Name: Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction (Video) | JoVE Journal
Uploaded: 2017-07-08T13:00:00.000+00:00
Duration: 17 min 5 s
Description: Learn calcium dye loading into frog nerve endings via nerve stump for measuring fast calcium transients in neuromuscular junctions. This optical method enables studying drug effects on presynaptic calcium signaling.

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5 minutes

This research was performed under the Russian Government's Program for Competitive Growth of the Kazan Federal University and a grant from the Russian Foundation for Basic Research (16-04-01051; 16-34-00817; 15-04-02983). We thank four anonymous reviewers for providing helpful comments on earlier drafts of the manuscript. We express our gratitude to Yuliya Aratskaya for the voice recording. We are grateful to Dr. Victor Ilyin for the many helpful comments and the help with the finale editing of the manuscript.

Experiments were performed on isolated nerve-muscle preparations of the musculus cutaneous pectoris from the Rana ridibunda frog. The size of animals of both genders was about 5-9 cm. 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 Directive 86/609/EEC and was approved by the Ethical Committee of the Kazan Medical University.
1. Preparation of the Solutions
<ol>
	<li>Preparation of Ringer's solution.
	<ol>
		<li>Prepare Ringer's solution: 113.0 mM NaCl, 2.5 mM KCl, 3.0 mM NaHCO3, and 1.8 mM CaCl2. Adjust the pH to 7.2-7.4.</li>
		<li>Prepare Ringer's solution with a low Ca2+ and high Mg2+ content: 113.0 mM NaCl, 2.5 mM KCl, 3.0 mM NaHCO3, 6.0 mM MgCl2, 0.9 mM CaCl2. Adjust the pH to 7.2-7.4.</li>
	</ol>
	</li>
	<li>Preparation of the dye-loading solution.
	<ol>
		<li>Prepare the water-based solution containing HEPES-Na at 10 mM (pH 7.2-7.4).</li>
		<li>Add 14 µL of the HEPES solution to a vial with the dye30. 
		NOTE: the Ca2+ indicator dye comes in a 500-µL vial with 500 µg of powder.</li>
		<li>Vortex and spin down to mix thoroughly.</li>
		<li>Dilute the solution to bring the final concentration of the Ca2+ indicator down to 30 mM. Avoid exposure to light and store at -20 °C.</li>
	</ol>
	</li>
</ol>
2. Dye-loading Procedure
<ol>
	<li>Dissect the cutaneous pectoris muscle with a piece of the pectoralis proprius nerve. 
	NOTE: The dissection procedure is available in a free download of the paper by Blioch et al., 196831.
	<ol>
		<li>For the dissection procedure, use two fine forceps and corneal scissors (see the Table of Materials). Transfer the dissected tissue into a silicon elastomer-coated Petri dish pre-filled with Ringer's solution and fix the tissue with fine stainless-steel pins such that it is slightly stretched in the dish.</li>
		<li>Re-fill the Petri dish with a fresh aliquot of the Ringer's solution. Remove the connective tissues. Do not damage the nerve.</li>
	</ol>
	</li>
	<li>Prepare the filling pipette: using a razor blade, cut out a ~2 mm-long piece of the conical part of a standard plastic 10 µL pipette tip.</li>
	<li>Prepare a piece of modeling clay to mount the filling pipette on the Petri dish.</li>
	<li>Connect the back of the filling pipette to a plastic syringe via silicone tubing and plastic connecting adapters made from pipette tips.</li>
	<li>Before the dye loading procedure, remove the Ringer's solution from the Petri dish using a plastic pipette. Dry the muscle-nerve preparation using a fine syringe; this will prevent the dilution of the Ca2+ dye upon the subsequent loading of the filling pipette.</li>
	<li>Remove the Ca2+ indicator vial from the freezer and allow it to thaw at room temperature in a dark place.</li>
	<li>Under stereomicroscope control with low magnification (10×), detect the junction between the muscle and the nerve. With fine tweezers and scissors, cut the pectoralis proprius nerve close to the muscle surface (see step 2.1). Leave a nerve stump about 2 mm long.</li>
	<li>Fix the filling pipette attached to tubing and the syringe on the Petri dish using modeling clay.</li>
	<li>Move the tip of the pipette close to the nerve stump.</li>
	<li>Without pinching it, gently aspirate the nerve stump into the tip of the filling pipette.</li>
	<li>Remove the suction tubing from the blunt end of the filling pipette.</li>
	<li>Carefully remove the excess solution from the filling pipette using a syringe with a long needle (see the Table of Materials). Do not pinch the nerve stump.</li>
	<li>Vertically elevate the tip of the filling pipette slightly, keeping the nerve stump aspirated in the tip.</li>
	<li>Insulate the aspirated part of the nerve stump from the outside of the filling pipette tip using petroleum jelly.</li>
	<li>Dry up the nerve stump insulated in the filling pipette if necessary: gently aspirate the excess of solution from the filling pipette using a syringe with a long needle.</li>
	<li>Draw 0.5 µL of the dye-loading solution (see step 1) using a pipette with a long pipette tip.</li>
	<li>Gently insert the pipette tip with loading solution into the filling pipette. Eject the mixture directly onto the nerve stump.</li>
	<li>Seal the open end of the filling pipette with petroleum jelly.</li>
	<li>Add a small aliquot of Ringer's solution to the Petri dish to keep the preparation wet.</li>
	<li>Incubate the preparation at room temperature under dark and wet conditions for 5 h.</li>
	<li>Remove the filling pipette with loading solution, rinse the preparation with the Ringer's solution, and keep it overnight in the refrigerator at 8 °C.</li>
</ol>
3. Preparing the Tissue for Microscopy
<ol>
	<li>Mount the preparation into the silicon elastomer-coated chamber and fix it with steel micro-needles such that it is slightly stretched.</li>
	<li>Rinse the tissue with an aliquot of fresh Ringer's solution.</li>
	<li>Use a suction electrode to stimulate the nerve; construction of the electrode is available from the free download of the paper by Kazakov et al., 201532. Position the electrode tip close to the cut end of the nerve and aspirate the nerve stump into the electrode orifice.</li>
	<li>Mount the preparation chamber on the microscope stage. Place the temperature probe and the inlet and outlet firings in the chamber.</li>
	<li>Connect the power cord to the Peltier element.</li>
	<li>To superfuse the preparation, use a simple gravity-driven system. To remove the excess solution, turn on the perfusion suction pump.</li>
	<li>Switch on the thermo-controller unit.</li>
	<li>Set the temperature control to 20 °C.</li>
	<li>Mount the ultraviolet protection shield.</li>
	<li>Connect the stimulating wire electrode to the electric stimulator and observe the muscular contractions under the microscope with a 4x objective lens.</li>
	<li>Fill up the perfusion system with the Ringer's solution with low-Ca2+ and high-Mg2+ content. 
	NOTE: This solution is used to prevent muscular contractions. A decrease in the concentration of external calcium and an elevation of external magnesium result in the reduction of the amplitude of Ca2+ transients.However, based on previous experience, 0.9 mM CaCl2 and 6 mM MgCl2 are still sufficient to reliably resolve the amplitude of Ca2+ transients. It is worth mentioning that there exist some other ways to diminish muscular contractions without reducing the Ca2+ concentration.For example, the use of d-tubocurarine or alpha-bungarotoxin, specific blockers of nicotinic acetylcholine receptors, would completely or partially block muscle twitches17,27,28,30.However, the addition of these toxins can also affect presynaptic calcium entry33. To avoid this, µ-conotoxin GIIIA can be used27.</li>
	<li>Switch on the pump and start the superfusion of the preparation with the Ringer's solution with low Ca2+ and high Mg2+.</li>
	<li>Switch to the 40× objective lens on the microscope.</li>
	<li>Switch on the monochromator (see the Table of Materials).</li>
	<li>Select an emission wavelength of 488 nm and a continuous mode of illumination in the monochromator control software.</li>
	<li>Under high magnification in fluorescence mode, ensure that the nerve terminals were loaded with the dye.</li>
</ol>
<img alt="Neuronal fluorescence microscopy, cellular structure, dendrites visualization, microscopy imaging." class="xfigimg" src="/files/ftp_upload/55122/55122fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1: Nerve and terminals with loaded Ca2+ indicator. (A) Nerve filled with Ca2+ indicator after the loading procedure. Scale bar = 200 µm. (B) Nerve endings filled with Ca2+ indicator. Scale bar = 20 µm. (C) Ca2+-indicator fluorescence is clearly visible in the nerve ending. Scale bar = 20 µm. <a href="//cloudfront.jove.com/files/ftp_upload/55122/55122fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="17">
	<li>Allow the preparation to equilibrate for at least 30 min in the low-Ca2+ and high-Mg2+ solution.</li>
</ol>
4. Video Capture with the Digital CCD Camera
Note: The details of capturing fluorescence signals are specific for each microscope and camera type, but the key consideration is the image capturing speed.
<ol>
	<li>Use 1 kHz as the minimum capture frequency for recordings of single Ca2+ transients in the NMJ. 
	NOTE: Fast digital CCD cameras are necessary for fluorescence imaging (see the Table of Materials). The data acquisition system and software (see the Table of Materials) were used here for the synchronization of the camera, monochromator, and stimulator. In brief, this protocol permits the generation of synchronization pulses on digital outputs of the data acquisition system to open the shutter, capture the video signal, and initiate stimulation. All temporal parameters can be set in the protocols and/or on the apparatuses. A typical protocol is a series of 500 frames acquired at 1 kHz (80 x 80 pixels). Illumination with excitation light can bleach out the Ca2+ indicator and photodamage the cell tissue. Thus, avoid long exposures to excitation light. In this protocol, the shutter is open only for the time needed to capture the video. Acquire twenty series per specific nerve terminal. The aim here is to monitor the same sites in the control group and after drug delivery.</li>
	<li>Under the 4X objective lens of a microscope, use the bright-field regime to visualize the muscle and nerve branches.</li>
	<li>Switch to the 40X objective lens and, using the epifluorescence regime and an excitation wavelength of 488 nm, search for dye-loaded nerve endings. Identify a nerve-ending region of interest.</li>
	<li>On the trinocular tube of the microscope, select the light path exchange levels: 100% light to camera.</li>
	<li>Start the acquisition software for the CCD camera.</li>
	<li>Under "Live" mode, find the ROI and adjust the focus.</li>
	<li>Select the menu "Change" settings.</li>
	<li>Use the "Basic Configuration" at 1,000 frames per second (fps), with a resolution of 80 x 80.</li>
	<li>Set the number of input frames to 500.</li>
	<li>Enter the name of the experiment.</li>
	<li>Choose "External Trigger."</li>
	<li>Set the pre-trigger time to 10 ms.</li>
	<li>Set the number of repeats to 20.</li>
	<li>In the monochromator control software, select an emission wavelength of 488 nm and "external trigger illumination" mode.</li>
	<li>Run the data acquisition software.</li>
	<li>Load the stimulation protocol.</li>
	<li>Before recording the video, capture the dark frame using the video acquisition software.</li>
	<li>Run the stimulation protocol.</li>
	<li>Select the ROI and check the recorded signal.</li>
</ol>
5. Data Analysis
NoOTE: For data analysis, use the CCD camera software and ImageJ; the data is represented as a curve in a spreadsheet program. In the CCD camera software, average 20 repeats and export the results to an ImageJ support file. In ImageJ, select the ROI and background. Subtract the background from the ROI. Represent the data as a ratio: (ΔF/F0 -1) x 100%, where's ΔF is the intensity of fluorescence during stimulation and F0 is the intensity of fluorescence at rest.
<ol>
	<li>In the acquisition software for the CCD camera, click on File &gt; Average Files. Select the files and average them.</li>
	<li>Save the averaged file as a .fit file by clicking "Save as Fit file."</li>
	<li>Run the ImageJ software. Perform the following steps:</li>
	<li>Click Image &gt; adjust &gt; brightness/contrast.</li>
	<li>Click Image &gt; stacks &gt; tools &gt; stack sorter.</li>
	<li>Click Analysis &gt; tools &gt; ROI manager.</li>
	<li>Drag and drop the averaged .fit file into the ImageJ window.</li>
	<li>Zoom in on the window for a better view.</li>
	<li>By moving the cursor, select the last frame and delete it (this is the dark frame)</li>
	<li>Select a rectangular ROI over the area believed to be the background. Add it to the ROI manager</li>
	<li>Measure the background by clicking More &gt; Multi Measure. Note the MEAN. Copy the data, export it to a spreadsheet program, and calculate the average value of the threshold for a ratio.</li>
	<li>Subtract the threshold from the stacks by clicking Process &gt; Main &gt; Subtract. Enter the averaged value of the threshold.</li>
	<li>Select a rectangular ROI around a nerve terminal. Add it to the ROI manager.</li>
	<li>Measure by clicking More &gt; Multi Measure. Note the MEAN. Copy and export it to a spreadsheet program.</li>
	<li>Average the offset of the signals. 
	NOTE: Use the first several dozen points demonstrating base dye fluorescence without stimulation; this is the fluorescence at rest.</li>
	<li>Divide the signals by the fluorescence at rest.</li>
	<li>Subtract "1" and multiply by 100%.</li>
	<li>Plot the signal and calculate the amplitude of the Ca2+ transient.</li>
</ol>

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<li>Kazakov, A., Aleksandrov, M., Zhilyakov, N. V., Khaziev, E. F., Samigullin, D. V. <a target="_blank" href="http://research-journal.org/en/biology-en/prostoj-vsasyvayushhij-elektrod-dlya-elektricheskoj-stimulyacii-biologicheskix-obektov/">A simple suction electrode for electrical stimulation of biological objects.</a> Mezhdunarodnyj nauchno-issledovatel'skij zhurnal. 40 (9), In Russian 13-16 (2015).</li>
<li>Khaziev, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acetylcholine-induced+inhibition+of+presynaptic+calcium+signals+and+transmitter+release+in+the+frog+neuromuscular+junction%5BTitle%5D)+AND+%22Front+Physiol%22%5BJournal%5D)">Acetylcholine-induced inhibition of presynaptic calcium signals and transmitter release in the frog neuromuscular junction.</a> Front Physiol. 7 (621), 1-10 (2016).</li>
<li>Bélair, E. L., Vallée, J., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+in+vivo+modulation+of+synaptic+efficacy+at+the+neuromuscular+junction+of+Rana+pipiens+frogs%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Long-term in vivo modulation of synaptic efficacy at the neuromuscular junction of Rana pipiens frogs.</a> J Physiol. 569 (1), 163-178 (2005).</li>
<li>Samigullin, D., Fatikhov, N., Khaziev, E., Skorinkin, A., Nikolsky, E., Bukharaeva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+presynaptic+calcium+currents+and+endogenous+calcium+buffers+at+the+frog+neuromuscular+junction+with+two+different+calcium+fluorescent+dyes%5BTitle%5D)+AND+%22Front+Synaptic+Neurosci%22%5BJournal%5D)">Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.</a> Front Synaptic Neurosci. 6 (29), 1-10 (2015).</li>
<li>Angleson, J. K., Betz, W. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11152728">Intraterminal Ca2+ and spontaneous transmitter release at the frog neuromuscular junction.</a> J Neurophysiol. 85 (1), 287-294 (2001).</li>
<li>Shahrezaei, V., Cao, A., Delaney, K. R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/17182774">Ca2+ from one or two channels controls fusion of a single vesicle at the frog neuromuscular junction.</a> J Neurosci. 26 (51), 13240-132499 (2006).</li>
<li>Troncone, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promiscuous+and+reversible+blocker+of+presynaptic+calcium+channels+in+frog+and+crayfish+neuromuscular+junctions+from+Phoneutria+nigriventer+spider+venom%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Promiscuous and reversible blocker of presynaptic calcium channels in frog and crayfish neuromuscular junctions from Phoneutria nigriventer spider venom.</a> J Neurophysiol. 90 (5), 3529-3537 (2003).</li>
<li>Samigullin, D. V., Khaziev, E. F., Zhilyakov, N. V., Sudakov, I. A., Bukharaeva, E. A., Nikolsky, E. E. <a target="_blank" href="https://link.springer.com/article/10.1007/s12668-016-0318-6">Calcium transient registration in response to single stimulation and during train of pulses in mouse neuromuscular junction.</a> BioNanoSci. 7 (1), 162-166 (2016).</li>
</ol>

The authors have nothing to disclose.

In this paper, we presented the method of performing Ca2+-sensitive dye loading into frog nerve endings through the nerve stump. By the end of the loading procedure, all terminals in the proximal part of the nerve have significant levels of fluorescence. It has been estimated that the intra-terminal concentration of the probe varies between 40 and 150 &#181;M17.
The incubation procedure is conducted in two steps: at room temperature and then at a lower temperature in a refrigerator. It is important to control the time of tissue incubation with the dye at room temperature. Depending on the actual length of the nerve stump, the specific dye, and the temperature, the incubation time may vary. If overexposed, terminals in the proximal parts close to the nerve stump can be overloaded. However, in the middle part of the nerve, it is still possible to find terminals that are satisfactorily loaded. During the long incubation in the refrigerator, the dye is evenly distributed over the nerve endings.
Our own observations33,35, as well as the data of other researchers30, prove the lack of any appreciable influence of the loading procedure on the amplitude of the postsynaptic response or on the frequency of miniature end-plate potentials. Good longevity was documented in the loaded preparations. There are some important points we would like to draw attention to. It is very essential to place the nerve stump into the dye-loading solution withinseveral minutes after excision to enable the dye to enter into the axons of the cut nerve; delays can cause ineffective loading, presumably due to the resealing of nerve axons27,36. Some investigators immerse the nerve stump in 100 mM EDTA (a Ca2+- and Mg2+-chelator) immediately after the excision of the nerve to prevent the cut axons from resealing. The buffer is removed after 1-2 min and replaced with a dye-loading solution37. The use of a petroleum-jelly well instead of plastic tubing for the loading procedure permits the use of a shorter nerve stump. While using this approach, the nerve is cut after it is immersed in the HEPES solution with dye, and the axons do not reseal because of the lack of divalent ions in the dye solution27,28.
In our study, we used the water-soluble salt form of the Ca2+ indicator instead of dextran. The dextran conjugates diffuse in the axon more slowly than the salt forms. However, the use of the dextran conjugate reduces dye compartmentalization and handling by the nerve and NMJs. Calcium Green 1-3,000 MW dextran conjugate has a good diffusion rate and demonstrates reduced compartmentalization38.
It is very important to avoid a long period of fluorescent illumination of the tissue, because this affects its health and survival. We use Nomarski optics in the visible-light channel to search for nerve terminals. During the recording, we limit the illuminated field using a diaphragm.
It is noteworthy that this loading method is suitable only for preparations that can withstand long incubations. To reduce the dye-loading time when studies are being conducted on more fragile tissues (e.g., synapses of warm-blooded animals), it is necessary to downscale the nerve stump length and to use micropipettes for loading29,39.
This loading technique is well suited for imaging changes in cytosolic Ca2+, with fluorescent indicators under both single-nerve stimulation and rhythmic synaptic activity17,27,35. The analysis of the Ca2+-transient amplitude can be used to study the influence of different substances on the calcium entry that participates in neurotransmitter release33.

Calcium ions (Ca2+) participate in many neuronal signaling processes, including the initiation, maintenance, and plasticity of mediator release1,2,3,4,5. Upon the arrival of the action potential, extracellular Ca2+ enters the nerve terminal and initiates neurotransmitter release. In some synapses, the calcium current can be measured directly by electrophysiological methods6,7,8. In the case of the neuromuscular junction (NMJ), one cannot use direct patch clamp and two-electrode voltage clamp techniques due to the minute size of the nerve endings.
Recordings of inward Ca2+ currents from the nerve endings in the NMJ can be done by indirect electrophysiological methods9,10. However, these methods require the pretreatment of the synapse by sodium and potassium ion channel blockers. Optical methods do not require the pharmacological separation of ionic currents in the nerve terminal and permit recordings of Ca2+ influx, triggered by action potentials, and the subsequent elevation of Ca2+ ions in the axoplasm11,12,13,14. These methods are based on recordings of changes in the fluorescence of specific Ca2+-sensitive dyes upon the binding of free Ca2+ ions15,16,17,18,19.
Ca2+ indicators can be loaded into the cells through a variety of methods, depending on the purpose of the experiment. Researchers use the bath application of membrane-permeable dye forms20,21, loading via patch pipette22, or microinjection23,24,25. However, all these methods have some limitations in the case of the NMJ due to its peculiarities in synaptic architectonics. For the NMJ, the most convenient and successful method is to load the dye through the nerve stump, a forward-filling method26,27,28,29. This technique can be used for loading various fluorescence dyes into the peripheral nerve endings. This method was successfully used for Drosophila nerve terminals28, the lizard motor nerve28, and frog motor nerve terminals17,26,27,30. Depending on the object under study, methodical details can vary. A glass micro-pipette can be employed for small nerves from larvae28. Several researchers have described a method27,28 in which a freshly cut end of nerve innervating a muscle is immersed into a well pre-filled with a dye. The preparation is then left for several hours to soak the dye. The dye is soaked up by the axons and transported to the nerve terminals. In this paper, we describe a method of loading a fluorescence indicator into frog motor nerve terminals through the nerve stump. Our protocol uses a plastic pipette tip for the incubation of the tissue with a dye. We also describe how to acquire and analyze Ca2+ fluorescence transients.

<table><tbody><tr><td>Ca2+ indicator</td><td>Molecular Probes, USA</td><td>Oregon Green 488 BAPTA-1 hexapotassium salt, O6806</td><td>500 &amp;mu;g</td></tr><tr><td>Silicone Elastomer</td><td>Dow Corning, USA</td><td>Sylgard 184 elastomer</td><td></td></tr><tr><td>Pipette</td><td>Biohit, Russia</td><td>720210</td><td>0.5-10&amp;micro;L</td></tr><tr><td>Pipette tip</td><td>Fisher Scientific, USA</td><td>02-707-175</td><td>10&amp;micro;L</td></tr><tr><td>Pipette tip</td><td>Biohit, Russia</td><td>781349</td><td>10&amp;micro;L</td></tr><tr><td>Razor Blade</td><td>Fisher Scientific, USA</td><td>12-640</td><td></td></tr><tr><td>Minutien Pins</td><td>Fine scince tools, Canada</td><td>26002-20</td><td></td></tr><tr><td>Corneal Mini-Scissors</td><td>MT MEDI CORP, Canada</td><td>S-1111</td><td></td></tr><tr><td>Jeweler Forceps</td><td>MT MEDI CORP, Canada</td><td>F-9610</td><td></td></tr><tr><td>Jeweler Forceps</td><td>MT MEDI CORP, Canada</td><td>F-9611</td><td></td></tr><tr><td>Modelling clay</td><td>local producer</td><td></td><td>can be replaced by any local producer</td></tr><tr><td>Petroleum jelly</td><td>local producer</td><td></td><td>can be replaced by any local producer</td></tr><tr><td>Microspin FV 2400&amp;nbsp;</td><td>Biosan, Latva</td><td>BS-010201-AAA</td><td></td></tr><tr><td>Multi-spin MSC 3000</td><td>Biosan, Latva</td><td>BS-010205-AAN</td><td></td></tr><tr><td>Single-use hypodermic needles</td><td>Bbraun</td><td>100 Sterican</td><td>0.4&amp;times;40mm</td></tr><tr><td>Syrynge</td><td>local producer</td><td></td><td>0.5 ml</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma-Aldrich, USA</td><td>H0887</td><td>100ml</td></tr><tr><td>Microscope, BX51</td><td>Olympus, Japan</td><td></td><td></td></tr><tr><td>Data acquisition system&amp;nbsp;</td><td>Molecular Devices, USA</td><td>Digitdata 1550</td><td></td></tr><tr><td>software</td><td>Molecular Devices, USA</td><td>pClamp software, Version 10</td><td>protocol can be download from : http://kpfu.ru/portal/docs/F_230007060/Video.capture.with.&lt;br/&gt; RedShirt.Neuro.CCD.camera.pro</td></tr><tr><td>Bath and bath temperature controler</td><td>Experimental Builder</td><td></td><td>can be replaced by any chamber with temperature control. For example from https://www.warneronline.com/&amp;nbsp;</td></tr><tr><td>Monochromator</td><td>Till Photonics, Germany</td><td>Polychrome V</td><td>no longer available, can be replaced by other&amp;nbsp;&amp;nbsp; sutable stable light source 488 nm</td></tr><tr><td>monochromator control software</td><td>Till Photonics, Germany</td><td>Polycon</td><td></td></tr><tr><td>Digital CCD camera</td><td>Redshirt imaging, USA</td><td>Neuro CCD SMQ</td><td></td></tr><tr><td>Model 2100 Isolated Pulse Stimulator</td><td>A-M Systems, USA</td><td></td><td></td></tr><tr><td>Acquisition software for CCD camera</td><td>Redshirt imaging, USA</td><td>Turbo SM software</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health, USA</td><td></td><td>http://rsb.info.nih.gov/ij/download.html</td></tr><tr><td>Spreadsheet program</td><td>Microsoft, USA</td><td>Microsoft Office Excel&amp;nbsp;</td><td></td></tr></tbody></table>

loading a calcium dye into frog nerve endings through the nerve stump: calcium transient registration in the frog neuromuscular junction

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

After the dye loading and upon motor nerve stimulation, an increase in the amplitude of the fluorescent signal (Ca2+ transient) can be detected in the nerve terminals (see Figure 2). Parameters of Ca2+ transients are presented in Table 1. Quantitatively, the parameters of the Ca2+ transients measured in our study are close to the data obtained by other scientists at synapses of cold-blooded animals15,34. The parameters of the Ca2+ transients depend on the rate of binding of Ca2+ with the dye and the subsequent dissociation. The rate of entry of Ca2+ into the nerve ending, interaction with the dye, and diffusion in the cytoplasm all affect the rise time of the Ca2+ transient. The decay time of the fluorescent signal depends on the affinity of the dye, the speed of the Ca2+ interaction with intracellular buffers, and the removal by ion pumps35. The amplitude analysis of Ca2+ transients can be used to study the influence of various substances on the calcium entry that participates in neurotransmitter release33.
<img alt="Transient absorption graph, ∆F/F₀ vs. time, showing photonic excitation response in milliseconds." class="xfigimg" src="/files/ftp_upload/55122/55122fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2: The averaged Ca2+ transient measured in the frog NMJ. The Ca2+ transient was calculated based on the average of signals from 13 frog NMJs. <a href="https://cloudflare.jove.com/files/ftp_upload/55122/55122fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak ΔF/F (%)</td>
			<td>Rise time 20%-80% (ms)</td>
			<td>τ (ms)</td>
		</tr>
		<tr>
			<td>12,6 ±1,1 (n = 13)</td>
			<td>4.6 ±0.5 (n = 13)</td>
			<td>115.3 ±8.3 (n = 13)</td>
		</tr>
	</tbody>
</table>
Table 1: The averaged parameters of theCa2+ transient. The data are presented as the mean ± SE; n is the number of measurements in distinct NMJs. The peak ΔF/F is the averaged amplitude of ΔF/F.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Hello my name is Dimitry Samigullin I work in the laboratory of Biophysics of Synaptic Processes of Kazan Institute of Biochemistry and Biophysics, the Russian Academy of Science. In our laboratory we study vast ways of calcium signalization in the Neuromuscular Junction. One of the most approachable methods to measure the synaptic level of calcium is an optical recording.Here we describe the method of the loading of a calcium sensitive dye, through a nerve stump, in the frog nerve endings. Also, we present the protocol to measure, and analysis of, a vast calcium transient in frog terminals. Using this method it is possible to study the influence of active drugs on the presynaptic level of the calcium during synaptic activity.Preparing of the dye loading solution. Fluorescein's Calcium Dye Oregon Green BAPTA, comes in 500 microliters via a small amount 500 micrograms of powder. Add 14 microliters of solution, containing HEPES, to the powder.Vortex loading solution, and spin down to mix thoroughly. Wrap the vial with the loading solution with foil, to avoid exposure of light. Store the loading solution at minus 20 degrees Celsius, in a freezer.Dye loading procedure. Dissect a cutaneous pectoris muscle from the animal. Mount dissected tissue in a silver lined Petri dish.Stretch the tissue with fine stainless steel pins. Add Ringer's solution to the Petri dish. Using a razor blade, cut a small piece, around 2 millimeters of the ten microliters pipette tube from the thin end.Prepare a piece of plasticine for holding the filling pipette on the Petri dish. Connect the filling pipette with a plastic syringe through a silicone tube and adapters made of pipette tubes. Remove temporary Ringer's solution from the Petri dish by a plastic pipette.Dry preparation with a syringe. That will prevent suction of the solution in the filling pipette. Remove the vial with loading solution from the freezer and allow to thaw at room temperature in a dark place.With a fine tweezer and scissors, under visual control, with sterile microscope, cut the nerve close to the muscle. Leave around two millimeters of nerve stump. Fix the filling pipette with a tube and the syringe, on the Petri dish using plasticine.Move it close to the nerve branch. Gently aspirate the nerve stump in the filling pipette. Remove the suction tube from the blunt end of the filling pipette.Dry the nerve stump in the filling pipette by the syringe. Move up the filling pipette with the nerve stump. Isolate the nerve stump from the outside by Vaseline.One more time, dry the nerve stump in the filling pipette by the syringe. Draw the zero point five microliters of loading solution using a pipette with a long pipette tip. Gently insert the pipette tip with the loading solution into the filling pipette.Eject the mixture directly to the nerve stump. Seal the open end of the filling pipette by Vaseline. Add small amount of solution into the Petri dish.Incubate the preparation at room temperature in a darkened and wet location for five hours. After the incubation procedure, take the preparation, and remove the filling pipette with the loading solution. Place the preparation in a bath and wash it out with Ringer's solution.Keep the bath with the preparation overnight in a fridge at eight degrees Celsius temperature. Prepare the tissue for microscopy. Take the preparation from the bath.Mount preparation in a silver lined chamber and slightly stretch the tissue. Rinse the tissue with fresh Ringer's solution. Take the Suction Stimulation Electrode.Place the Stimulation Suction Electrode in the chamber with its tip close to the cut end of the nerve. Draw the nerve to the electrode. Construction of the electrode is described in Khazokoff's article, a link to the article is in the materials.Fix the preparation chamber on the microscope stage. Place the temperature probe. Connect inflow tubes in a power cord for Peltier element in the preparation chamber.Place outflow tube in the preparation chamber. Turn on a perfusion pump. Switch on the term controller unit.Set the temperature on the controller unit to reach 20 degrees Celsius. Install ultra-violet protection shield. Set up delay duration and the period on the stimulator.Connect a wire from the suction electrode with the stimulator. Turn it on. Under a low magnification objective, check the muscular contraction by turning the stimulus on.Fill up the perfusion system with a Ringer's solution with low calcium and high magnesium. Set the perfusion rate of about two milliliters per minute. Replace the objective for 40 times magnification.Switch on the Polychrome V.Select emission wavelength and continuous mode illumination. Open the shutter. Under 40 times magnification objective, in fluorescence mode illuminations ensure that the terminals are loaded.Find the terminal and focus on it. The loaded nerve and well-loaded motor nerve terminals, are represented on the figure. Video capture with Red-Short Neuro CCD Camera.Run Turbo-SM Software. Select, Change Settings. Input number of frames is 500.Enter the experiment name. Basic configuration is 1000 frames per second. 80 by 80.Choose external trigger. Enter pre-trigger time. Minus ten milliseconds.Enter number of repeats, 20. In Polychrome software, select external trigger illumination mode. Run Clampex software.Load stimulation protocol. The protocol is available for downloading from our lab page on Kazan Federal University website. Before recording capture a dark frame in Turbo-SM software.On the trinocular cube select light path exchange levels. 100 percent for camera. Run the stimulation protocol.Select the region of interest and check the signal. In response to the nerve stimulation we record changing of fluorescence intensity that reflects calcium entry into the nerve ending. Data analysis.In Turbo-SM software, select file. Then, select, average files. Select files.Enter name of average file, and average them. Save the average file as fit file. Select, save as fit file.Enter the file name and save it. Run Image J, and open the instruments below. Analysis, tools, ROI Manager.Find saved average file in fit format. Drag and drop it to Image J.Zoom the window for a better view. Select a rectangular region of interest over what you believe to be background.Add it to ROI manager. In ROI manager, select, more.Multi-measure. Load down mean.Copy data, export to Excel. In Excel, use average function to calculate average value of background for a ratio. Without closing Excel, return to the Image J.Close the window with background calculation without saving.Use ROI Manager, delete rectangular background drawing. Open instrument process, math, subtract. Enter calculated averaged value of threshold from Excel, and subtract the ground from stacks.Select a rectangular region of interest around a nerve terminal. Add it to ROI Manager. In ROI Manager, select, more, Multi-measure, load down, mean.Copy and paste in Excel window. Use Excel instrument plot graph. Select a flat area before starting the signal.Load the graph with the details from the first values of fluorescence at the rest, to the beginning of the signal. Then determine the numbers of points to average rest fluorescence values. Average these values, then find the average number of the fluorescence at the rest.Divide signals by fluorescence at the rest. Subtract one and multiply by 100 percent. Plot the signal and calculate the amplitude of calcium transient.Time parameters of fluorescence signals depend on the speed of the forward and or reverse reactions of calcium-dye-complex formation. The rise time of calcium signal represents dynamics of calcium input. It's diffusion in the terminal, and calcium interaction with dye.The decay of calcium transient depends on the dye affinity and rate of calcium buffering and its withdrawal. Analysis of calcium transient amplitude can be used to study influence of different active substances on the calcium entry that participate in neurotransmitter release. This loading technique is well suitable for the imaging of changes in cytosomal calcium.With fluorescent indicators and the both single nerve stimulation, and rhythmic synaptic activity. Analysis of calcium transient amplitude can be used to study the influence of different substances on calcium entry that participates in neurotransmitter release. There are some important points we want to pay attention to.Incubation procedure includes two steps. Incubation at room temperature, and in a fridge. It is important to control the time incubation with the dye at room temperature.Depending on the length of the nerve stump, the dye and the temperature in the room, incubation time might vary. If you overexpose the pre-operation terminals and proximal parts, close to the nerve stump, can be overloaded. Due to the long incubation in the fridge, dye is evenly distributed along the nerve endings.It is essential to place the nerve into the dye loading solution, within several minutes after cutting, to allow the dye to enter into the nerve, since longer time intervals produced ineffective loading, presumably due to receiving closed nerve. To reduce dye compartmentalization it is possible to use the extended form of the dye. It is very important to prevent the long fluorescent illumination of the tissue because it affects its survival.We use Nomarski optics in the visible light channel to search for the terminals. During the recording, we limit the illuminated view, by the diaphragm. Long duration of loading process can be reduced using shutter stamp of the nerve.In this case, glass micro-bypass can be used.

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Research

JoVE Journal

Neuroscience

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Loading Drosophila Nerve Terminals with Calcium Indicators

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

In this video, Drosophila larvae nerve fibers are immersed in FM dye, which binds to cell membrane lipids. Suction microelectrode and electrical pulses stimulate the endocytosis process, incorporating the FM dye into newly formed endocytic vesicles. This method, which visualizes presynaptic stages of neurotransmission, is useful for studying synaptic function and neurotransmitter release.

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

Source: Heredia, D. J., et. al. Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm. J. Vis. Exp. (2018) This video demonstrates fluorescence imaging of calcium dynamics at neuromuscular junctions (NMJs) in a transgenic mouse expressing genetically encoded calcium indicators. A NJM is identified by fluorescently tagged acetylcholine receptors in the diaphragm with the phrenic nerve. An image splitter is used to visualize multiple signals...

Fluorescence Imaging of Calcium Dynamics at Neuromuscular Junctions in the Diaphragm of a Transgenic Mouse

Source: Zhilyakov, N. V. et. al., Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy. J. Vis. Exp. (2021)This video demonstrates the imaging technique of recording fast calcium transients at nerve endings using confocal microscopy. By stimulating the nerve and capturing fluorescence changes from calcium-binding dyes, it visualizes dynamic calcium influx and clearance at the nerve ending, offering precise insights into...

Imaging Fast Calcium Transients in Mouse Nerve Endings Using Confocal Microscopy

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca2+ Imaging of the Enteric Nervous System

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

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.

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

Learn calcium dye loading into frog nerve endings via nerve stump for measuring fast calcium transients in neuromuscular junctions. This optical method enables studying drug effects on presynaptic calcium signaling.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction (Video) | JoVE Journal

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

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