Sub-millisecond ligand probing of cell receptors with multiple solution exchange


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Preparation of the rapid solution application pipette for RASE.
Figure 2: The arrangement of multiple solution supply and exchange.
Figure 3: Calibration and adjustment of rapid solution application.
Figure 4: Examples of protocol implementation in single-channel recordings and in dose-response rapid receptor probing.
Figure 5: Examples of protocol implementations in testing mGuR-NMDAR interaction on the millisecond time scale.


  1. 1

    Clements, J.D., Lester, R.A., Tong, G., Jahr, C.E. & Westbrook, G.L. The time course of glutamate in the synaptic cleft. Science 258, 1498–1501 (1992).

    CAS  Article  Google Scholar 

  2. 2

    Lisman, J.E., Raghavachari, S. & Tsien, R.W. The sequence of events that underlie quantal transmission at central glutamatergic synapses. Nat. Rev. Neurosci. 8, 597–609 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Zheng, K., Scimemi, A. & Rusakov, D.A. Receptor actions of synaptically released glutamate: the role of transporters on the scale from nanometers to microns. Biophys. J. 95, 4584–4596 (2008).

    CAS  Article  Google Scholar 

  4. 4

    Krishtal, O.A. & Pidoplichko, V.I. A receptor for protons in the nerve cell membrane. Neuroscience. 5, 2325–2327 (1980).

    CAS  Article  Google Scholar 

  5. 5

    Franke, C., Hatt, H. & Dudel, J. Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. Neurosci. Lett. 77, 199–204 (1987).

    CAS  Article  Google Scholar 

  6. 6

    Raman, I.M. & Trussell, L.O. The kinetics of the response to glutamate and kainate in neurons of the avian cochlear nucleus. Neuron 9, 173–186 (1992).

    CAS  Article  Google Scholar 

  7. 7

    Albuquerque, E.X. et al. Functional properties of the nicotinic and glutamatergic receptors. J. Recept. Res. 11, 603–625 (1991).

    CAS  Article  Google Scholar 

  8. 8

    Brett, R.S., Dilger, J.P., Adams, P.R. & Lancaster, B. A method for the rapid exchange of solutions bathing excised membrane patches. Biophys. J. 50, 987–992 (1986).

    CAS  Article  Google Scholar 

  9. 9

    Lester, R.A.J. & Jahr, C.E. NMDA channel behavior depends on agonist affinity. J. Neurosci. 12, 635–643 (1992).

    CAS  Article  Google Scholar 

  10. 10

    Tong, G. & Jahr, C.E. Multivesicular release from excitatory synapses of cultured hippocampal-neurons. Neuron 12, 51–59 (1994).

    CAS  Article  Google Scholar 

  11. 11

    Raman, I.M., Zhang, S. & Trussell, L.O. Pathway-specific variants of AMPA receptors and their contribution to neuronal signaling. J. Neurosci. 14, 4998–5010 (1994).

    CAS  Article  Google Scholar 

  12. 12

    Colquhoun, D., Jonas, P. & Sakmann, B. Action of brief pulses of glutamate on AMPA/kainate receptors in patches from different neurones of rat hippocampal slices. J. Physiol. 458, 261–287 (1992).

    CAS  Article  Google Scholar 

  13. 13

    Spruston, N., Jonas, P. & Sakmann, B. Dendritic glutamate-receptor channels in rat hippocampal CA3 and CA1 pyramidal neurons. J. Physiol. 482, 325–352 (1995).

    CAS  Article  Google Scholar 

  14. 14

    Sachs, F. Practical limits on the maximal speed of solution exchange for patch-clamp experiments. Biophys. J. 77, 682–690 (1999).

    CAS  Article  Google Scholar 

  15. 15

    Veselovsky, N.S., Engert, F. & Lux, H.D. Fast local superfusion technique. Pflugers Arch. 432, 351–354 (1996).

    CAS  Article  Google Scholar 

  16. 16

    Niu, L. et al. Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 93, 12964–12968 (1996).

    CAS  Article  Google Scholar 

  17. 17

    Maconochie, D.J. & Knight, D.E. A method for making solution changes in the sub-millisecond range at the tip of a patch pipette. Pflugers Arch. 414, 589–596 (1989).

    CAS  Article  Google Scholar 

  18. 18

    Savtchenko, L.P., Sylantyev, S. & Rusakov, D.A. Central synapses release a resource-efficient amount of glutamate. Nat. Neurosci. 16, 10–12 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Sylantyev, S. et al. Electric fields due to synaptic currents sharpen excitatory transmission. Science 319, 1845–1849 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Sylantyev, S., Savtchenko, L.P., Ermolyuk, Y., Michaluk, P. & Rusakov, D.A. Spike-driven glutamate electrodiffusion triggers synaptic potentiation via a Homer-dependent mGluR-NMDAR link. Neuron 77, 528–541 (2013).

    CAS  Article  Google Scholar 

  21. 21

    Wlodarczyk, A.I. et al. GABA-independent GABAA receptor openings maintain tonic currents. J. Neurosci. 33, 3905–3914 (2013).

    CAS  Article  Google Scholar 

  22. 22

    Marty, A. & Neher, E. Tight-seal whole-cell recording. In Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 31–52 (Springer, 2009).

  23. 23

    Molleman, A Patch Clamping: An Introductory Guide To Patch-Clamp Electrophysiology (J. Wiley & Sons, 2003).

  24. 24

    Penner, R. A practical guide to patch clamping. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 3–30 (Springer, 2009).

  25. 25

    Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100 (1981).

    CAS  Article  Google Scholar 

  26. 26

    Brown, J., Hainsworth, A., Stefani, A. & Randall, A. Whole-cell patch-clamp recording of voltage-sensitive Ca2+ channel currents in single cells: heterologous expression systems and neurones. in Calcium Signaling Protocols (eds. Lambert, D.G. & Rainbow, R.D.) 123–148 (Humana Press, 2013).

Download references


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).

Author information




S.S. carried out the experimental studies; D.A.R. and S.S. designed the protocol, analyzed the data and wrote the paper.

Corresponding authors

Correspondence to Sergiy Sylantyev or Dmitri A Rusakov.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Calibration of the rapid solution application pipette with an open patch electrode.

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)

Preparing an outside-out patch for the RASE protocol.

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 RASE protocol applied to the nucleated patch pulled from the same cell as an outside-out patch.

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sylantyev, S., Rusakov, D. Sub-millisecond ligand probing of cell receptors with multiple solution exchange. Nat Protoc 8, 1299–1306 (2013).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.