Skip to main content
SpringerLink
Log in

Combining Multiphoton Excitation Microscopy with Fast Microiontophoresis to Investigate Neuronal Signaling

  • Protocol
  • First Online:
Book cover Multiphoton Microscopy

Part of the book series: Neuromethods ((NM,volume 148))

Abstract

Multiphoton excitation (MPE) microscopy allows subcellular structural and functional imaging of neurons and can be combined with techniques for activating postsynaptic receptors at spatial and temporal scales that mimic normal synaptic transmission. Here, we describe procedures for combining MPE imaging of dye-filled neurons with fast microiontophoresis, by which neurotransmitter agonists can be applied from high-resistance micropipettes with subcellular resolution. With adequate compensation of the pipette capacitance, the effective time constant of the pipette is reduced, and this permits application of very brief pulses of receptor agonist (≤1 ms). The consequent high temporal and spatial resolution leads to the high specificity required for single-synapse investigations. This chapter includes detailed procedures for electrophysiological whole-cell recording, structural and functional (Ca2+) MPE imaging of dye-filled neurons, targeting a microiontophoresis pipette to a specific subcellular compartment of a dye-filled neuron under visual control, and capacitance compensation of the microiontophoresis pipette, as well as examples of experimental results that can be obtained.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Silver RA, MacAskill AF, Farrant M (2016) Neurotransmitter-gated ion channels in dendrites. In: Stuart G, Spruston N, Häusser M (eds) Dendrites, 3rd edn. Oxford University Press, New York, pp 217–257

    Chapter  Google Scholar 

  2. Kew JNC, Davies CH (eds) (2010) Ion channels. From structure to function. Oxford University Press, New York

    Google Scholar 

  3. Zheng J, Trudeau MC (2015) Handbook of ion channels. CRC Press, Boca Raton

    Book  Google Scholar 

  4. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76

    Article  CAS  Google Scholar 

  5. Higley MJ, Sabatini BL (2012) Calcium signaling in dendritic spines. Cold Spring Harb Perspect Biol 4:a005686

    Article  Google Scholar 

  6. Müller C, Beck H, Coulter D, Remy S (2012) Inhibitory control of linear and supralinear dendritic excitation in CA1 pyramidal neurons. Neuron 75:851–864

    Article  Google Scholar 

  7. Bootman MD, Berridge MJ, Putney JW, Roderick HL (eds) (2012) Calcium signaling. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  8. Nguyen Q-T, Clay GO, Nishimura N, Schaffer CB, Schroeder LF, Tsai PS, Kleinfeld D (2008) Pioneering applications of two-photon microscopy to mammalian neurophysiology. In: Masters BR, So PTC (eds) Handbook of biomedical nonlinear optical microscopy. Oxford University Press, New York, pp 715–734

    Google Scholar 

  9. Yasuda R, Nimchinsky EA, Scheuss V, Pologruto TA, Oertner TG, Sabatini BL, Svoboda K (2004) Imaging calcium concentration dynamics in small neuronal compartments. Sci STKE 2004(219):pl5

    PubMed  Google Scholar 

  10. Grimes WN, Li W, Chávez AE, Diamond JS (2009) BK channels modulate pre- and postsynaptic signaling at reciprocal synapses in retina. Nat Neurosci 12:585–592

    Article  CAS  Google Scholar 

  11. Stone TW (1985) Microiontophoresis and pressure ejection. IBRO handbook series: Methods in the neurosciences. General ed: Smith AD. Wiley, Chichester

    Google Scholar 

  12. Lalley PM (1999) Microiontophoresis and pressure ejection. In: Windhorst U, Johansson H (eds) Modern techniques in neuroscience research. Springer-Verlag, Berlin, pp 193–212

    Chapter  Google Scholar 

  13. Liu G, Choi S, Tsien RW (1999) Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices. Neuron 22:395–409

    Article  CAS  Google Scholar 

  14. Murnick JG, Dubé G, Krupa B, Liu G (2002) High-resolution iontophoresis for single-synapse stimulation. J Neurosci Meth 116:65–75

    Google Scholar 

  15. Müller C, Remy S (2013) Fast micro-iontophoresis of glutamate and GABA: a useful tool to investigate synaptic integration. J Vis Exp (77). https://doi.org/10.3791/50701

  16. Castilho Á, Ambrósio AF, Hartveit E, Veruki ML (2015) Disruption of a neural microcircuit in the rod pathway of the mammalian retina by diabetes mellitus. J Neurosci 35:3344–3355

    Article  Google Scholar 

  17. Geiger JRP, Bischofberger J, Vida I, Fröbe U, Pfitzinger S, Weber HJ, Haverkampf K, Jonas P (2002) Patch-clamp recording in brain slices with improved slicer technology. Pflügers Arch 443:491–501

    Article  CAS  Google Scholar 

  18. Bischofberger J, Engel D, Li L, Geiger JRP, Jonas P (2006) Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nat Prot 1:2075–2081

    Google Scholar 

  19. Davie JT, Kole MHP, Letzkus JJ, Rancz EA, Spruston N, Stuart GJ, Häusser M (2006) Dendritic patch-clamp recording. Nat Prot 1:1235–1247

    Google Scholar 

  20. Tsai PS, Kleinfeld D (2009) In vivo two-photon laser scanning microscopy with concurrent plasma-mediated ablation: principles and hardware realization. In: Frostig RD (ed) In vivo optical imaging of brain function, 2nd edn. CRC Press, Boca Raton, pp 59–115

    Chapter  Google Scholar 

  21. Mainen ZF, Maletic-Savatic M, Shi SH, Hayashi Y, Malinow R, Svoboda K (1999) Two-photon imaging in living brain slices. Methods 18:231–239

    Article  CAS  Google Scholar 

  22. Dodt H-U, Frick A, Kampe K, Zieglgänsberger W (1998) NMDA and AMPA receptors on neocortical neurons are differentially distributed. Eur J Neurosci 10:3351–3357

    Article  CAS  Google Scholar 

  23. Bers DM, Patton CW, Nuccitelli R (2010) A practical guide to the preparation of Ca2+ buffers. In: Whitaker M (ed) Calcium in living cells. Methods in cell biology, vol 99. Wilson L, Matsudaira P (series eds). Academic Press, Burlington, pp 1–26

    Google Scholar 

  24. Euler T, Hausselt SE, Margolis DJ, Breuninger T, Castell X, Detwiler PB, Denk W (2009) Eyecup scope–optical recordings of light stimulus-evoked fluorescence signals in the retina. Pflügers Arch 457:1393–1414

    Google Scholar 

  25. Pologruto TA, Sabatini BL, Svoboda K (2003) ScanImage: flexible software for operating laser scanning microscopes. Biomed Eng Online 2:13

    Article  Google Scholar 

  26. Langer D, van ’t Hoff M, Keller AJ, Nagaraja C, Pfäffli OA, Göldi M, Kasper H, Helmchen F (2013) HelioScan: a software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility. J Neurosci Meth 215:38–52

    Google Scholar 

  27. Nguyen Q-T, Driscoll J, Dolnick EM, Kleinfeld D (2009) MPScope 2.0: a computer system for two-photon laser scanning microscopy with concurrent plasma-mediated ablation and electrophysiology. In: Frostig RD (ed) In vivo optical imaging of brain function, 2nd edn. CRC Press, Boca Raton, pp 117–142

    Chapter  Google Scholar 

  28. Brown KT, Flaming DG (1986) Advanced micropipette techniques for cell physiology. IBRO handbook series: Methods in the neurosciences. General ed: Smith AD. Wiley, Chichester

    Google Scholar 

  29. Dutta-Moscato J (2007) Microiontophoresis as a technique to investigate spike timing dependent plasticity. MSc thesis, University of Pittsburgh

    Google Scholar 

  30. Nelson R, Kolb H (1985) A17: a broad-field amacrine cell in the rod system of the cat retina. J Neurophysiol 54:592–614

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by The Research Council of Norway (NFR 182743, 189662, 214216 to EH; NFR 213776, 261914 to MLV).

Author information

Authors and Affiliations

  1. Department of Biomedicine, University of Bergen, Bergen, Norway

    Espen Hartveit & Margaret Lin Veruki

Authors
  1. Espen Hartveit
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margaret Lin Veruki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Espen Hartveit .

Editor information

Editors and Affiliations

  1. Department of Biomedicine, University of Bergen, Bergen, Norway

    Espen Hartveit

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Hartveit, E., Veruki, M.L. (2019). Combining Multiphoton Excitation Microscopy with Fast Microiontophoresis to Investigate Neuronal Signaling. In: Hartveit, E. (eds) Multiphoton Microscopy. Neuromethods, vol 148. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9702-2_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9702-2_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9701-5

  • Online ISBN: 978-1-4939-9702-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation