Skip to main content

Advertisement

SpringerLink
Log in

Calcium Transient Registration in Response to Single Stimulation and During Train of Pulses in Mouse Neuromuscular Junction

  • Published:
BioNanoScience Aims and scope Submit manuscript

Abstract

Calcium (Ca2+) is a key ion involved in transmitter release in chemical synapses. Optical recording of fluorescence changes of Ca2+ indicators is one of the most frequently used methods to measure intracellular Ca2+ dynamics. This technique is based on use of Ca2+-binding fluorescent dyes which change their emission intensity after binding to Ca2+. The most crucial step in this type of experiments is loading of Ca2+ dye. In this paper, we present the method of Ca2+-sensitive dye loading to mammalian nerve endings through the stump of the nerve. We represent Ca2+ transient registered parameters in response to a single motor nerve stimulus. The study of Ca2+ dynamics during high frequency stimulation close to real pattern of synaptic transmission allows us to understand such fundamental process as synaptic plasticity. We describe the results obtained during the registration of Ca2+ transient caused by the rhythmic motor nerve stimulation. Intracellular level of Ca2+ estimated by the amplitude of Ca2+ transient rises with the increase of stimulation frequency. The amplitude of Ca2+ transient decreases after blocking of voltage dependent Ca2+ channels by cadmium. The obtained data showed that detected increase of fluorescence intensity is induced by Ca2+ influx through the voltage-gated Ca2+ channels to the nerve ending during an action potential. This dye-loading method is suitable for registration of presynaptic Ca2+ dynamics under both single nerve stimulus and rhythmic activity.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Katz, B., & Miledi, R. (1965). The effect of calcium on acetylcholine release from motor nerve terminals. Proc Roy Soc London B Biol Science, 161, 496–503.

    Article  Google Scholar 

  2. Smith, S. J., & Augustine, G. J. (1988). Calcium ions, active zones and synaptic transmitter release. Trends in Neurosciences, 11, 458–464.

    Article  Google Scholar 

  3. Jappy, D., Valiullina, F., Draguhn, A., Rozov, A. (2016). GABABR-dependent long-term depression at hippocampal synapses between CB1-positive interneurons and CA1 pyramidal cells. Frontiers in Cellular Neuroscience, 10, 4. doi:10.3389/fncel.2016.00004.

    Article  Google Scholar 

  4. Neher, E. (1995). The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology, 34, 1423–1442.

    Article  Google Scholar 

  5. Khaziev, E. F., Fatikhov, N. F., Samigullin, D. V., Barrett, G. L., Bukharaeva, E. A., Nikolsky, E. E. (2012). Decreased entry of calcium into motor nerve endings upon activation of presynaptic cholinergic receptors. Doklady Biological Sciences, 446, 283–285.

    Article  Google Scholar 

  6. Mintz, I. M., Sabatini, B. L., Regehr, W. G. (1995). Calcium control of transmitter release at a cerebellar synapse. Neuron, 15, 675–688.

    Article  Google Scholar 

  7. Molgó, J., & Mallart, A. (1985). Effects of Anemonia sulcatatoxin II on presynaptic currents and evoked transmitter release at neuromuscular junctions of the mouse. Pflügers Archiv, 405(4), 349–353.

    Article  Google Scholar 

  8. Slutsky, I., Rashkovan, G., Parnas, H., Parnas, I. (2002). Ca2 + -independent feedback inhibition of acetylcholine release in frog neuromuscular junction J. Neuroscience, 22(9), 3426–3433.

    Google Scholar 

  9. Tsien, R. Y. (1989). Fluorescent indicators of ion concentrations. Methods in Cell Biology, 30, 127–156.

    Article  Google Scholar 

  10. Macleod, T. G. (2012). Direct injection of indicators for calcium imaging at the Drosophila larval neuromuscular junction. Cold Spring Harbor Protocols, 2012, 797–801.

    Google Scholar 

  11. Regehr, W. G. (2005). Monitoring presynaptic calcium dynamics with membrane-permeant indicators. In R. Yuste & A. Konnerth (Eds.), Imaging in neuroscience and development: a laboratory manual (pp. 307–314). New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  12. Petrov, K., Girard, E., Nikitashina, A., Colasante, C., Bernard, V., Nurullin, L., et al. (2014). Schwann cells sense and control acetylcholine spillover at the neuromuscular junction by α7 nicotinic receptors and butyrylcholinesterase. The Journal of Neuroscience, 34(36), 11870–11873.

    Article  Google Scholar 

  13. Peng, Y. Y., & Zucker, R. S. (1993). Release of LHRH is linearly related to the time integral of presynaptic Ca2+ elevation above a threshold level in bullfrog sympathetic ganglia. Neuron, 10, 465–473.

    Article  Google Scholar 

  14. Macleod, T. G. (2012). Forward-filling of dextran-conjugated indicators for calcium imaging at the drosophila larval neuromuscular junction. Cold Spring Harbor Protocols, 2012, 791–796.

    Google Scholar 

  15. Zhilyakov, N., Khaziev, E., Sudakov, I., Kazakov, A., Aleksandrov, M., Samigullin, D. (2016). Loading mammalian nerve endings with calcium dye through the nerve stump. Inter Res J, 3(45), 12–16.

    Google Scholar 

  16. David, G., & Barrett, E. F. (2003). Mitochondrial Ca2+ uptake prevents desynchronization of quantal release and minimizes depletion during repetitive stimulation of mouse motor nerve terminals. Journal of Physiology, 548(Pt 2), 425–438.

    Article  Google Scholar 

  17. Soga-Sakakibara, S., Kubota, M., Suzuki, S., Akita, T., Narita, K., Kuba, K. (2010). Calcium dependence of the priming, activation and inactivation of ryanodine receptors in frog motor nerve terminals. European Journal of Neuroscience, 32(6), 948–962.

    Article  Google Scholar 

  18. Kazakov, A., Aleksandrov, M., Zhilyakov, N., Khaziev, E., Samigullin, D. (2015). A simple suction electrode for electrical stimulation of biological objects. Inter Res J, 40(9), 13–16.

    Google Scholar 

  19. Vyshedskiy, A., & Lin, J. W. (2000). Presynaptic Ca2+ influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution. Journal of Neurophysiology, 83, 552–562.

    Google Scholar 

  20. Samigullin, D., Fatikhov, N., Khaziev, E., Skorinkin, A., Nikolsky, E., Bukharaeva, E. (2015). Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes. Frontiers Synaptic Neuroscience, 6, 29. doi:10.3389/fnsyn.2014.00029.

    Article  Google Scholar 

  21. Regehr, W. G., & Atluri, P. P. (1995). Calcium transients in cerebellar granule cell presynaptic terminals. Biophysical Journal, 68, 2156–2170.

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the Russian Government’s Program for Competitive Growth of Kazan Federal University and a grants from the Russian Foundation for Basic Research (16-04-01051; 16-34-00817; 15-04-02983).

Author information

Authors and Affiliations

  1. Open Laboratory of Neuropharmacology, Kazan Federal University, Kazan, Russia

    Dmitry V. Samigullin, Eduard F. Khaziev, Nikita V. Zhilyakov, Ellya A. Bukharaeva & Eugeny E. Nikolsky

  2. Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, Russia

    Dmitry V. Samigullin, Eduard F. Khaziev, Nikita V. Zhilyakov, Igor A. Sudakov, Ellya A. Bukharaeva & Eugeny E. Nikolsky

  3. Department of Radiophotonics and Microwave Technologies, Kazan National Research Technical University named after A.N.Tupolev, Kazan, Russia

    Dmitry V. Samigullin & Eduard F. Khaziev

  4. Department of Medical and Biological Physics, Kazan State Medical University, Kazan, Russia

    Eugeny E. Nikolsky

Authors
  1. Dmitry V. Samigullin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eduard F. Khaziev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nikita V. Zhilyakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Igor A. Sudakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ellya A. Bukharaeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eugeny E. Nikolsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dmitry V. Samigullin.

Ethics declarations

The experimental protocol met the requirements of the European Communities Council Directive 86/609/EEC and approved by the Ethical Committee of Kazan Medical University.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Samigullin, D.V., Khaziev, E.F., Zhilyakov, N.V. et al. Calcium Transient Registration in Response to Single Stimulation and During Train of Pulses in Mouse Neuromuscular Junction. BioNanoSci. 7, 162–166 (2017). https://doi.org/10.1007/s12668-016-0318-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12668-016-0318-6

Keywords

Search

Navigation