Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, United States.
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, United States.
J Neurosci Methods. 2021 Jan 15;348:109008. doi: 10.1016/j.jneumeth.2020.109008. Epub 2020 Nov 23.
Whole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode's aperture and a cell's membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane.
We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette's tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings deep in the brain of the living mouse.
This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26 % (n = 17/64) to 48 % (n = 32/66). Whole-cell recording yield improved from 10 % (n = 9/88) to 24 % (n = 18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex.
COMPARISON WITH EXISTING METHOD(S): Current methods of in vivo whole-cell patch clamping do not synchronize the position of the pipette to motion of the cell.
This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.
全细胞膜片钳记录技术是在行为任务、感觉刺激和光遗传学操作过程中测量单个细胞亚阈电生理特性的金标准方法。然而，这些记录需要在玻璃微电极尖端和细胞膜之间形成一个紧密的千兆欧姆密封。这些密封很难形成，尤其是在体内，部分原因是它们强烈依赖于玻璃微电极尖端和细胞膜之间的距离。
我们阐明并利用了这种依赖性，开发了一种自主的方法，用于将玻璃微电极尖端的位置和同步放置到附近运动神经元的细胞膜上，从而实现高产量的密封形成，并随后在活体小鼠的大脑深处进行记录。
这种同步程序将丘脑中的高阻封接（距软脑膜表面下>3 毫米）的报告产量从 26%（n=17/64）提高到了 48%（n=32/66）。当在高阻封接形成过程中使用运动补偿时，全细胞膜片钳记录的产量从 10%（n=9/88）提高到了 24%（n=18/76）。作为其应用的一个例子，我们利用这个系统研究了感觉环境和腹后内侧核（VPM）投射同步对桶状皮层细胞内动力学的作用。
目前的活体全细胞膜片钳记录方法没有将微电极的位置与细胞的运动同步。
与之前报道的方法相比，这种方法大大提高了皮层下全细胞膜片钳记录的产量，从而使得活体小鼠大脑中的全脑电生理学成为可能。
