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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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....

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

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 .......

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

NameCompanyCatalog NumberComments
Capillary GlassClark Electromedical instruments, UKGC150-10
Confocal and multiphoton microscope system Leica TCS SP5 MPLeica Microsystems , Heidelberg, Germany
Flaming/Brown Micropipette Puller P 97Sutter Instrument, USAP-97
Flow regulatorKD Medical GmbH Hospital Products, GermanyKD REGDisposable infusion set with Flow regulator
HEPESSigma-Aldrich, USAH0887100mL
Illumination system Leica CLS 150XLeica Microsystems, Germany
ImageJNational Institutes of Health, USAhttp://rsb.info.nih.gov/ij/download.html
Las AF softwareLeica Microsystems, Heidelberg, Germany
Las X softwareLeica Microsystems, Heidelberg, Germanyhttps://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/
Magnetic Holder with Suction TubingBIOSCIENCE TOOLS, USAMTH-S
Microspin FV 2400Biosan, LatviaBS-010201-AAA
Minutien PinsFine science tools, Canada26002-20
Multi-spin MSC 3000Biosan, LatviaBS-010205-AAN
Oregon Green 488 BAPTA-1 pentapotassium saltMolecular Probes, USAO6806500 μg
PipetteBiohit, Russia7202100.5-10 µL
Pipette tipBiohit, Russia78134910 µL
Plasticinelocal producer
Single-use hypodermic needlesBbraun100 Sterican0.4×40 mm
Spreadsheet programMicrosoft, USAMicrosoft Office Excel
Stereomicroscope, Leica M80Leica Microsystems , Germany
Suction electrodeKazakov 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 elastomerDow Corning, USA
Syringelocal producer0.5 mL
Syringelocal producer60 mL

References

  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.

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