Acosta-García Ma Cristina, Morales-Reyes Israel, Jiménez-Anguiano Anabel, Batina Nikola, Castellanos N P, Godínez-Fernández R
Posgrado en Biología Experimental, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, México City, Mexico.
Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, México City, Mexico.
Heliyon. 2018 Mar 1;4(2):e00550. doi: 10.1016/j.heliyon.2018.e00550. eCollection 2018 Feb.
This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.
本文展示了通过使用金基底培养NG108 - 15细胞来同时记录电活动和潜在离子电流。通过扫描电子显微镜(SEM)对在两种不同基底(塑料培养皿和金基底)上生长的细胞进行了定量表征,并通过光学显微镜和原子力显微镜(AFM)进行了定性表征。如在SEM图像上进行的测量所示,在金基底和培养皿上培养的细胞表面积之间未观察到显著差异。我们还通过标准膜片钳实验,通过分析静息电位、膜电阻、离子电流等特征,评估了细胞的电生理兼容性。在两种基底上生长的细胞在对电压的依赖性以及Na +和K +电流密度的大小方面均未显示出显著差异;然而,与在培养皿上生长的细胞相比,在金基底上培养的细胞显示出较低的膜电容。通过使用两个独立的膜片钳放大器,我们能够用传统的膜片钳技术并同时通过金基底记录膜电流。此外,所提出的技术使我们能够同时记录电活动(如动作电位发放)和潜在的膜离子电流。金的优异导电性使得克服传统电生理实验中发现的重要困难成为可能,例如由电解浴溶液的电阻所带来的困难。我们得出结论,本文所介绍的技术构成了对电活动和潜在离子电流同时记录问题的一种解决方案,几十年来,该问题一直仅得到部分解决。