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