assessing a functional neuromuscular junction via simultaneous optical stimulation and video recording

Assessing a Functional Neuromuscular Junction Via Simultaneous Optical Stimulation and Video Recording

Prepare a 4-to-1 mix of 3 milligrams per milliliter collagen I and solubilized basement membrane matrix. Then, resuspend the myoblasts in this freshly prepared collagen mixture.
Next, add 15 microliters of this cell-collagen suspension to each muscle chamber of the bioreactor, making sure to spread the suspension across both pillars using the pipette tip. Allow the cell gel mixture to polymerize at 37 degrees Celsius for 30 minutes. Then, fill each bioreactor well with 450 microliters of myotonic growth media.
On day 11 of motor neuron differentiation, transfer the cells and media to a 50-milliliter tube, and allow the neurospheres to settle to the bottom for 5 minutes. Aspirate the supernatant and resuspend the cells in 15 milliliters of motor neuron suspension culture medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial-cell-derived neurotrophic factor.
On day 14 of the differentiation protocols, seed the motor neurons into the platform. Prepare a 4-to-1 gel mixture of 2 milligrams per milliliter collagen I and solubilized basement membrane matrix. Use a 400-nanometer cell strainer to select large neurospheres, and resuspend them in the prepared gel mixture.
Carefully, aspirate media from the reservoir and the neurosphere well. Then, add 15 microliters of the gel mixture into the neurosphere channel. Load a 10-microliter pipette with 10 microliters of gel, and pick one neurosphere. Deposit the neurosphere into the neurosphere channel, ensuring that it is in the chamber. Then, slowly raise the pipette while releasing the remaining gel once the neurosphere is deposited.
Allow the gel to polymerize for 30 minutes at 37 degrees Celsius. Then, add 450 microliters of NbActiv4 supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial-cell-derived neurotrophic factor to the reservoirs.
For imaging, use an inverted fluorescent microscope with a scientific complementary metal oxide semiconductor camera software. Set the binning to 2-by-2, exposure to 20 milliseconds, rolling shutter on, readout rate to 540 megahertz, dynamic range to 12-bit and gain 1, and sensor mode to overlap. Use a 2x objective on the microscope to image the microtissues, and attach a live-cell chamber to the microscope stage.
Select the region of interest that contains the innervated skeletal tissues to minimize the file size and processing time. Then, place a 594-nanometer long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera. Next, place a rectangular four-well plate containing four bioreactors into the live-cell cell chamber. Then, click Live View, and center, and focus the image with the desired ROI.
Download the custom macrocode from the GitHub folder to control the stage position, the Arduino board, and video acquisition. Then, set the output movie as day_tissuegroup_tissuename_experiment.ND2. Run the macrocode with the desired x- and y-coordinates set on the stage, and acquire a fast time-lapse lapse with 1,700 frames at 50 frames per second.
After imaging, replace the media and return the samples to the incubator. Allow at least 24 hours between image acquisition sessions to avoid tissue fatigue.

assessing-functional-neuromuscular-junction-via-simultaneous-optical

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

EoE Sub Articles

eoe sub articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Bioreactor preparation
<ol>
	<li>Make bioreactor molds
	<ol>
		<li>Download a bioreactor CAD (Computer-aided design) file from the Supplementary CAD File or create a custom design.</li>
		<li>Generate a CNC (Computer Numerical Control) toolpath from the 3D model using CAM (Computer-aided manufacturing) software.</li>
		<li>Machine acetal molds using a CNC milling machine.</li>
	</ol>
	</li>
	<li>Fabrication of bioreactors
	<ol>
		<li>Mix a 10:1 base to curing agent mixture of polydimethylsiloxane (PDMS, 77 g of mixture per 4 platforms/molds).</li>
		<li>Place the mixture into a vacuum chamber, close all the valves, turn on the vacuum, and de-gas the mixture for at least 30 min until no air bubbles remain. Pour the mixture into molds and de-gas the molds in the vacuum chamber for 1 h.</li>
		<li>Close the molds with the top half of the mold in the correct orientation. Place a steel hexagonal rod over the center and clamp on both sides.</li>
		<li>Refill the top with PDMS.</li>
		<li>Cure the molds in a 65 &#176;C oven for at least 4 h.</li>
		<li>Remove the platforms from the molds after cooling down to room temperature.</li>
		<li>Clean the devices in an ultrasonic bath using 1 h cycles of dish soap, 400 mL of 100% isopropanol, and distilled water.</li>
		<li>Dry overnight at 65 &#176;C.</li>
		<li>Soak glass coverslips in 1% nonionic surfactant polyol for 30 min, ensuring that they do not stack so that they are all properly coated.</li>
		<li>Rinse well with distilled water and dry overnight at 65 &#176;C.</li>
		<li>Treat the glass coverslips and PDMS platform with a plasma cleaner on high with 6 L/min of oxygen for 1-2 min. Once treated, bond the two together by pressing the PDMS down onto the coverslip for at least 30 s.</li>
		<li>Autoclave the bonded devices before adding cells.</li>
	</ol>
	</li>
</ol>
2. Building an optical stimulation setup
<ol>
	<li>Using a 30 mm cage cube system, attach a 573 nm dichroic mirror in the center. Couple a red 627 nm LED (Light Emitting Diode) with a 594 nm long-pass excitation filter and attach it to the top side of the cube, with the LED facing the mirror. Then attach a blue 488 nm LED with a 546 nm short-pass excitation filter to the adjacent side of the cube, with the LED facing the mirror as shown in the schematic in Figure 1A.</li>
	<li>Attach a ring-actuated iris diaphragm to the bottom of the cage cube to control the size of the illuminated area.</li>
	<li>Power each LED with a T-Cube LED driver and control via an Arduino Uno Rev3 board. Connect the side input of the LED driver to a power source plug, connect the middle output to the Arduino, and connect the side output directly to the LEDs.</li>
	<li>Connect the Arduino Uno to a computer with a USB cable.</li>
	<li>Download files from GitHub link (https://github.com/ofvila/NMJ-function-analysis).</li>
	<li>Open the &#34;Optical_Stimulation_Ramp_Arduino.ino&#34; file from the GitHub folder.</li>
	<li>Set starting parameters: Steps = 30; startFrequency = 0.5; endFrequency = 3; pulseLength = 100.</li>
	<li>Ensure that pin output 4 is assigned to the Blue LED driver and pin output 2 is assigned to the Red LED driver. Check the middle wires of the LED driver are connected to the ground and to the respective pin output.</li>
	<li>Compile and load the &#34;Optical_Stimulation_Ramp_Arduino.ino&#34; program to Arduino.</li>
	<li>Connect each LED driver to its corresponding channel.</li>
</ol>
3. Cell culture setup (day -21-0)
<ol>
	<li>Primary skeletal muscle cells
	<ol>
		<li>Thaw and expand primary skeletal myoblasts (obtained from Cook Myosite) for a maximum of six passages using Myotonic growth medium (+ supplement). Maintain cells in an incubator set to 37 &#176;C and 5% CO2.</li>
		<li>Change the media every 2 days.</li>
		<li>Once cells are about 60% confluent, pass them using 0.05% Trypsin-EDTA (1x, for 5 min at 37 &#176;C). Collect the dissociated cells and add fresh media to neutralize the trypsin.</li>
		<li>Spin down the cells at 300 x g and aspirate the supernatant above the cell pellet.</li>
		<li>Resuspend the cells with MGM and seed 1:3 with 60 mL per triple-layer flask.</li>
	</ol>
	</li>
	<li>ChR2-hiPSC 
	NOTE: All cell lines for this work were created and used in compliance with the institutional guidelines of Columbia University, NY, USA. In this protocol, ChR2-expressing hiPSCs were generated via CRISPR-Cas9 genome editing using previously described methods18, but any stable optogenetic cell lines (lentiviral, piggyBac, etc.) can be used in the same way. Constitutive promoters expressed in both iPSCs and motoneurons were chosen (CAG). The cells were found to have healthy karyotypes, as noted in previous publications.
	<ol>
		<li>Coat 6-well plates with 1 mL/well of solubilized basement membrane matrix diluted in DMEM/F12 (1:80) and incubate the plates at room temperature for 1 h.</li>
		<li>Seed iPSCs on coated 6-well plates with 2 mL of feeder-free cell culture medium (iPSC media), exchanging 2 mL of media every other day and passaging every 5-7 days. Maintain cells in an incubator set to 37&#176;C and 5% CO2.</li>
		<li>Passaging
		<ol>
			<li>Dissociate the stem cells by incubating them with 1 mL of enzyme-free stem cell-releasing reagent for 4 min and mechanically shearing them with a wide-tip P1000 pipette.</li>
			<li>Seed cells at a ratio of 1:24 or 1:48 in iPSC media with 2 &#956;M of Y-27632 dihydrochloride in 2 mL/well onto coated 6-well plates.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Skeletal muscle tissue seeding (day -3)
<ol>
	<li>Isolate and count 30 x 106 myoblasts using an automated cell counter.</li>
	<li>Resuspend the myoblasts in a 4:1 mix of 3 mg/mL collagen I and solubilized basement membrane matrix (800 &#956;L of collagen mixture + 200 &#956;L of basement membrane matrix to reach a final volume of 1 mL). For the collagen component, add the accompanying neutralizing agent (9:1 mix of collagen to neutralizing agent) and dilute the mixture with 1x PBS to achieve a volume of 800 &#956;L.</li>
	<li>Add 15 &#956;L of cell-collagen suspension to each muscle chamber of the bioreactor, being sure to spread the suspension across both pillars using the pipette tip (Figure 2).</li>
	<li>Let the cell-gel mixture polymerize at 37 &#176;C for 30 min and then fill each bioreactor well with 450 &#956;L of Myotonic growth media.</li>
	<li>After 3 days (once the gel has compacted), begin myotube differentiation.</li>
</ol>
5. Myotube differentiation (days 0-14)
<ol>
	<li>Begin myotube infusion by switching tissues to 450 &#956;L of fusion-inducing media (FS, Table 1) for 7 days, changing the media every other day. 
	NOTE: Try to begin myotube differentiation on the same day as motoneuron differentiation from hiPSCs in order to seed motoneurons at the end of both cell types&#39; differentiation schedules.</li>
	<li>On day 7 change the media to 450 &#956;L of maturation media Ia (MMIa, Table 1).</li>
	<li>On day 9, change to 450 &#956;L of maturation media Ib (MMIb, Table 1).</li>
	<li>On day 11, change the media to 450 &#956;L of NbActiv4. Keep changing the media every 2 days until the motoneurons are seeded (Step 7).</li>
</ol>
6. Motoneuron differentiation (days 0-14)
<ol>
	<li>On day 0, transfer 4 x 106 ChR2- hiPSCs to an ultra-low attachment Petri dish with 15 mL of motoneuron suspension culture medium (MSCM, Table 1). Supplement MSCM with 3 &#956;M CHIR99021, 0.2 &#956;M LDN193189, 40 &#956;M SB431542 hydrate and 5 &#956;M Y-27632 dihydrochloride.</li>
	<li>On day 2, isolate the neurospheres (NS) with a 37 &#956;M reversible strainer and replate in 15 mL of MSCM with 3 &#956;M CHIR99021, 0.2 &#956;M LDN193189, 40 &#956;M SB431542 hydrate, and 0.1 &#956;M retinoic acid. After day 2, the NS should be visible in the Petri dish without using a microscope.</li>
	<li>On day 4, transfer the cells and media to a 50 mL tube and allow the neurospheres to settle to the bottom (5 min). Aspirate the supernatant and resuspend the cells in 15 mL of MSCM supplemented with 0.5 &#956;M SAG, 0.2 &#956;M LDN193189, 40 &#956;M SB431541, and 0.1 &#956;M retinoic acid.</li>
	<li>On day 7, repeat Step 6.3. but resuspend the cells in 15 mL of MSCM supplemented with 0.5 &#956;M SAG and 0.1&#956;M retinoic acid.</li>
	<li>On day 9, repeat Step 6.3. but resuspend cells in 15 mL of MSCM supplemented with 10 &#956;M DAPT.</li>
	<li>On day 11, repeat Step 6.3. but resuspend the cells in 15 mL of MSCM supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.</li>
	<li>On day 14, seed the motoneurons into the platform.</li>
</ol>
7. Seeding motoneurons aggregates in the bioreactor (day 14)
<ol>
	<li>Prepare a 4:1 gel mixture of 2 mg/mL collagen I and Matrigel (800 &#956;L of collagen mixture + 200 &#956;L of Matrigel to reach a final volume of 1 mL). For the collagen component, add the accompanying neutralizing agent (9:1 mix of collagen to neutralizing agent) and dilute the mixture with 1x PBS to achieve a final volume of 800 &#956;L.</li>
	<li>Use a 400 nm cell strainer to select large neurospheres and resuspend them in the gel mixture.</li>
	<li>Aspirate media from the reservoir and carefully from the neurosphere well (Figure 2).</li>
	<li>Add 15 &#956;L of gel mixture into the neurosphere channel.</li>
	<li>Load a 10 &#956;L pipette with 10 &#956;L of gel and then pick one neurosphere.</li>
	<li>Deposit the NS into the neurosphere channel and ensure that the NS is in the chamber. Slowly raise the pipette while releasing the remaining gel once the NS is deposited. If unsure that the NS was correctly deposited, check its location using a microscope.</li>
	<li>Allow the gel to polymerize for 30 min at 37 &#176;C.</li>
	<li>To the reservoirs, add 450 &#956;L of NbActiv4 supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.</li>
	<li>Change the media every other day to allow for axonal growth from NS to muscle tissue.</li>
</ol>
8. Simultaneous optical stimulation and video recording of NMJ function (day 24+)
<ol>
	<li>For imaging, use an inverted fluorescent microscope with a scientific complementary metal-oxide-semiconductor (sCMOS) camera.</li>
	<li>Set the camera software binning to 2x2, exposure to 20 ms, rolling shutter ON, readout rate to 540 MHz, dynamic range: 12-bit &#38; gain 1, and sensor mode: overlap.</li>
	<li>Use 2x objective on the microscope to image the microtissues.</li>
	<li>Attach a live-cell chamber (37 &#176;C, 5% CO2) to the microscope stage.</li>
	<li>Select the region of interest (ROI) that contains the innervated skeletal tissues tissue to minimize the file size and processing time.</li>
	<li>Place a 594 nm long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera.</li>
	<li>Place a rectangular 4-well plate containing 4 bioreactors (24 tissues) into the live-cell chamber.</li>
	<li>Click Live View. Center and focus the image with the desired ROI.</li>
	<li>Upload the custom macro code from the GitHub folder (https://github.com/ofvila/NMJ-function-analysis) to control the stage position, the Arduino board, and video acquisition.</li>
	<li>Set output movie as day_tissue group_tissue name_experiment.nd2.</li>
	<li>Run the macro code with the desired X,Y coordinates set on the stage and acquire a fast time lapse with 1700 frames at 50 frames/s.</li>
	<li>Replace the media after imaging and return the samples to the incubator. Allow at least 24 h between image acquisition sessions to avoid tissue fatigue.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SkMDC</td>
			<td>Cook Myosite</td>
			<td>P01059-14M</td>
			<td></td>
		</tr>
		<tr>
			<td>Media and Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>12634-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin solution</td>
			<td>Millipore Sigma</td>
			<td>A9576-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>G-5 Supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17503-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>10131-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Recombinant Human</td>
			<td>Millipore Sigma</td>
			<td>91077C-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stem Cell Technologies</td>
			<td>100-0276</td>
			<td></td>
		</tr>
		<tr>
			<td>MyoTonic Growth Media Kit</td>
			<td>Cook Myosite</td>
			<td>MK-4444</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>NBactiv4 500 mL</td>
			<td>BrainBits LLC</td>
			<td>Nb4-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A13710-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 mm cage cube system</td>
			<td>ThorLabs</td>
			<td>CM1-DCH, CP33, ER1-P4 and ER2-P4</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;m Reversible Strainer, large</td>
			<td>Stem Cell Technologies</td>
			<td>27250</td>
			<td></td>
		</tr>
		<tr>
			<td>546 nm short-pass excitation filter</td>
			<td>Semrock</td>
			<td>FF01-546/SP-25</td>
			<td></td>
		</tr>
		<tr>
			<td>573 nm dichroic mirror</td>
			<td>Semrock</td>
			<td>FF573-Di01&#8211;25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>594 nm long- pass emission filter</td>
			<td>Semrock</td>
			<td>BLP01-594R-25</td>
			<td></td>
		</tr>
		<tr>
			<td>594 nm long-pass excitation filter</td>
			<td>Semrock</td>
			<td>BLP01-594R-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue (470nm) Rebel LED on a SinkPAD-II 10mm Square Base - 65 lm @ 700mA</td>
			<td>LuxeonStarLEDs</td>
			<td>SP-05-B4</td>
			<td></td>
		</tr>
		<tr>
			<td>Carclo 29.8&#176; Frosted 10 mm Circular Beam Optic - Integrated Legs</td>
			<td>LuxeonStarLEDs</td>
			<td>10413</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 60 mm Ultra-Low Attachment Culture Dish</td>
			<td>Corning</td>
			<td>3261</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat sink</td>
			<td>LuxeonStarLEDs</td>
			<td>LPD-19-10B</td>
			<td></td>
		</tr>
		<tr>
			<td>Optics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 400 &#181;m, 25 pack, sterile</td>
			<td>PluriSelect</td>
			<td>43-50400-03</td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 500 &#181;m, 25 pack, sterile</td>
			<td>PluriSelect</td>
			<td>43-50500-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Red (627nm) Rebel LED on a SinkPAD-II 10mm Square Base - 65 lm @ 700mA</td>
			<td>LuxeonStarLEDs</td>
			<td>SP-05-R5</td>
			<td></td>
		</tr>
		<tr>
			<td>ring-actuated iris diaphragm</td>
			<td>ThorLabs</td>
			<td>SM1D12D</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Cube LED drivers</td>
			<td>ThorLabs</td>
			<td>LEDD1B, KPS101</td>
			<td></td>
		</tr>
		<tr>
			<td>Molds</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female Threaded Hex Standoffs, 3 1/2&#34; 10-32, Partially Threaded 1/2&#34;</td>
			<td>McMaster</td>
			<td>91920A046</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-Profile C-Clamp</td>
			<td>McMaster</td>
			<td>1705A12</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 3&#8242;,5&#8242;-cyclic monophosphate</td>
			<td>Millipore Sigma</td>
			<td>A9501-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR 99021, 10 mg</td>
			<td>Tocris</td>
			<td>4423-10</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT 10 mg</td>
			<td>R&#38;D Systems</td>
			<td>2634-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Human CNTF, research grade, 5 &#181;g</td>
			<td>Miltenyl Biotec</td>
			<td>130-096-336</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Vitronectin Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>2349-VN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Vitronectin Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>2349-VN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF1 Recombinant Human Protein</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0078</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin mouse protein, natural</td>
			<td>ThermoFisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Agrin Protein</td>
			<td>R&#38;D Systems</td>
			<td>6624-AG-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GDNF Protein, CF 50ug</td>
			<td>R&#38;D Systems</td>
			<td>212-GD-050/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Neurotrophin 3 100 ug</td>
			<td>Cell Sciences</td>
			<td>CRN500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Neurotrophin-4</td>
			<td>Cell Sciences</td>
			<td>CRN501B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Sonic Hedgehog/Shh (C24II) N-Terminus</td>
			<td>R&#38;D Systems</td>
			<td>1845-SH-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Murine/Rat BDNF 50 ug</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid, 50 mg</td>
			<td>Millipore Sigma</td>
			<td>R2625-50</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG Smoothened Agonist</td>
			<td>Millipore Sigma</td>
			<td>566660</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542 10 mg</td>
			<td>Stem Cell Technologies</td>
			<td>72234</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS LDN-193189</td>
			<td>Miltenyl Biotec</td>
			<td>130-103-925</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin from human plasma</td>
			<td>Millipore Sigma</td>
			<td>V8379-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-actinin mAb (Mouse IgG1)</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Acetyltransferase (ChAT) (Goat)</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmin mAb (Mouse IgG1)</td>
			<td>Dako</td>
			<td>M076029-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain (MHC) (Mouse IgG2b)</td>
			<td>DSHB</td>
			<td>MF20</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Uno R3</td>
			<td>Arduino</td>
			<td>A000066</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated stage</td>
			<td>Applied scientific instrumentation</td>
			<td>MS- 2000 XYZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Expanded plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001 (115V)</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Countess Automated Cell Counter</td>
			<td>Marshal Scientific</td>
			<td>I-CACC</td>
			<td></td>
		</tr>
		<tr>
			<td>IX-81 Inverted fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX-ILL100LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Series Stage Top Incubator System</td>
			<td>Tokai Hit STX</td>
			<td>TOKAI-HIT-STXG</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyla 4.2 sCOMS Camera</td>
			<td>Andor Technology</td>
			<td>ZYLA-4.2P-CL10</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Software (IDE)</td>
			<td>Arduino</td>
			<td>IDE 1.8.19</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercam</td>
			<td>Mastercam</td>
			<td>Mastercam for Solidworks</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Matlab</td>
			<td>R2021b</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS elements</td>
			<td>Nikon</td>
			<td>Basic Research</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks 3D CAD</td>
			<td>Solidworks</td>
			<td>Solidworks Standard</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source:&#160; Liberman, M., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63759">Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction.</a> J. Vis. Exp. (2022).
This video demonstrates a method to assess functional neuromuscular junctions. Using a bioreactor containing muscle tissue and motor neurons, along with fluorescent microscopy, the light-sensitive channels in the neurons are stimulated to generate electrical signals, causing muscle fiber contractions.

<img alt="Figure 1" src="/files/ftp_upload/22952/22952_Figure1.jpg" /> 
Figure 1. Stimulation, recording, and analysis of NMJ function. (A) Schematic of LED setup and light path. (B) Optical set-up with an inverted microscope, live cell chamber, automated stage, and sCMOS camera. (C) Algorithm for the batch processing of optical stimulation videos of NMJ cultures. (D) The representative frame of the video generated by the MATLAB code and the corresponding contractility trace graph. The blinking blue box in the video and the vertical lines in the graph indicate when the blue light stimulation occurs.
<img alt="Figure 2" src="/files/ftp_upload/22952/22952_Figure2.jpg" /> 
Figure 2. Co-culture systems for the generation of 3D NMJs. (A) Schematic of the microfluidic platform. (B) Schematic of the open-well PDMS bioreactor. (C) Innervated skeletal muscle tissue in the microfluidic platform. (D) Innervated tissue in the open-well bioreactor. Scale bar 0.5 mm.
Table 1. Myogenic and neural media formulations. Organized list of growth factors and supplements for media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Fusion-Inducing Medium (FS)</td>
			<td></td>
			<td colspan="2">Motoneuron Suspension Culture Medium (MSCM)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Concentration</td>
			<td></td>
			<td>Component</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>100%</td>
			<td></td>
			<td>Neurobasal</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>500 &#956;g/mL</td>
			<td></td>
			<td>Advanced DMEM/F12</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>10 ng/mL</td>
			<td></td>
			<td>B27</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>10 ng/mL</td>
			<td></td>
			<td>N2</td>
			<td>1x</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Glutamax</td>
			<td>1x</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2-Phospho L-Ascorbic Acid</td>
			<td>0.5 &#956;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2-Mercaptoethanol</td>
			<td>0.1 mM</td>
		</tr>
	</tbody>
</table>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Maturation Medium Ia (MMIa)</td>
			<td></td>
			<td colspan="2">Maturation Medium Ib (MMIb)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Concentration</td>
			<td></td>
			<td>Component</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>50%</td>
			<td></td>
			<td>Neurobasal A</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td>50%</td>
			<td></td>
			<td>Glutamax</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>1x</td>
			<td></td>
			<td>B27</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>G-5</td>
			<td>1x</td>
			<td></td>
			<td>GDNF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>1x</td>
			<td></td>
			<td>BDNF</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>10 ng/mL</td>
			<td></td>
			<td>Shh</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>20 ng/mL</td>
			<td></td>
			<td>Retinoic Acid</td>
			<td>0.1 &#956;M</td>
		</tr>
		<tr>
			<td>Shh</td>
			<td>50 ng/mL</td>
			<td></td>
			<td>IGF-1</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>0.1 &#956;M</td>
			<td></td>
			<td>cAMP</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>IGF-1</td>
			<td>10 ng/mL</td>
			<td></td>
			<td>human CNTF</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>1 &#956;M</td>
			<td></td>
			<td>Neurotropin-3</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>human CNTF</td>
			<td>5 ng/mL</td>
			<td></td>
			<td>Neurotropin-4</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>Neurothropin-3</td>
			<td>20 ng/mL</td>
			<td></td>
			<td>Vitronectin</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>Neurothropin-4</td>
			<td>20 ng/mL</td>
			<td></td>
			<td>Mouse laminin</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>100 ng/mL</td>
			<td></td>
			<td>Agrin</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>Mouse laminin</td>
			<td>4 &#956;g/mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agrin</td>
			<td>100 ng/mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Bioreactor preparation

	Make ...

Begin with a bioreactor with a muscle chamber containing collagen-embedded muscle tissue supported over the chamber&#39;s pillars.
The neurosphere channel contains a neurosphere in a collagen matrix. The motor neurons of the neurosphere express light-sensitive channels.
Add a differentiation medium containing neural growth factors that stimulate the neurons to extend their projections.
Regularly exchange the medium to ensure continuous neuron extension toward the muscle tissue, forming neuromuscular junctions.
Observe the bioreactor under a microscope equipped with a camera and a blue light source.
Apply blue light pulses to open the light-sensitive channels in the neurons, allowing ions from the medium to flow and stimulate the neurons to generate electrical signals.
These signals travel via the neuronal projections to the neuromuscular junctions, transmitting the electrical signal to the muscle tissue.
Record a video simultaneously. A gradual contraction of the muscle fiber confirms the development of a functional neuromuscular junction.

31 Aufrufe. Beurteilung einer funktionellen neuromuskulären Verbindung durch gleichzeitige optische Stimulation und Videoaufzeichnung

Serie: Beurteilung einer funktionellen neuromuskulären Verbindung durch gleichzeitige optische Stimulation und Videoaufzeichnung - Experiment

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

In vitro Neuromuskuläre Junction Induziert von humaninduzierten Pluripotenten Stammzellen

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Forschung

JoVE Journal

Entwicklungsbiologie

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.

Neuromuskuläre Synapse Messfunktionalität

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

Neurowissenschaften

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Assessing a Functional Neuromuscular Junction Via Simultaneous Optical Stimulation and Video Recording

Transkript

Assessing a Functional Neuromuscular Junction Via Simultaneous Optical Stimulation and Video Recording