Constructing a Rapid Solution Exchange System

  • David M. MacLeanEmail author
Part of the Neuromethods book series (NM, volume 106)


The detailed study of ligand-gated channels often requires the reproducible, rapid, and temporally precise application of defined concentrations of ligands. There are numerous different systems for ligand application offering trade-offs between cost, ease of use, volume of compound needed, etc. When studying the extremely rapid kinetics of activation and desensitization in ionotropic ligand-gated receptors (iGluR), a prime consideration is how fast solutions can be exchanged. Of the available systems, rapid piezo-driven translation of parallel solutions streams over an outside-out patch offers the fastest and most versatile option, with open tip current rise-times in the 100–300 μs range, and occasionally as fast 20 μs (Bowie and Lange, J Neurosci 22:3392–3403, 2002; Carbone and Plested, Neuron 74:845–857, 2012; Maclean and Bowie, J Physiol 589:5383–5390, 2011; Robert and Howe, J Neurosci 23:847–858, 2003), and pulse durations ≤1 ms (Bowie and Lange, J Neurosci 22:3392–3403, 2002; Carbone and Plested, Neuron 74:845–857, 2012; Robert and Howe, J Neurosci 23:847–858, 2003). Indeed, under ideal conditions agonist applications as short as 100 μs may be possible (Auzmendi et al., PLoS One 7:e42275, 2012). However, from the experimenter’s perspective a “fast” system may also mean a system which rapidly changes test solutions, i.e., how fast can one switch between the various agonists or concentrations being tested. And while the former sense of “fast” is certainly more important in that it is necessary for certain experiments, time spent optimizing the latter yields substantial rewards. With fast switching amongst test solutions, one can complete experiments quicker, easier, and with fewer stressful moments hoping your best patch of the week hangs on for that last data point. This protocol outlines the construction of a double or multi-barrel solution exchange system which can rapidly jump between solutions (10–90 % rise-time 50–300 μs) but can also quickly switch amongst several test solutions (<0.5 s). This protocol can also be modified for constructing application pipettes for whole-cell recording.

Key words

Rapid perfusion Theta tube Outside-out patch Electrophysiology Ligand-gated ion channel Ionotropic ligand-gated receptors 



This work was supported in part by an NSF grant MCB-1110501 to Dr. Vasanthi Jayaraman and an American Heart Association Postdoctoral Fellowship to Dr. MacLean. I wish to thank Dr. Andrew Plested for sharing samples and specifications of multi-barrel glass, Drs. James Howe, Derek Bowie, and Bryan Daniels as well as Patricia Brown and George Dawe for discussions on application pipette construction, Drs. Howe, Popescu, and Hansen for comments on the manuscript, and Dr. Bowie for instruction in an earlier version of this method.


  1. 1.
    Clements JD et al (1992) The time course of glutamate in the synaptic cleft. Science 258(5087):1498–1501CrossRefPubMedGoogle Scholar
  2. 2.
    Scimemi A, Beato M (2009) Determining the neurotransmitter concentration profile at active synapses. Mol Neurobiol 40(3):289–306PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Fenwick EM, Marty A, Neher E (1982) A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J Physiol 331:577–597PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Krishtal OA, Pidoplichko VI (1980) A receptor for protons in the nerve cell membrane. Neuroscience 5(12):2325–2327CrossRefPubMedGoogle Scholar
  5. 5.
    Franke C, Hatt H, Dudel J (1987) Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. Neurosci Lett 77(2):199–204CrossRefPubMedGoogle Scholar
  6. 6.
    Barberis A (2012) Fast perfusion methods for the study of ligand-gated ion channels. Neuromethods 67:173–187CrossRefGoogle Scholar
  7. 7.
    Jonas P (1995) Fast application of agonists to isolated membrane patches. In: Sakmann B, Neher E (eds) Single-channel recording. Plenum, New York, pp 231–243CrossRefGoogle Scholar
  8. 8.
    Tang CM (2001) Rapid solution application methods. Curr Protoc Neurosci Chapter 6:Unit 6.9PubMedGoogle Scholar
  9. 9.
    Colquhoun D, Jonas P, Sakmann B (1992) Action of brief pulses of glutamate on AMPA/kainate receptors in patches from different neurones of rat hippocampal slices. J Physiol 458:261–287PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Raman IM, Trussell LO (1992) The kinetics of the response to glutamate and kainate in neurons of the avian cochlear nucleus. Neuron 9(1):173–186CrossRefPubMedGoogle Scholar
  11. 11.
    Barberis A, Sachidhanandam S, Mulle C (2008) GluR6/KA2 kainate receptors mediate slow-deactivating currents. J Neurosci 28(25):6402–6406CrossRefPubMedGoogle Scholar
  12. 12.
    Robert A, Howe JR (2003) How AMPA receptor desensitization depends on receptor occupancy. J Neurosci 23(3):847–858PubMedGoogle Scholar
  13. 13.
    Perrais D, Coussen F, Mulle C (2009) Atypical functional properties of GluK3-containing kainate receptors. J Neurosci 29(49):15499–15510CrossRefPubMedGoogle Scholar
  14. 14.
    Bowie D, Lange GD (2002) Functional stoichiometry of glutamate receptor desensitization. J Neurosci 22(9):3392–3403PubMedGoogle Scholar
  15. 15.
    Carbone AL, Plested AJ (2012) Coupled control of desensitization and gating by the ligand binding domain of glutamate receptors. Neuron 74(5):845–857CrossRefPubMedGoogle Scholar
  16. 16.
    Rosenmund C, Stern-Bach Y, Stevens CF (1998) The tetrameric structure of a glutamate receptor channel. Science 280(5369):1596–1599CrossRefPubMedGoogle Scholar
  17. 17.
    Maclean DM, Bowie D (2011) Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation. J Physiol 589(Pt 22):5383–5390PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Schwenk J et al (2009) Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors. Science 323(5919):1313–1319CrossRefPubMedGoogle Scholar
  19. 19.
    Tomita S et al (2005) Stargazin modulates AMPA receptor gating and trafficking by distinct domains. Nature 435(7045):1052–1058CrossRefPubMedGoogle Scholar
  20. 20.
    von Engelhardt J et al (2010) CKAMP44: a brain-specific protein attenuating short-term synaptic plasticity in the dentate gyrus. Science 327(5972):1518–1522CrossRefGoogle Scholar
  21. 21.
    Zhang W et al (2009) A transmembrane accessory subunit that modulates kainate-type glutamate receptors. Neuron 61(3):385–396PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    MacLean DM et al (2014) Stargazin promotes closure of the AMPA receptor ligand-binding domain. J Gen Physiol 144:503Google Scholar
  23. 23.
    Auzmendi J et al (2012) Achieving maximal speed of solution exchange for patch clamp experiments. PLoS One 7(8):e42275PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Sachs F (1999) Practical limits on the maximal speed of solution exchange for patch clamp experiments. Biophys J 77(2):682–690PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Sirrieh RE, MacLean DM, Jayaraman V (2013) Amino-terminal domain tetramer organization and structural effects of zinc binding in the N-methyl-D-aspartate (NMDA) receptor. J Biol Chem 288(31):22555–22564PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Zhang W et al (2008) Structural and single-channel results indicate that the rates of ligand binding domain closing and opening directly impact AMPA receptor gating. J Neurosci 28(4):932–943CrossRefPubMedGoogle Scholar
  27. 27.
    Jones MV et al (1998) Defining affinity with the GABAA receptor. J Neurosci 18(21):8590–8604PubMedGoogle Scholar
  28. 28.
    Plested AJ, Mayer ML (2009) AMPA receptor ligand binding domain mobility revealed by functional cross linking. J Neurosci 29(38):11912–11923PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of TexasHoustonUSA

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