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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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><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 &amp;mu;g</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>Biohit, Russia</td><td>781349</td><td>10 &amp;micro;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&amp;times;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.&amp;nbsp;- 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...

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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. Schatting van een presynaptisch calciumgehalte is een belangrijke taak bij het bestuderen van synaptische transmissie. Het bestuderen van calciumsignalering is ook belangrijk voor het vinden van manieren om neurodegeneratieve ziekten te behandelen. Het belangrijkste voordeel van de gepresenteerde laadtechniek is dat de kleuring alleen is uitgevoerd voor de cellen van belang.Onze methode voor het registreren van calciumintransitie biedt een extreem goede ruimtelijk-temporele resolutie. ...

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

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Biology

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 ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

Source:&#160;<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/61481/quantifying-spontaneous-ca2-fluxes-and-their-downstream-effects-in-primary-mouse-midbrain-neurons'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Bancroft, E. A.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Quantifying Spontaneous Ca<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:0.6em;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>2+<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2020)</a> &#160;This video demonstrates the real-time visualization of calcium influx in ventral midbrain neurons derived from mouse embryos. Using a viral vector to express a calcium indicator, the neurons are imaged under a confocal microscope to track changes in fluorescence intensity during spontaneous and neurotransmitter-induced calcium activity.

Source:&#160;Bancroft, E. A.,&#160;et al. Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Structural & Functional Brain Imaging

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

At 20 times magnification, locate the end plate band at the center of the muscle by looking for fluorescently labeled alpha-bungarotoxin neuromuscular junctions under green yellow light excitation. Switch to blue light excitation to image calcium responses in muscle, motor neuron, or Schwann cells. In this example, GCaMP 3 is expressed in muscle cells.If desired, set up the image splitter with bandpass filters and dichroic single edge filter for the dual wavelength imaging. Perform experiments with the brightness bar on the lookup table bar, set to 110% of the level at which the genetically encoded calcium indicator expressing tissue exhibits saturation at 20 times magnification, without binning in response to potassium chloride.Record at 20 frames per second so not to miss any fast events. Stimulate with 1 to 45 seconds of 20 to 40 hertz of nerve stimulation by delivering a train of impulses using a suction electrode. And collect dynamic fluorescent calcium responses in one cell subtype, together with the static fluorescently labeled alpha-bungarotoxin neuromuscular junction signal.

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

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