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Neurociência

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Published: December 01, 2021
doi:
10.3791/63308
, , , ,
1Laboratory of Biophysics of Synaptic Processes,Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 2Department of Radiophotonics and Microwave Technologies,Kazan National Research Technical University named after A.N. Tupolev, 3Department of Normal Physiology,Kazan State Medical University

Summary

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Abstract

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice’s motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

Introduction

The problem of measuring fast calcium waves in excitable cells is one of the most important and challenging aspects of studying signal transmission in the central and peripheral nervous systems. Calcium ions play an important role in triggering neurotransmitter release, synaptic plasticity, and modulation of the activity of various intracellular proteins1,2,3,4,5. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases6. To measure changes in the calcium levels, fluorescent calcium-sensitive dyes are commonly used, and changes in their fluorescence level are analyzed7,8,9.

Loading of calcium dyes into cells can be achieved in different ways. Predominantly, cell-permeant dyes are used10,11. However, in such a case, it is not only difficult to control the concentration of a dye inside the cell, but it is also hard to select target cells for loading. This method is not applicable for studying peripheral nerve endings since the dye enters postsynaptic cells. Instead, cell impermeant dyes are more suitable for such preparations. In this case, the dyes are delivered to the cells by microinjection or through a patch pipette12,13,14. There is also a method of loading through a nerve stump. The latter method is most suitable for neuromuscular junction preparations15,16,17,18,19,20. It allows performing staining for only cells of interest. Although this method does not provide an accurate evaluation of the concentration of the dye in the target cell, the concentration can be estimated approximately by comparing the level of fluorescence of the cells at rest in solutions with a known concentration of calcium21. In this study, a modification of this method applied to synapses of mammals is presented.

Calcium entry during the depolarizing phase of the action potential is a fast process, especially in the neuromuscular junction; therefore, for its registration, appropriate equipment is required1. A recent study using a voltage-sensitive fluorescent dye demonstrated that the duration of the action potential in the peripheral synapse of a mouse is approximately 300 µs22. Calcium transient, evaluated using calcium-sensitive dyes in the peripheral synapses of the frog, has a longer duration: the rise time is about 2-6 ms and the decay time is about 30-90 ms, depending on the calcium dye used23,24. To measure fast processes with the help of fluorescent dyes, CCD or CMOS cameras are generally used, with fast and sensitive CCD matrices. However, these cameras have the disadvantage of low resolution, limited by the size of the sensitive elements of the matrix25,26,27,28. The fastest cameras with sufficient sensitivity to record both action potentials and calcium transients in response to low frequency stimulation of cells have a scanning frequency of 2,000 Hz, and a matrix with a dimension of 80 x 8029. To obtain signals with a higher spatial resolution, confocal microscopy is used, especially if it is necessary to assess some volumetric changes in the signal30,31,32. But it should be kept in mind that confocal microscopy has a high scanning speed in line scan mode, but there are still significant limitations on the speed of recordings of fast processes when building a spatial image33. There are confocal microscopes based on rotating Nipkow disks (slit-scanning microscopy) and Multipoint-Array Scanners, which have a higher scanning speed. At the same time, they are inferior to the classical confocal microscopes in confocal image filtering (pinholes crosstalk for microscopes with a Nipkow disk)32,34,35. Confocal imaging with resonance scanning can also provide a high spatio-temporal resolution required for high temporal measurements36. However, take into account that the registration of weak fluorescent responses at a high scanning speed when using resonance scanners requires highly sensitive detectors such as hybrid detectors36.

This article presents a method for increasing the temporal resolution of signals recorded with the Laser Scanning Confocal Microscopy (LSCM) while maintaining the spatial resolution37. The current method is a further development of the methods described earlier and transferred to the LSCM platform38,39,40. This approach does not require changes in the microscope hardware and is based on the application of an algorithm for recording periodically evoked fluorescent signals with a time shift relative to the moment of stimulation.

Protocol

Experiments were performed on isolated nerve-muscle preparations of levator auris longus (m. LAL) from the Mice BALB/C (20-23 g, 2-3 months old)41. The experimental procedures were performed in accordance with the guidelines for the use of laboratory animals of the Kazan Federal University and the Kazan Medical University, in compliance with the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol met the requirements of the European Communities Council Directiv…

Representative Results

After loading the preparation with dye according to the presented technique, most of the synapses located close to the nerve stump had a sufficient level of fluorescence (see Figure 2). After loading preparation with the dye andapplying the described method of registration and image processing, calcium transients with the desired spatial and temporal resolution were obtained (see Figure 4). The calcium transient has been recovered by the proposed method (see <st…

Discussion

The method for loading Ca2+-sensitive dye into mouse nerve endings through the nerve stump and for registering a fast calcium transient using a confocal microscope is presented in this article. As a result of the implementation of this loading method, most of the synapses located close to the nerve stump had a sufficient level of fluorescence to enable registration of the entry of calcium into the nerve endings in response to low-frequency stimulation of the motor nerve.

Unlike the …

Declarações

The authors have nothing to disclose.

Acknowledgements

Fluorescence studies of this work were carried out with the financial support of the Russian Science Foundation Grant (project No. 19-15-00329). The method was developed under financing from the government assignment for FRC Kazan Scientific Center of RAS АААА-А18-118022790083-9. The research was developed with the use of the equipment of the Federal Research Center "Kazan Scientific Center of RAS". The authors would like to thank Dr. Victor I. Ilyin for critical reading of this manuscript.

Materials

Capillary Glass Clark Electromedical instruments, UK GC150-10
Confocal and multiphoton microscope system Leica TCS SP5 MP Leica Microsystems , Heidelberg, Germany
Flaming/Brown Micropipette Puller P 97 Sutter Instrument, USA P-97
Flow regulator KD Medical GmbH Hospital Products, Germany KD REG Disposable infusion set with Flow regulator
HEPES Sigma-Aldrich, USA H0887 100mL
Illumination system Leica CLS 150X Leica Microsystems, Germany
ImageJ National Institutes of Health, USA http://rsb.info.nih.gov/ij/download.html
Las AF software Leica Microsystems, Heidelberg, Germany
Las X software Leica Microsystems, Heidelberg, Germany https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/
Magnetic Holder with Suction Tubing BIOSCIENCE TOOLS, USA MTH-S
Microspin FV 2400 Biosan, Latvia BS-010201-AAA
Minutien Pins Fine science tools, Canada 26002-20
Multi-spin MSC 3000 Biosan, Latvia BS-010205-AAN
Oregon Green 488 BAPTA-1 pentapotassium salt Molecular Probes, USA O6806 500 μg
Pipette Biohit, Russia 720210 0.5-10 µL
Pipette tip Biohit, Russia 781349 10 µL
Plasticine local producer
Single-use hypodermic needles Bbraun 100 Sterican 0.4×40 mm
Spreadsheet program Microsoft, USA Microsoft Office Excel
Stereomicroscope, Leica M80 Leica Microsystems , Germany
Suction electrode Kazakov A. SIMPLE SUCTION ELECTRODE FOR ELECTRIC STIMULATION OF BIOLOGICAL OBJECTS / A. Kazakov, M. Alexandrov, N. V. Zhilyakov et al. // International research journal. - 2015. – No. 9 (40) Part 3. – P. 13-16. http://research-journal.org/biology/prostoj-vsasyvayushhij-elektrod-dlya-elektricheskoj-stimulyacii-biologicheskix-obektov/
Sylgard 184 elastomer Dow Corning, USA
Syringe local producer 0.5 mL
Syringe local producer 60 mL
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Referências

  1. Llinas, R., Steinberg, I. Z., Walton, K. Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate. Proceedings of the National Academy of Sciences of the United States of America. 73 (8), 2918-2922 (1976).
  2. Augustine, G. J. How does calcium trigger neurotransmitter release. Current Opinion in Neurobiology. 11 (3), 320-326 (2001).
  3. Burnashev, N., Rozov, A. Presynaptic Ca2+ dynamics, Ca2+ buffers and synaptic efficacy. Cell Calcium. 37 (5), 489-495 (2005).
  4. Schneggenburger, R., Neher, E. Presynaptic calcium and control of vesicle fusion. Current Opinion in Neurobiology. 15 (3), 266-274 (2005).
  5. Pang, Z. P., Südhof, T. C. Cell biology of Ca2+-triggered exocytosis. Current Opinion in Cell Biology. 22 (4), 496-505 (2010).
  6. Leal, S. S., Gomes, C. M. Calcium dysregulation links ALS defective proteins and motor neuron selective vulnerability. Frontiers in Cellular Neuroscience. 9, 225 (2015).
  7. Grynkiewicz, G., Poenie, M., Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).
  8. Tsien, R. Y. Fluorescent indicators of ion concentrations. Methods in Cell Biology. 30, 127-156 (1989).
  9. Adams, S. R. How calcium indicators work. Cold Spring Harbor Protocols. 2010 (3), (2010).
  10. Macleod, G. T. Topical application of indicators for calcium imaging at the Drosophila larval neuromuscular junction. Cold Spring Harbor Protocols. 2012 (7), 786-790 (2012).
  11. Regehr, W. G. Monitoring presynaptic calcium dynamics with membrane-permeant indicators. Imaging in Neuroscience and Development: A Laboratory Manual. , 307-314 (2005).
  12. Eilers, J., Konnerth, A. Dye loading with patch pipettes. Cold Spring Harbor Protocols. 2009 (4), 5201 (2009).
  13. Coleman, W. L., et al. Synapsin II and calcium regulate vesicle docking and the cross-talk between vesicle pools at the mouse motor terminals. Journal of Physiology. 586 (19), 4649-4673 (2008).
  14. Macleod, G. T. Direct injection of indicators for calcium imaging at the drosophila larval neuromuscular junction. Cold Spring Harbor Protocols. 2012 (7), 797-801 (2012).
  15. Peng, Y. Y., Zucker, R. S. Release of LHRH is linearly related to the time integral of presynaptic Ca+ elevation above a threshold level in bullfrog sympathetic ganglia. Neuron. 10 (3), 465-473 (1993).
  16. Tsang, C. W., Elrick, D. B., Charlton, M. P. α-Latrotoxin releases calcium in frog motor nerve terminals. The Journal of Neuroscience. 20 (23), 8685-8692 (2000).
  17. Newman, Z., et al. Endocannabinoids mediate muscarine-induced synaptic depression at the vertebrate neuromuscular junction. The European Journal of Neuroscience. 25 (6), 1619-1630 (2007).
  18. Macleod, G. T. Forward-filling of dextran-conjugated indicators for calcium imaging at the drosophila larval neuromuscular junction. Cold Spring Harbor Protocols. 2012 (7), 791-796 (2012).
  19. Rossano, A. J., Macleod, G. T. Loading drosophila nerve terminals with calcium indicators. Journal of Visualized Experiments: JoVE. (6), e250 (2007).
  20. Wu, L. G., Betz, W. J. Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction. Neuron. 17 (4), 769-779 (1996).
  21. Suzuki, S., et al. Ca2+ dynamics at the frog motor nerve terminal. Pflugers Archiv: European Journal of Physiology. 440 (3), 351-365 (2000).
  22. Ojala, K. S., et al. A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs. Journal of Biological Chemistry. 296, 100302 (2021).
  23. Samigullin, D., et al. Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes. Frontiers in Synaptic Neuroscience. 6, 29 (2015).
  24. DiGregorio, D. A., Vergara, J. L. Localized detection of action potential-induced presynaptic calcium transients at a Xenopus neuromuscular junction. The Journal of Physiology. 505, 585-592 (1997).
  25. Bullen, A., Patel, S. S., Saggau, P. High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators. Biophysical Journal. 73 (1), 477-491 (1997).
  26. Bullen, A., Saggau, P. High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes. Biophysical Journal. 76 (4), 2272-2287 (1999).
  27. Bullen, A., Saggau, P. Optical recording from individual neurons in culture. Modern Techniques in Neuroscience Research. (4), 89-126 (1999).
  28. Bullen, A., Saggau, P. Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy. Pflugers Archiv European Journal of Physiology. 436 (5), 788-796 (1998).
  29. Wilson, T. Optical aspects of confocal microscopy. Confocal Microscopy. , 93-141 (1990).
  30. Cox, G. Biological confocal microscopy. Materials Today. 5 (3), 34-41 (2002).
  31. Mukhitov, A., Arkhipova, S., Nikolsky, E. Modern Light Microscopy in Biological and Medical Research. Nauka. , (2011).
  32. Mertz, J. Optical sectioning microscopy with planar or structured illumination. Nature Methods. 8 (10), 811-819 (2011).
  33. Webb, R. H. Confocal optical microscopy. Reports on Progress in Physics. 59 (3), 427-471 (1996).
  34. Toomre, D., Pawley, J. B. Disk-scanning confocal microscopy. Handbook of Biological Confocal Microscopy: Third Edition. , 221-238 (2006).
  35. Venkateswarlu, K., et al. Three-dimensional imaging and quantification of real-time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy. Biotechnology and Bioengineering. 117 (10), 3108-3123 (2020).
  36. Arkhipov, A. Y., Khaziev, E. F., Skorinkin, A. I., Bukharaeva, E. A., Samigullin, D. V. Enhancement of the temporal resolution of fluorescent signals acquired by the confocal microscope. Microscopy and Microanalysis. 26 (2), 204-210 (2020).
  37. Rama, S. Shift and mean algorithm for functional imaging with high spatio-temporal resolution. Frontiers in Cellular Neuroscience. 9, (2015).
  38. Chan, K. G., Streichan, S. J., Trinh, L. A., Liebling, M. Simultaneous temporal superresolution and denoising for cardiac fluorescence microscopy. IEEE Transactions on Computational Imaging. 2 (3), 348-358 (2016).
  39. Veeraraghavan, A., Reddy, D., Raskar, R. Coded strobing photography: compressive sensing of high speed periodic videos. IEEE Transactions on Pattern Analysis and Machine Intelligence. 33 (4), 671-686 (2011).
  40. Angaut-Petit, D., Molgo, J., Connold, A. L., Faille, L. The levator auris longus muscle of the mouse: A convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins. Neuroscience Letters. 82 (1), 83-88 (1987).
  41. Macleod, G. T. Calcium imaging at the Drosophila larval neuromuscular junction. Cold Spring Harbor Protocols. 7 (7), 758-766 (2012).
  42. Samigullin, D. V., Khaziev, E. F., Zhilyakov, N. V., Bukharaeva, E. A., Nikolsky, E. E. Loading a calcium dye into frog nerve endings through the nerve stump: calcium transient registration in the frog neuromuscular junction. Journal of Visualized Experiments: JoVE. (125), e55122 (2017).
  43. Samigullin, D. V., et al. Calcium transient registration in response to single stimulation and during train of pulses in mouse neuromuscular junction. BioNanoScience. 7 (1), 162-166 (2017).
  44. Luo, F., Dittrich, M., Stiles, J. R., Meriney, S. D. single-pixel optical fluctuation analysis of calcium channel function in active zones of motor nerve terminals. Journal of Neuroscience. 31 (31), 11268-11281 (2011).
  45. Luo, F., Dittrich, M., Cho, S., Stiles, J. R., Meriney, S. D. Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction. Journal of Neurophysiology. 113 (7), 2480-2489 (2015).
  46. Wright, M., Kim, A., Son, Y. -. J. Subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice. Journal of Visualized Experiments: JoVE. (55), e3124 (2011).
  47. Burke, S. R. A., Reed, E. J., Romer, S. H., Voss, A. A. Levator Auris Longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions. Journal of Visualized Experiments: JoVE. (135), e57482 (2018).
  48. Kazakov, A., Alexandrov, M., Zhilyakov, N. V., Khaziev, E. F., Samigullin, D. V. A simple suction electrode for electrical stimulation of biological objects. Meždunarodnyj naučno-issledovatel’skij žurnal (International Research Journal). 9 (40), 13-16 (2015).
  49. Bowman, W. C. Neuromuscular block. British Journal of Pharmacology. 147, 277-286 (2006).
  50. Hill, J. M., Alewood, P. F., Craik, D. J. Three-dimensional solution structure of µ-conotoxin GIIIB, a specific blocker of skeletal muscle sodium channels. Bioquímica. 35 (27), 8824-8835 (1996).
  51. Land, B. R., Johnson, B. R., Wyttenbach, R. A., Hoy, R. R. Tools for physiology labs: Inexpensive equipment for physiological stimulation. Journal of Undergraduate Neuroscience Education. 3 (1), 30-35 (2004).
  52. Samigullin, D. V., Zhilyakov, N. V., Khaziev, E. F., Bukharaeva, E. A., Nikolsky, E. E. Calcium transient and quantal release in mouse neuromuscular junction under extracellular calcium concentration change. BioNanoScience. 8 (4), 984-987 (2018).
  53. Khaziev, E., et al. acetylcholine-induced inhibition of presynaptic calcium signals and transmitter release in the frog neuromuscular junction. Frontiers in Physiology. 7, 621 (2016).
  54. Zhilyakov, N., Arkhipov, A., Malomouzh, A., Samigullin, D. Activation of neuronal nicotinic receptors inhibits acetylcholine release in the neuromuscular junction by increasing ca2+ flux through cav1 channels. International Journal of Molecular Sciences. 22 (16), 9031 (2021).
  55. Sabatini, B. L., Regehr, W. G. Optical measurement of presynaptic calcium currents. Biophysical Journal. 74 (3), 1549-1563 (1998).
  56. McArdle, J. J., et al. Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena. Journal of Neuroscience Methods. 4 (2), 109-115 (1981).

Tags

Calcium TransientsNeuromuscular JunctionConfocal MicroscopyPresynaptic CalciumSynaptic TransmissionCalcium SignalingNeurodegenerative DiseasesLoading TechniqueSpatial-temporal ResolutionConfocal MicroscopeNerve TrunkNerve StumpDye Loading Solution
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Zhilyakov, N. V., Arkhipov, A. Y., Khaziev, E. F., Mukhamedyarov, M. A., Samigullin, D. V. Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy. J. Vis. Exp. (178), e63308, doi:10.3791/63308 (2021).
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