Automated patch-clamp electrophysiology

Measurements were performed with the IonFlux instrument (Molecular Devices, Sunnyvale, CA), which utilizes microfluidic compound delivery on timescales below 100 ms, facilitating fast activating ligand gated ion channel measurements. Using this platform, a large number of cells (20 per trap channel) can be held under voltage clamp and exposed to a series of compound concentrations within a short time period in parallel across a plate. Continuous recording coupled with fast solution exchange enables high-throughput. The IonFlux 16 plate layout consists of units of twelve wells; two wells contain intracellular solution (cytosolic), one contains cells diluted in extracellular solution, eight contain the compounds of interest diluted in extracellular solution, and one well is for waste collection. Cells are captured from suspension by applying suction to microscopic channels in ensemble recording arrays. Once the array is fully occupied, the applied suction breaks the cell membranes of captured cells, establishing a whole cell voltage clamp. For compound applications, pressure is applied to the appropriate compound wells, introducing the compound into the extracellular solution that is rapidly flowing over the cells. To record GABA_AR-mediated currents, cell arrays were voltage clamped at a holding potential of −80 mV. IonFlux software (IonFlux App) was used for data acquisition and exported into Excel (Microsoft) for data analysis. The data were uploaded to GraphPad Prism 5 (GraphPad Software) for visualization, determination of standard deviations, and non-linear regression analysis using Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) for concentration-dependent response curves.