Fluorescence studies of this work were carried out with the financial support of the Russian Science Foundation Grant (project No. 19-15-00329). The method was developed under financing from the government assignment for FRC Kazan Scientific Center of RAS &#1040;&#1040;&#1040;&#1040;-&#1040;18-118022790083-9. The research was developed with the use of the equipment of the Federal Research Center &#34;Kazan Scientific Center of RAS&#34;. The authors would like to thank Dr. Victor I. Ilyin for critical reading of this manuscript.

Experiments were performed on isolated nerve-muscle preparations of levator auris longus (m. LAL) from the Mice BALB/C (20-23 g, 2-3 months old)41. The experimental procedures were performed in accordance with the guidelines for the use of laboratory animals of the Kazan Federal University and the Kazan Medical University, in compliance with the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol met the requirements of the European Communities Council Directive 86/609/EEC and was approved by the Ethical Committee of the Kazan Medical University.
1. Preparation of the Ringer&#39;s and Filing solutions
<ol>
	<li>Prepare the Ringer&#39;s solution for mammalian muscle by mixing the following ingredients: NaCl (137 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), NaH2PO4 (1 mM), NaHCO3 (11.9 mM), and glucose (11 mM). Bubble through the solution with 95% O2 and 5% CO2 and adjust its pH to 7.2-7.4 by adding HCl/NaOH if necessary.</li>
	<li>Prepare the dye loading solution.
	<ol>
		<li>Prepare HEPES (10 mM) solution with pH in the 7.2-7.4 range. 500 &#181;g of commercial dye comes in a 500 &#181;L vial. Dissolve the dye in 14 &#181;L of the HEPES solution to obtain a dye concentration of 30 mM. Shake well and centrifuge until totally dissolved.</li>
		<li>Dilute the solution of Ca2+ indicator with HEPES solution down to 1 mM concentration. Keep it in a freezer (-20 &#176;C) and avoid exposure to light.</li>
	</ol>
	</li>
</ol>
2. Dye loading procedure
NOTE: The dye loading procedure is performed according to the protocol for loading through the nerve stump, adapted from the protocols previously published19,42,43,44,45,46.
<ol>
	<li>Dissect LAL muscle according to the dissection procedure for this preparation as described in the previously published protocols47,48.
	<ol>
		<li>Fix the tissue slightly stretched (no more than 30% from initial length) in the elastomer-coated Petri dish with fine stainless-steel pins and add Ringer&#39;s solution until the muscle is fully covered. 
		NOTE: The Petri dish was pre-filled with elastomer according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the Filling Pipette
	<ol>
		<li>Using a micropipette puller (see Table of Materials), prepare a micropipette with a fine tip which is as sharp as possible for the intracellular recordings. Use capillaries without internal filaments (1.5 mm in outer diameter and 0.86 or 1.10 mm in inner diameter).</li>
		<li>Break off the micropipette tip after scoringthe taper with an abrasive, leaving the tip open to about 100 &#181;m in diameter. Fire-polish the tip down to limit when the internal diameter shrinks from &#62;80 &#181;m to 12-13 &#181;m. Attach a silicone tube to one side of the Filling Pipette and a syringe (without a needle) to the other side.</li>
	</ol>
	</li>
	<li>Under a stereomicroscope, find the place where the nerve trunk turns into separate nerve branches. Place the Filling Pipette with the mounted tube and the syringe on the Petri dish using wax. Move the pipette tip until it stands above the nerve.</li>
	<li>With fine scissors, cut the nerve close to the muscle fiber, leaving a small piece of the nerve stump about 1 mm long. Gently aspirate the nerve stump together with some Ringer&#39;s solution, without pinching it, into the tip of the Filling Pipette. Remove the silicone tube from the Filling Pipette.</li>
	<li>Draw some amount of the dye loading solution (~0.3 &#181;L) using a syringe with a long filament. This volume corresponds to approximately 3 cm of the filament. 
	NOTE: Initially, it is necessary to make a filament from a pipette tip with a volume of 10 &#181;L by pulling on the fire using an alcohol lamp or a gas burner.</li>
	<li>Gently insert the filament tip with loading solution into the Filling Pipette. Release the mixture directly onto the nerve stump. Incubate the preparation at room temperature in dark for 30 min.</li>
	<li>After that, rinse the preparation with fresh Ringer&#39;s solution and incubate at 25 &#176;C for up to 2 h in a glass beaker with 50 mL (or more) Ringer&#39;s solution (preparation must be covered with the solution). During this time, the dye will reach the synapses.</li>
</ol>
3. Video capture with confocal microscopy
NOTE: Registration of calcium transients is performed with a laser scanning confocal microscope (LSCM) (see Table of Materials). To register fast calcium transients, an original protocol that permitted recordings of signals with a sufficient spatial and temporal resolution was used. The method has been described thoroughly in the publication by Arkhipov et al37. The microscope was equipped with a 20x water immersion objective (1.00 NA). The 488 nm laser line was attenuated to 10% intensity and emission fluorescence was collected from 503 to 558 nm.
<ol>
	<li>Mount the preparation into the silicon elastomer-coated experimental chamber and fix it, slightly stretched, with a set of steel micro-needles. Rinse the preparation extensively with Ringer&#39;s solution. 
	NOTE: A simple custom-made perfusion experimental chamber made of organic glass with the bottom of the chamber covered with an elastomer (prepared in accordance with the manufacturer&#39;s instructions; see Table of Materials) was used. The chamber has a solution supply tube. The solution is pumped out via a syringe needle, mounted on a magnetic holder (see Table of Materials). As an experimental chamber, a Petri dish could be used (like the one used for incubation of the preparation) but with attached supply and suction tubes.</li>
	<li>Install suction electrode which will be used to stimulate the nerve. 
	NOTE: Construction of the electrode is similar to what was published in the 2015 paper by Kazakov et al49. Place and fix the electrode by waxing beside the bath. Move the tip close to the nerve stump and aspirate it into the electrode.</li>
	<li>Mount the preparation chamber onto the microscope stage and place the inlet and outlet fittings into the chamber.</li>
	<li>To perfuse the preparation, use a simple gravity-flow-driven system. Turn on the perfusion suction pump to remove the excess solution.</li>
	<li>Plug the stimulating suction electrode into an electric stimulator and ensure that muscular contractions occur after stimuli. See section 3.9-3.12 for stimulation conditions and recording.</li>
	<li>Fill up the perfusion system with the Ringer&#39;s solution with d-tubocurarine (10 &#181;M). 
	NOTE: This solution helps to prevent muscular contractions. D-tubocurarine or alpha-bungarotoxin-specific blockers of nicotinic acetylcholine receptors on the postsynaptic membrane would completely or partially block muscle contractions50. Also, for preventing muscular contractions, specific blockers of postsynaptic sodium channels such as &#181;-conotoxin GIIIB could be used51.</li>
	<li>Switch on the perfusion suction pump and start perfusion of the preparation with the Ringer&#39;s solution containing d-tubocurarine.</li>
	<li>Set imaging parameters in the LSCM software as follows.
	<ol>
		<li>In the LSCM software (LAS AF; see Table of Materials), choose Electrophysiology. 
		NOTE: In this mode, when an image is captured at the time point, a synchronizing pulse is sent to the stimulator with the help of the trigger box. This elicits action potential generation in the preparation (Figure 1; stimulator unit).</li>
		<li>Select Acquisition Mode. For triggering the stimulator using the microscope sync pulse, in the Job menu settings, select the Trigger settings. Set the Trigger Out On Frame field to the out1 channel.</li>
		<li>Use the following settings: Scanning Mode: XYT, Frequency of Scanning: 1400 Hz, Zoom Factor: 6.1, Pinhole: fully open. Ensure that sequential trans-passing Bidirectional X mode is on.</li>
		<li>Set minimum time to form a frame at 52 ms and frames to be collected in a raw video at 20 frames. 
		NOTE: These settings permit image capturing with a resolution of 128 x 128 pixels while taking a single frame every 52 ms.</li>
		<li>Set excitation wavelength of the argon laser at 488 nm with 8% of output power.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63308/63308fig01.jpg" /> 
Figure 1: The schematic of the experimental setup. 1. Laser Scanning Confocal Microscope (LSCM). 2. Synchronization module of LSCM (trigger box). 3. Stimulator. 4. Isolation unit. 5. The biological sample. 6. Suction electrode for electrical stimulation of nerve. 7. Perfusion systems (7a: perfusate reservoir, 7b: dropper, 7c: flow regulator, 7d: vacuum flask). Arrows point to the direction of propagation of synchronizing pulse. <a href="https://www.jove.com/files/ftp_upload/63308/63308fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6" style="margin-left: 40px;">
	<li>Press the Live Mode button to switch to Live mode, which helps to get a preview of nerve terminals loaded with the dye.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63308/63308fig02.jpg" /> 
Figure 2: Mouse nerve and terminals loaded with the Ca2+ indicator. <a href="https://www.jove.com/files/ftp_upload/63308/63308fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="9">
	<li>Stimulation unit 
	NOTE: In this work, the stimulator described in the article by Land et al.52 was used. This device allows for setting temporal parameters of stimulation via the MatLab software.
	<ol>
		<li>Create a new file, paste the code from the above-mentioned article to the MatLab code window, and save the file. Click on Run, so a window with stimulation parameters appears. Set the delay time and duration of the stimulus. 
		NOTE: The delay determines the temporal resolution of the reconstituted fluorescent signal. The electric pulse of 0.2 ms duration is delayed, and then sent to the isolation unit. The latter forms the amplitude and polarity of the stimulating pulse and electrically isolates the biological object from the recording equipment.</li>
		<li>To stimulate the nerve, select supramaximal amplitude of the stimulating impulse (25%-50% greater than the maximum stimulation intensity necessary to activate all the nerve fibers). 
		NOTE: The presented method is based on a special algorithm for recordings of single fast fluorescent signals using LSCM with the minimized sweep. At each step of the developed algorithm, the recorded fluorescent signal is shifted from the previous one by a time interval that is shorter than the microscope sweep. The value of time shifts determines the temporal resolution of the required signal. The number of steps (shifts) in the algorithm depends on the required temporal resolution and original temporal resolution. With this method of registration, the stimulation of the preparation is carried out with a frequency of 0.25 Hz.</li>
	</ol>
	</li>
	<li>In the Live mode, search for the ROI and obtain the best focus. Run the data acquisition software.</li>
	<li>Shift the delay on the stimulator by 2 ms less relative to the previous value and run the data acquisition software.</li>
	<li>Repeat step 3.11 26 times to acquire 26 sequences, with each sequence shifted by 2 ms from the previous one.</li>
</ol>
4. Video processing
NOTE: A series of video images acquired by the confocal microscope is exported in the TIFF format with the free software LAS X (see Table of Materials). This series was divided into frames and exported to a folder. For generating the image sequence with higher time resolution, the ImageJ software, which has an open initial code for the analysis and processing of the data, was used. The algorithm of signals processing is represented schematically in Figure 3.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63308/63308fig03.jpg" /> 
Figure 3: Scheme for compiling a high-resolution video file (2 ms on frame) from original video files with a low temporal resolution (52 ms on frame). The original video files and the corresponding signals are colored in black, magenta, and green. The compiled video file and the resulting signal are colored red. The scheme on the right, line by line, shows the video images obtained with a confocal microscope. On the left, the corresponding signals of fluorescence change from the selected ROI. The topmost line is formed frame by frame from the received frames according to the scheme. The result is a video image consisting of the entire array of frames so that there is a delay time of 2 ms between frames instead of 52 ms. Each line corresponds to an offset of the stimulation signal by (n - 1) * t, where t is time shift (2 ms), and n is the number of shift iterations. k denotes the number of frames in the original video files (lines 2-4) and depends on the duration of the recorded signal. In this case, to register a signal with a duration of 1 s, it is necessary to select k = 20 (52 ms * 20 = 1040 ms). t0 is the required delay before stimulation. To calculate the number of shift iterations n, the initial temporal resolution between frames (52 ms) must be divided by the required temporal resolution (2 ms). In this case, n = 26, which corresponds to 26 registered sweeps. As a result of the performed manipulations, a video image consisting of n * k = 520 frames is obtained. <a href="https://www.jove.com/files/ftp_upload/63308/63308fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Run LAS X software. Open the project which was created during performing experiment. Click on Export, and then on Save As to save frames in .tiff format in the destination folder.</li>
	<li>Run the ImageJ software. Click on File &#62; Import &#62; Image Sequence.</li>
	<li>In the Open Image Sequence window, choose the destination folder and open the first frame.</li>
	<li>In the Sequence Options window, in the Starting Image field, set the frame number to 1 for the first frame. In the Increment field, set the value equal to the number of frames in the initial signal recording (20 for the present case) and click on OK.</li>
	<li>To save the generated file of stitched first frames in a separate folder, click on File &#62; Save &#62; Folder.</li>
	<li>Repeat steps 4.3-4.5 for the next 19 frames. In the Sequence Options window, set the corresponding frame number in the Starting Image field.</li>
	<li>To generate the full high-time-resolution video, stitch all the frames together. To do this, click on File &#62; Import &#62; Image Sequence and select 1 in the Starting Image and Increment fields. The result will be the final video with increased temporal resolution. Save the file in .tiff or any other suitable format.</li>
</ol>
5. Video analysis
NOTE: In ImageJ, select ROI and background. Subtract background from ROI. Data is represented as the ratio, (&#916;F / F0 - 1) * 100%, where F0 is the intensity of fluorescence at rest and &#916;F is the intensity of fluorescence during stimulation.
<ol>
	<li>Click on Image &#62; Stacks &#62; Tools &#62; Stack Sorter. Then, click on Analysis &#62; Tools &#62; ROI Manager.</li>
	<li>Drag and drop the .tiff file saved in step 4.7 into the ImageJ window. Expand the image for a better view. To improve image visualization, click on Image &#62; Adjust &#62; Brightness/Contrast &#62; Auto. This step will not affect the data.</li>
	<li>Set the background close to the nerve terminal by drawing ROI. Add it to the ROI manager. Calculate the background by clicking on More &#62; Multi Measure. Copy mean values, paste to the Spreadsheet program, and calculate the average.</li>
	<li>Subtract the calculated average value from the stacks by clicking on Process &#62; Main &#62; Subtract. Enter the value.</li>
	<li>Draw ROI around a nerve terminal via a polygon line. Add it to the ROI manager.</li>
	<li>Measure the intensity of the nerve terminal: Click on More &#62; Multi Measure. Copy mean values and paste them to the Spreadsheet program.</li>
	<li>Calculate the average offset of signals. 
	NOTE: Use the corresponding points depending on the delay time before stimulation. This step establishes the F0 value that will be used in subsequent calculations.</li>
	<li>Divide the signal values by the average offset value. 
	NOTE: After this step, the signal does not contain the contribution of the background and raw fluorescence to the amplitude values for the selected ROI.</li>
	<li>Subtract 1 from values obtained in step 5.8, and then multiply by 100%.</li>
	<li>Plot a graph of Ca2+- transient and calculate the amplitude.</li>
</ol>

The authors have nothing to disclose.

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

The problem of measuring fast calcium waves in excitable cells is one of the most important and challenging aspects of studying signal transmission in the central and peripheral nervous systems. Calcium ions play an important role in triggering neurotransmitter release, synaptic plasticity, and modulation of the activity of various intracellular proteins1,2,3,4,5. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases6. To measure changes in the calcium levels, fluorescent calcium-sensitive dyes are commonly used, and changes in their fluorescence level are analyzed7,8,9.
Loading of calcium dyes into cells can be achieved in different ways. Predominantly, cell-permeant dyes are used10,11. However, in such a case, it is not only difficult to control the concentration of a dye inside the cell, but it is also hard to select target cells for loading. This method is not applicable for studying peripheral nerve endings since the dye enters postsynaptic cells. Instead, cell impermeant dyes are more suitable for such preparations. In this case, the dyes are delivered to the cells by microinjection or through a patch pipette12,13,14. There is also a method of loading through a nerve stump. The latter method is most suitable for neuromuscular junction preparations15,16,17,18,19,20. It allows performing staining for only cells of interest. Although this method does not provide an accurate evaluation of the concentration of the dye in the target cell, the concentration can be estimated approximately by comparing the level of fluorescence of the cells at rest in solutions with a known concentration of calcium21. In this study, a modification of this method applied to synapses of mammals is presented.
Calcium entry during the depolarizing phase of the action potential is a fast process, especially in the neuromuscular junction; therefore, for its registration, appropriate equipment is required1. A recent study using a voltage-sensitive fluorescent dye demonstrated that the duration of the action potential in the peripheral synapse of a mouse is approximately 300 &#181;s22. Calcium transient, evaluated using calcium-sensitive dyes in the peripheral synapses of the frog, has a longer duration: the rise time is about 2-6 ms and the decay time is about 30-90 ms, depending on the calcium dye used23,24. To measure fast processes with the help of fluorescent dyes, CCD or CMOS cameras are generally used, with fast and sensitive CCD matrices. However, these cameras have the disadvantage of low resolution, limited by the size of the sensitive elements of the matrix25,26,27,28. The fastest cameras with sufficient sensitivity to record both action potentials and calcium transients in response to low frequency stimulation of cells have a scanning frequency of 2,000 Hz, and a matrix with a dimension of 80 x 8029. To obtain signals with a higher spatial resolution, confocal microscopy is used, especially if it is necessary to assess some volumetric changes in the signal30,31,32. But it should be kept in mind that confocal microscopy has a high scanning speed in line scan mode, but there are still significant limitations on the speed of recordings of fast processes when building a spatial image33. There are confocal microscopes based on rotating Nipkow disks (slit-scanning microscopy) and Multipoint-Array Scanners, which have a higher scanning speed. At the same time, they are inferior to the classical confocal microscopes in confocal image filtering (pinholes crosstalk for microscopes with a Nipkow disk)32,34,35. Confocal imaging with resonance scanning can also provide a high spatio-temporal resolution required for high temporal measurements36. However, take into account that the registration of weak fluorescent responses at a high scanning speed when using resonance scanners requires highly sensitive detectors such as hybrid detectors36.
This article presents a method for increasing the temporal resolution of signals recorded with the Laser Scanning Confocal Microscopy (LSCM) while maintaining the spatial resolution37. The current method is a further development of the methods described earlier and transferred to the LSCM platform38,39,40. This approach does not require changes in the microscope hardware and is based on the application of an algorithm for recording periodically evoked fluorescent signals with a time shift relative to the moment of stimulation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Capillary Glass</td>
			<td>Clark Electromedical instruments, UK</td>
			<td>GC150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal and multiphoton microscope system Leica TCS SP5 MP</td>
			<td>Leica Microsystems , Heidelberg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller P 97</td>
			<td>Sutter Instrument, USA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow regulator</td>
			<td>KD Medical GmbH Hospital Products, Germany</td>
			<td>KD REG</td>
			<td>Disposable infusion set with Flow regulator</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich, USA</td>
			<td>H0887</td>
			<td>100mL</td>
		</tr>
		<tr>
			<td>Illumination system Leica CLS 150X</td>
			<td>Leica Microsystems, Germany</td>
			<td></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>Las AF software</td>
			<td>Leica Microsystems, Heidelberg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Las X software</td>
			<td>Leica Microsystems, Heidelberg, Germany</td>
			<td></td>
			<td>https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/</td>
		</tr>
		<tr>
			<td>Magnetic Holder with Suction Tubing</td>
			<td>BIOSCIENCE TOOLS, USA</td>
			<td>MTH-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspin FV 2400</td>
			<td>Biosan, Latvia</td>
			<td>BS-010201-AAA</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine science tools, Canada</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-spin MSC 3000</td>
			<td>Biosan, Latvia</td>
			<td>BS-010205-AAN</td>
			<td></td>
		</tr>
		<tr>
			<td>Oregon Green 488 BAPTA-1 pentapotassium salt</td>
			<td>Molecular Probes, USA</td>
			<td>O6806</td>
			<td>500 &#956;g</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Biohit, Russia</td>
			<td>720210</td>
			<td>0.5-10 &#181;L</td>
		</tr>
		<tr>
			<td>Pipette tip</td>
			<td>Biohit, Russia</td>
			<td>781349</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Plasticine</td>
			<td>local producer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use hypodermic needles</td>
			<td>Bbraun</td>
			<td>100 Sterican</td>
			<td>0.4&#215;40 mm</td>
		</tr>
		<tr>
			<td>Spreadsheet program</td>
			<td>Microsoft, USA</td>
			<td>Microsoft Office Excel</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope, Leica M80</td>
			<td>Leica Microsystems , Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction electrode</td>
			<td>Kazakov A. SIMPLE SUCTION ELECTRODE FOR ELECTRIC STIMULATION OF BIOLOGICAL OBJECTS / A. Kazakov, M. Alexandrov, N. V. Zhilyakov et al. // International research journal.&#160;- 2015. - No. 9 (40) Part 3. - P. 13-16.</td>
			<td></td>
			<td>http://research-journal.org/biology/prostoj-vsasyvayushhij-elektrod-dlya-elektricheskoj-stimulyacii-biologicheskix-obektov/</td>
		</tr>
		<tr>
			<td>Sylgard 184 elastomer</td>
			<td>Dow Corning, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>local producer</td>
			<td></td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>local producer</td>
			<td></td>
			<td>60 mL</td>
		</tr>
	</tbody>
</table>

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.

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

Watch this Scientific Journal Video about Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy at JoVE.com

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

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1.9K Views.  Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS. 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.

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

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

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.

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 ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...