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 Directiv...

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.

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	<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 ...

Estimation of a presynaptic calcium level is a key task in studying synaptic transmission. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The main advantage of the presented loading technique is that the staining has been performed only for the cells of interest.Our method for recording calcium intransience provides extremely good spatial-temporal resolution. The presented registration method can be helpful to neurophysiologists who need to register fast periodic processes using a confocal microscope. The most difficult step in the loading technique is holding the nerve inside the pipette.The diameter of the pipette must be perfectly fit, so make some several ones. Knowing all the steps in the protocol are complicated and demonstrating each of them will help a lot. Begin by fixing the levator auris longus muscle tissue in a slightly stretched manner in the elastomer coated Petri dish with the fine stainless steel pins and add Ringer's solution to the dish until the muscle is fully covered.Next, use a micropipette puller to prepare a micropipette with a fine sharp tip for the intracellular recordings. Use capillaries without internal filaments. After scoring the taper with an abrasive, break off the micropipette tip, leaving a tip open to about 100 micrometers in diameter.Then, fire polish the tip until the internal diameter shrinks from 80 micrometers to 12 micrometers. When done, attach a silicone tube to one side and a syringe without a needle to the other side of the filling pipette. Under a stereo microscope, find where the nerve trunk turns into separate nerve branches.Place the filling pipette on the Petri dish using wax and then move the pipette tip until it stands above the nerve. Cut the nerve close to the muscle fiber with fine scissors, leaving a small piece of the nerve stump, about one millimeter long. Then, aspirate the nerve stump with some Ringer's solution into the tip of the filling pipette, without pinching the nerve.Once done, remove the silicone tube from the filling pipette. Later, use a syringe with a long filament to draw the dye loading solution. Then, insert the filament tip with the loading solution into the filling pipette and release the mixture onto the nerve stump, followed by incubating the nerve preparation at room temperature in the dark for 30 minutes.After the incubation, rinse the nerve preparation with fresh Ringer's solution before treating it with 50 milliliters of Ringer's solution in a glass beaker at 25 degrees Celsius for two hours. For the confocal microscopy, mount the nerve preparation into a silicon elastomer coated experimental chamber and fix it, slightly stretched, with a set of steel microneedles. Then, rinse the preparation extensively with the Ringer's solution.Install a suction electrode on the chamber. After mounting the preparation chamber onto the microscope stage, place the inlet and outlet fittings into the chamber. Turn on a gravity flow driven system to remove the excess solution from the preparation.Once done, plug the stimulating suction electrode into an electric stimulator and ensure that muscular contractions occur after stimuli. Then, fill up the perfusion system with the Ringer's solution containing 10 micromolar d-tubocurarine and switch on the perfusion suction pump to start perfusion. In the laser scanning confocal microscope, or LSCM software, choose electrophysiology and acquisition mode to set imaging parameters.In the job menu settings, select the trigger settings to trigger the stimulator with the microscope sync pulse and set the trigger out on frame field to the OUT 1 channel. Turn the scanning mode to XYT at a frequency of scanning of 1, 400 hertz. The zoom factor should be 6.1 with the pinhole fully open.Ensure that sequential, transpassing, bidirectional X mode is on, then, set the minimum time to form a frame at 52 milliseconds and collect frames in a raw video at 20 frames. Set excitation wavelength of the argon laser at 488 nanometers with 8%of output power. When the parameters are set, press the live mode button to get a live preview of nerve terminals loaded with the dye.In the live mode, search for the region of interest, or ROI, to obtain the best focus. Then, shift the delay on the stimulator by two milliseconds less relative to the previous value and run the data acquisition software to acquire 26 sequences by shifting each sequence two milliseconds from the previous one. To process the captured videos, run Leica Application Suite X, or LAS X software, and open the project created.Then, click on export and save as to save frames in TIF format in the destination folder. Next, run the ImageJ software and click on file, import, and image sequence. In the open image sequence window, choose the destination folder and open the first frame.From the sequence options, go to the starting image field and set the frame number to one for the first frame. Then, in the increment field, set the value equal to the number of frames in the initial signal recording before clicking okay. To save the generated file of stitched first frames in a separate folder, click on the file, save, and folder tabs in order.Repeat the procedures for 19 frames by setting the corresponding frame number in the starting image field. When all the images are obtained, click on file, import, and image sequence to select one in the starting image and increment fields. The final video with all the frames stitched together at increased temporal resolution can be seen.Then, save the video file in TIF format. To analyze the stitched video, click on image, stacks, tools, and stack sorter. Then, go to analysis, tools, and ROI manager.Drag and drop the TIF file saved previously into the ImageJ window and then expand the image for a better view. To improve the image visualization, click on image, adjust, to turn brightness contrast to auto. Set the background close to the nerve terminal by drawing ROI and adding value to the ROI manager.Then, calculate the background by clicking on more and multi measure. Copy mean values to paste to the spreadsheet program. Then, calculate the average value.Next, subtract the calculated average value from the stacks by clicking on process, main, and subtract. Enter the obtained value and draw ROI around a nerve terminal via a polygon line before adding it to the ROI manager. Measure the transient intensity of the nerve terminal by clicking more and multi measure tabs.Then, copy the mean intensity values and paste them to the spreadsheet program to calculate the average offset of signals. Divide the signal values by the average offset value. From the obtained value, subtract one and then multiply by 100%followed by plotting a calcium transient graph to determine the amplitude.After loading the nerve preparation with a dye, most of the synapse is located close to the nerve stump, indicated a sufficient level of fluorescence. After the image processing, calcium transience with the desired spatial and temporal resolution were obtained. One of the most important steps in this protocol is that the nerve stump must be placed in the dye containing solution in the first few minutes after cutting the nerve.This method can be used to load various dyes and drugs into motor neurons. Protocol of registration can be useful for assessing calcium in dendrites and spines in CNS preparations. Our technique allows to register a calcium signal with good spatial and temporal resolution.Studying calcium transience is a powerful tool in research regulation of a neurotransmitter release and synaptic plasticity.

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

Abstract