Automated fast perfusion of Xenopus oocytes for drug screening

Abstract

Fast ('concentration jump') applications of neurotransmitters are crucial for screening studies on ligand-gated ion channels. In this paper, we describe a method for automated fast perfusion of neurotransmitters (or drugs) during two-microelectrode voltage-clamp experiments on Xenopus oocytes. The oocytes are placed in a small bath chamber that is covered by a glass plate with two channels for the microelectrodes that are surrounded by a quartz funnel serving as a reservoir for test solutions. The oocytes are perfused in a vertical direction via the two channels in the plate. Automation of compound delivery is accomplished by means of a programmable pipetting workstation. A mean rise time for 10-90% current increase through muscle-type nACh channels of 55.0+/-1.3 ms (30 muM acetylcholine) was estimated. Automation, fast perfusion rates, and economical use of compounds ( approximately 100 mul/data point) make the system suitable for screening studies on ligand- and voltage-gated ion channels.

Fig. 1

Cross-section view (a) and top view (b) of the oocyte perfusion chamber. Two microelectrodes (1 and 2) are inserted via the sloping access inlets (8) through a glass cover plate (7) into the small (~15 μl) oocyte chamber. Drug is applied by the tip of the liquid handling arm (3) of a TECAN Miniprep 60 to a funnel reservoir made of quartz (6) surrounding the microelectrode access holes. Perfusion of the oocyte (10) that is placed on a cylindrical holding device (15)is enabled by means of the syringe pump (9) of the Miniprep 60 connected to the chamber body (11) via the outlet (12). Residual solution is removed from the funnel before drug application via the funnel outlets (4 and 5). In addition to the ground reference electrode (13), the cylindrical holder for the oocyte contains a reference electrode (14) that serves as an extracellular reference for the potential electrode. Salt bridges can be inserted into the side outlet for the ground electrode (13). c Schematic drawing of the solution flow inside the perfusion chamber and in the annular gap around the cylinder with oocyte. d Photo of the oocyte perfusion chamber. An oocyte (10) is placed on a cylinder and impaled with two microelectrodes (1,2) surrounded by the funnel (6)

Fig. 2

a Schematic diagram of the liquid handling system connected to the perfusion chamber. Neurotransmitters or drugs are applied via the tip of the movable liquid handling arm (LHA). Dashed lines and arrows illustrate LHA movements. LHA is connected to a pump (P1) allowing aspiration and delivery of the desired volumes from the rack with tested solutions (R) to the funnel reservoir (F) of the perfusion chamber. The second syringe pump (P2) is connected to the chamber drain (D). This pump is also connected to a distilled water station (DW). Pump P2 provides a suction pulse to the outlet, permitting fast perfusion of the chamber. Residual volumes of test solutions are removed from the chamber by means of a MiniWash pump module (MW) (TECAN). This module provides a standard suction pulse to the two funnel outlets (FO) that are operated ≈0.5 s before a new test solution is applied to the funnel that excluded mixing of test and wash solutions in the funnel. The outlets of the MiniWash pump module and the pump (P2) are connected to a waste container (W). Arrows indicate the direction of liquid flow in the tube system. b Schematic drawing of the tip of the liquid handling arm (left panel) and fragment of the tip with solutions inside (right panel). To shorten time between applications of different drugs or time between application of drug and washout, the drugs (D1 and D2) and the wash solution (WS) can be aspirated into the tip one after the other and separated from each other by air gaps (AG). c Block diagram of the protocol used for studies on ligand-gated channels. Each cycle (Cycle 1) starts with the standard cleaning step (Wash Tip) of the LHA tip in distilled water (Wash Tips procedure, see TECAN Gemini handbook). Control solutions for washout of drug and test solution (containing a drug or neurotransmitter) are subsequently aspirated [ASP(1) and ASP(2)] into the tip of LHA. Immediately before the test solution is applied, the residual solution is removed via the funnel outlets (residual solution removal, RSR) by means of MW. Application of test solution to the funnel (ATS) and immediate fast perfusion (FP) of the chamber are followed by the subsequent application of wash solution to the chamber (AWS) and rapid washout (FP). The next sequence of commands (Cycle 2) can then be executed. Control bath solution is aspirated and applied to the funnel, and the chamber is slowly perfused (SP, at 1–8 μl/s) to guarantee steady-state conditions throughout the experiment. Cycle 1 can be repeated user-defined depending on the number of drugs to be tested

Fig. 3

Subsequent oocyte perfusion by 1 μM (left trace) and 300 nM (right trace) ACh at different perfusion rates. ACh application (1) and washout (2) were performed at 350 μl/s (‘concentration jump mode’). Between the neurotransmitter applications, the chamber was perfused at low speed 8 μl/s [‘slow perfusion mode’,(3)]

Fig. 4

a Receptor-activated ionic currents in oocytes expressing nACh receptors (α-, β-, γ-, and δ-subunits). I_ACh were induced by subsequent applications of 100 nM, 300 nM, and 1 μM ACh to the same oocyte. b Upon application of 30 μM nACh with a chamber perfusion speed of 300 μl/s, I_ACh increased from 10 to 90% within 85 ms. c Upon application of 30 μM ACh with a chamber perfusion speed of 600 μl/s, I_ACh increased from 10 to 90% within 55 ms

Fig. 5

Estimation of the rate of solution exchange by ‘concentration jump’ application of high-potassium solution during K_v1.1 recordings. a The extracellular potassium concentration was rapidly increased (chamber perfusion speed of 600 μl/s) from 1 to 30 mM during a potassium outward current (voltage step from −80 to +20 mV) (thick line, 2). Superimposed current traces before (1) and after (3) the ‘concentration jump’ are shown as dashed lines. b The time of current decrease from 10 to 90% (t_10–90%) was taken as a measure of the volume rate of oocyte perfusion

Fig. 6

Timed applications of neurotransmitters. Fast perfusion of an oocyte expressing nACh receptors channels with 300 nM ACh for different time periods. I_ACh was induced by consecutive ACh applications for 2, 5, 10, and 20 s. Short periods of ACh application (<20 s) were achieved by successive application of neurotransmitter and wash (control) solution as illustrated in Fig. 2b