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&#39;s solution.
	<ol>
		<li>Prepare Ringer&#39;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&#39;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 &#181;L of the HEPES solution to a vial with the dye30. 
		NOTE: the Ca2+ indicator dye comes in a 500-&#181;L vial with 500 &#181;g 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 &#176;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&#39;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&#39;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 &#181;L 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&#39;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&#215;), 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 &#181;L 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&#39;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&#39;s solution, and keep it overnight in the refrigerator at 8 &#176;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&#39;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 &#176;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&#39;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, &#181;-conotoxin GIIIA can be used27.</li>
	<li>Switch on the pump and start the superfusion of the preparation with the Ringer&#39;s solution with low Ca2+ and high Mg2+.</li>
	<li>Switch to the 40&#215; 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="Figure 1" class="xfigimg" src="/files/ftp_upload/55122/55122fig1.jpg" /> 
Figure 1: Nerve and terminals with loaded Ca2+ indicator. (A) Nerve filled with Ca2+ indicator after the loading procedure. Scale bar = 200 &#181;m. (B) Nerve endings filled with Ca2+ indicator. Scale bar = 20 &#181;m. (C) Ca2+-indicator fluorescence is clearly visible in the nerve ending. Scale bar = 20 &#181;m. <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 &#34;Live&#34; mode, find the ROI and adjust the focus.</li>
	<li>Select the menu &#34;Change&#34; settings.</li>
	<li>Use the &#34;Basic Configuration&#34; 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 &#34;External Trigger.&#34;</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 &#34;external trigger illumination&#34; 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: (&#916;F/F0 -1) x 100%, where&#39;s &#916;F 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 &#62; Average Files. Select the files and average them.</li>
	<li>Save the averaged file as a .fit file by clicking &#34;Save as Fit file.&#34;</li>
	<li>Run the ImageJ software. Perform the following steps:</li>
	<li>Click Image &#62; adjust &#62; brightness/contrast.</li>
	<li>Click Image &#62; stacks &#62; tools &#62; stack sorter.</li>
	<li>Click Analysis &#62; tools &#62; 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 &#62; 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 &#62; Main &#62; 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 &#62; 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 &#34;1&#34; and multiply by 100%.</li>
	<li>Plot the signal and calculate the amplitude of the Ca2+ transient.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+suction+electrode+for+electrical+stimulation+of+biological+objects.">A simple suction electrode for electrical stimulation of biological objects.</a> Mezhdunarodnyj nauchno-issledovatel'skij zhurnal. 40 (9), 13-16 (2015).</li><li>Khaziev, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine-induced+inhibition+of+presynaptic+calcium+signals+and+transmitter+release+in+the+frog+neuromuscular+junction.">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>B&#233;lair, E. L., Vall&#233;e, J., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+modulation+of+synaptic+efficacy+at+the+neuromuscular+junction+of+Rana+pipiens+frogs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+presynaptic+calcium+currents+and+endogenous+calcium+buffers+at+the+frog+neuromuscular+junction+with+two+different+calcium+fluorescent+dyes.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraterminal+Ca2++and+spontaneous+transmitter+release+at+the+frog+neuromuscular+junction.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++from+one+or+two+channels+controls+fusion+of+a+single+vesicle+at+the+frog+neuromuscular+junction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+and+reversible+blocker+of+presynaptic+calcium+channels+in+frog+and+crayfish+neuromuscular+junctions+from+Phoneutria+nigriventer+spider+venom.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transient+registration+in+response+to+single+stimulation+and+during+train+of+pulses+in+mouse+neuromuscular+junction.">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

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="Figure 2" class="xfigimg" src="/files/ftp_upload/55122/55122fig2.jpg" /> 
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 &#916;F/F (%)</td>
			<td>Rise time 20%-80% (ms)</td>
			<td>&#964; (ms)</td>
		</tr>
		<tr>
			<td>12,6 &#177;1,1 (n = 13)</td>
			<td>4.6 &#177;0.5 (n = 13)</td>
			<td>115.3 &#177;8.3 (n = 13)</td>
		</tr>
	</tbody>
</table>
Table 1: The averaged parameters of theCa2+ transient. The data are presented as the mean &#177; SE; n is the number of measurements in distinct NMJs. The peak &#916;F/F is the averaged amplitude of &#916;F/F.

Watch this Scientific Journal Video about Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction at JoVE.com

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

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 ...

loading-calcium-dye-into-frog-nerve-endings-through-nerve-stump

Research

JoVE Journal

Neuroscience

7.7K Views.  Russian Academy of Sciences.  Hier beschrijven we een methode voor het laden van een calciumgevoelige kleurstof door de kikker zenuwstomp in de zenuwuitgangen. We presenteren ook een protocol voor de opname en analyse van snelle calcium transiënten in de perifere zenuw eind. 

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

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

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.

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Biology

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

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

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.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

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

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

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 labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

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

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

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

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.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

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

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

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 dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

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

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

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.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

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

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.

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.

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

In Vivo Calcium Imaging of Neuronal Ensembles in the Intact Dorsal Root Ganglia of a Mouse

Source: <a href='https://app.jove.com/v/64826/in-vivo-calcium-imaging-of-neuronal-ensembles-in-networks-of-primary-sensory-neurons-in-intact-dorsal-root-ganglia'>Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. Vis. Exp. (2023).</a>The video demonstrates an in vivo calcium imaging technique to monitor neuronal activity in the dorsal root ganglia (DRG) of a transgenic mouse. DRG neurons express a calcium indicator that increases fluorescence intensity upon binding to calcium. Mechanical or thermal stimuli applied to a hind paw generate action potentials that propagate to the DRG, inducing calcium influx in the neurons and resulting in increased fluorescence emission.

Source: Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

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

Source: Kopke, D. L., et al., <a href="https://app.jove.com/v/57765">FM dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation.</a> J. Vis. Exp. (2018)
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.

1. Larval Glue Dissection

	Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.
	...

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

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