The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10–15 s. This protocol, which has been validated in our recent studies and takes 20–60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.
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This work was supported by the Wellcome Trust, Medical Research Council (UK), the European Research Council (Advanced Grant), the European Commission COST Action BM1001 ECMNet, and the Biology and Biotechnology Research Council (UK).
The authors declare no competing financial interests.
The recording (Video 1) starts with the right and left θ-glass pipette channels ejecting distilled water and ACSF, respectively. The stream boundaries and the interface can be seen as image (phase) contrast. Solutions are swapped at three time points (min: sec): 1:11, 1:50 and 2:34. Starting from the 2:34 time point both channels eject ACSF (yielding an invisible interface and boundaries). Note that because of a reclined position of the pipette holder (Fig. 2c) the θ-glass pipette tip is out of focus when the recording pipette tip is in focus. The piezo-driven θ-glass pipette movements are not visible because they are too fast (∼150-200 μs) to be captured using the standard 24 frames per second rate of video recording. (AVI 5411 kb)
The pulling of an outside-out patch and transferring the patch pipette to the optimised XYZ position of the tip, as determined during the preceding calibration procedure. An acute cerebellar slice experiment, see 20 for details. (AVI 12629 kb)
The pulling of a nucleated patch from the same cell as that used in Supplementary Video 2, also depicting pipette positioning and the beginning of ligand application. An acute cerebellar slice experiment, see 20 for details. (AVI 17558 kb)
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Sylantyev, S., Rusakov, D. Sub-millisecond ligand probing of cell receptors with multiple solution exchange. Nat Protoc 8, 1299–1306 (2013). https://doi.org/10.1038/nprot.2013.075
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