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

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 proteins^1,2,3,4,5. 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.......

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 .......

Materials

Name	Company	Catalog Number	Comments
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