We gratefully acknowledge funding support by the NIH [grant numbers EB025765 and EB027062], DOD [award number W81XWH-18-1-0095], and the UCSF Health Innovation via Engineering (HIVE Fellowship). We gratefully acknowledge the Columbia University Stem Cell Core for their help and guidance with cell reprogramming.

All cell lines for this work were created and used in compliance with the institutional guidelines of Columbia University, NY, USA.
1. Bioreactor preparation
<ol>
	<li>Make bioreactor molds
	<ol>
		<li>Download a bioreactor CAD file from the Supplementary CAD File or create a custom own design.</li>
		<li>Generate a CNC toolpath from the 3D model using CAM 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, and ensure that the coverslips 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>Use a plasma cleaner on high with 6 L/min of oxygen for 1-2 min to the treat glass coverslips and PDMS platform. 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 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 3A.</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 by 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 PC by 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 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&#160;&#176;C and 5% CO2.</li>
		<li>Change the media every 2 days.</li>
		<li>Once cells are about 60% confluent, passage 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 karyotype, as noted in previous publications18, 19.
	<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 with 1 mL of enzyme-free stem cell releasing reagent for 4 min and mechanically shearing 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)
NOTE: Our motoneuron differentiation protocol was adapted from Maury et al8.
<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. NS should be visible in the Petri dish without using a microscope after day 2.</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 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>Add 450 &#956;L of NbActiv4 supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF to the reservoirs.</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>
9. Batch processing and analysis (day 24+)
<ol>
	<li>Movie processing
	<ol>
		<li>Use the custom MATLAB code to process the videos in batch analysis. The files can be downloaded from the GitHub folder (https://github.com/ofvila/NMJ-function-analysis). The functions are listed and explained in&#160;Table 2. 
		NOTE: The code is compatible with .nd2 and .czi formats. It requires parallel processing to be activated in MATLAB and needs the bioformats package.</li>
		<li>Run the recursiveOSAnalysis script to analyze the movies through parallel processing. Have all of the acquired videos in the same folder and select that folder when running the code.</li>
		<li>Adjust the post-analysis parameters as needed.
		<ol>
			<li>Change baselineTime (option 1) if spontaneous contraction is picked up at beginning of recording. This will show an initial reading way above 0 and will need the frame to be shifted to compensate.</li>
			<li>Change peakThreshold (option 2) if the contractions are not being registered. The code will detect contractions that are above 25% of the highest peak by default so that this value can be changed.</li>
			<li>Change minMinProminence and minMinWidth to adjust the sensitivity when detecting the start of each peak.</li>
		</ol>
		</li>
		<li>Generate video and graph output.
		<ol>
			<li>Run recursiveOSMovie to generate a video file for each tissue with its respective contractility trace.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
10. Perturbation of NMJ function (day 24+)
<ol>
	<li>Prepare treatment media by adding external effector to NbActiv4 basal media supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.
	<ol>
		<li>For the MG sera experiment, use 20% MG patient sera in NbActiv4 + BDNF + GDNF.</li>
		<li>For the BTX experiment, use 5 &#956;g/mL BTX in NbActiv4 + BDNF + GDNF.</li>
	</ol>
	</li>
	<li>Stimulate/image using Step 3.2. to record the baseline.</li>
	<li>Replace the media in tissues with treatment media (450 &#956;L/tissue).</li>
	<li>Incubate for the desired time (48 h for patient sera, 20 min for BTX).</li>
	<li>Stimulate/image again using Step 3.2. to record function after treatment.</li>
	<li>Remove the treatment media and allow the tissues to rest for the desired time (48 h after sera removal) 
	NOTE: Allow at least 24 h between stimulation and imaging to avoid tissue fatigue</li>
</ol>

<ol>
	<li>Al-bassam, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics,+incidence,+and+outcome+of+patients+admitted+to+the+intensive+care+unit+with+myasthenia+gravis.">Characteristics, incidence, and outcome of patients admitted to the intensive care unit with myasthenia gravis.</a> Journal of Critical Care. 45, 90-94 (2018).</li><li>Vila, O. F., Qu, Y., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+neuromuscular+junctions+and+their+potential+for+novel+drug+discovery+and+development.">In vitro models of neuromuscular junctions and their potential for novel drug discovery and development.</a> Expert Opinion on Drug Discovery. 15 (3), 307-317 (2020).</li><li>Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+the+neuromuscular+junction,+vitally+informative+for+understanding+function+and+the+molecular+mechanisms+of+congenital+myasthenic+syndromes.">Animal models of the neuromuscular junction, vitally informative for understanding function and the molecular mechanisms of congenital myasthenic syndromes.</a> International Journal of Molecular Sciences. 19 (5), 1326 (2018).</li><li>Jones, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+anatomy+of+the+human+neuromuscular+junction.">Cellular and molecular anatomy of the human neuromuscular junction.</a> Cell Reports. 21 (9), 2348-2356 (2017).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Rao, L., Qian, Y., Khodabukus, A., Ribar, T., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+human+pluripotent+stem+cells+into+a+functional+skeletal+muscle+tissue.">Engineering human pluripotent stem cells into a functional skeletal muscle tissue.</a> Nature Communications. 9 (1), 126 (2018).</li><li>Madden, L., Juhas, M., Kraus, W. E., Truskey, G. A., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+human+myobundles+mimic+clinical+responses+of+skeletal+muscle+to+drugs.">Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.</a> eLife. 4, 04885 (2015).</li><li>Maury, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+analysis+of+developmental+cues+efficiently+converts+human+pluripotent+stem+cells+into+multiple+neuronal+subtypes.">Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes.</a> Nature Biotechnology. 33 (1), 89-96 (2015).</li><li>Bianchi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+differentiation+of+functional+motor+neurons+from+human+iPSC+for+neural+injury+modelling.">Rapid and efficient differentiation of functional motor neurons from human iPSC for neural injury modelling.</a> Stem Cell Research. 32, 126-134 (2018).</li><li>Turan, S., Farruggio, A. P., Srifa, W., Day, J. W., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+correction+of+disease+mutations+in+induced+pluripotent+stem+cells+derived+from+patients+with+limb+girdle+muscular+dystrophy.">Precise correction of disease mutations in induced pluripotent stem cells derived from patients with limb girdle muscular dystrophy.</a> Molecular Therapy. 24 (4), 685-696 (2016).</li><li>Ebert, A. D., Liang, P., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+as+a+disease+modeling+and+drug+screening+platform.">Induced pluripotent stem cells as a disease modeling and drug screening platform.</a> Journal of Cardiovascular Pharmacology. 60 (4), 408-416 (2012).</li><li>Lin, C. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC-derived+functional+human+neuromuscular+junctions+model+the+pathophysiology+of+neuromuscular+diseases.">iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.</a> JCI Insight. 4 (18), (2021).</li><li>Centeno, E. G. Z., Cimarosti, H., Bithell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+versus+3D+human+induced+pluripotent+stem+cell-derived+cultures+for+neurodegenerative+disease+modelling.">2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling.</a> Molecular Neurodegeneration. 13 (1), 27 (2018).</li><li>Okano, T., Matsuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineered+skeletal+muscle:+preparation+of+highly+dense,+highly+oriented+hybrid+muscular+tissues.">Tissue engineered skeletal muscle: preparation of highly dense, highly oriented hybrid muscular tissues.</a> Cell Transplantation. 7 (1), 71-82 (1998).</li><li>Powell, C. A., Smiley, B. L., Mills, J., Vandenburgh, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stimulation+improves+tissue-engineered+human+skeletal+muscle.">Mechanical stimulation improves tissue-engineered human skeletal muscle.</a> American Journal of Physiology-Cell Physiology. 283 (5), 1557-1565 (2002).</li><li>Ronaldson-Bouchard, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+maturation+of+human+cardiac+tissue+grown+from+pluripotent+stem+cells.">Advanced maturation of human cardiac tissue grown from pluripotent stem cells.</a> Nature. 556 (7700), 239-243 (2018).</li><li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human-based+functional+NMJ+system+for+personalized+ALS+modeling+and+drug+testing.">A human-based functional NMJ system for personalized ALS modeling and drug testing.</a> Advanced Therapeutics. 3 (11), 2000133 (2020).</li><li>Vila, O. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+optogenetic+model+of+human+neuromuscular+junction.">Bioengineered optogenetic model of human neuromuscular junction.</a> Biomaterials. 276, 121033 (2021).</li><li>Vila, O. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+human+neuromuscular+function+through+optogenetics.">Quantification of human neuromuscular function through optogenetics.</a> Theranostics. 9 (5), 1232-1246 (2019).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nature Neuroscience. 8 (9), 1263-1268 (2005).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Sciences. 100 (24), 13940-13945 (2003).</li><li>Steinbeck, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+under+optogenetic+control+allows+modeling+of+human+neuromuscular+disease.">Functional connectivity under optogenetic control allows modeling of human neuromuscular disease.</a> Cell Stem Cell. 18 (1), 134-143 (2016).</li><li>Santhanam, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+derived+phenotypic+human+neuromuscular+junction+model+for+dose+response+evaluation+of+therapeutics.">Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics.</a> Biomaterials. 166, 64-78 (2018).</li><li>Phillips Ii, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+myasthenia+gravis.">The epidemiology of myasthenia gravis.</a> Annals of the New York Academy of Sciences. 998 (1), 407-412 (2003).</li><li>Sanders, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+change+in+acetylcholine+receptor+antibody+level+correlate+with+clinical+change+in+myasthenia+gravis.">Does change in acetylcholine receptor antibody level correlate with clinical change in myasthenia gravis.</a> Muscle &amp; Nerve. 49 (4), 483-486 (2014).</li><li>Vernino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+enigma+of+seronegative+myasthenia+gravis.">Unraveling the enigma of seronegative myasthenia gravis.</a> JAMA Neurology. 72 (6), 630-631 (2015).</li><li>Osaki, T., Uzel, S. G. M., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microphysiological+3D+model+of+amyotrophic+lateral+sclerosis+(ALS)+from+human+iPS-derived+muscle+cells+and+optogenetic+motor+neurons.">Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.</a> Science Advances. , (2018).</li><li>Paredes-Redondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+modeling+of+human+neuromuscular+circuits+in+Duchenne+muscular+dystrophy+with+CRISPR+and+pharmacological+corrections.">Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections.</a> Science Advances. 7 (37), (2021).</li></ol>

The authors declare no conflict of interest.

This system is an engineered 3D human tissue model that combines optogenetics and video processing to enable automated and unbiased evaluation of NMJ function. Using a standardized protocol, we have demonstrated the ability to measure changes in NMJ function during physiological development and characterize the damaging effects of pathologies such as neurotoxin exposure and myasthenia gravis patient sera.
Previous studies have reported the ability to model MG with optogenetic hPSC-derived moto...

The neuromuscular junction (NMJ) is the chemical synapse between motoneurons (MNs) and skeletal muscle cells (SkM) that allows for muscle contraction. Toxins, such as neurotoxin &#945;-bungarotoxin (BTX), or neuromuscular diseases (NMD) like myasthenia gravis (MG) can lead to degeneration of the NMJ and reductions in muscle control1. Bioengineered human tissue models better recapitulate the functional and physiological mechanisms of human NMJs and offer greater translational potential than animal models.
While animal models have advanced the understanding of the formation and function of the NMJ, there are significant differences between human and animal synapses that limit the translation of results to humans and make in vivo characterization of the NMJ challenging2,3,4. Studies have shown distinct physiological differences between mouse and human NMJs. Mice have larger NMJs and smaller active zone densities when compared to human NMJs4. Additionally, drug studies conducted in animal models do not always reflect the effects found in human clinical trials. Engineered human tissue models provide the opportunity to study the healthy development of the NMJ and the pathology of neuromuscular diseases and allow for drug screenings. Human induced pluripotent stem cells (hiPSCs)5 can be differentiated into a variety of cell types, including skeletal muscle cells6,7 and motoneurons8,9. hiPSCs can be generated easily from patient cells, allowing for better disease modeling10 and drug screening11,12 through patient-specific tissue models.
Two-dimensional (2D) monolayer co-cultures of SkMs and MNs lack the morphology, phenotype, organization, and functional behavior of physiological NMJs. NMJs randomly form in 2D culture, which inhibits the isolation of motor units for analysis, limits accurate functional measurements, and prevents their use for repeated, systematic experiments13. Three-dimensional (3D) tissue models of NMJs overcome many of these limitations, recapitulating the morphological and functional characteristics of physiological NMJs7,14,15,16,17. Using this model, the two tissue types are developed separately and then integrated by directing axonal growth, allowing for more organized NMJs to develop compared to 2D culture systems.
Our previous study demonstrated that combining optogenetics with tissue engineering can allow for accurate non-invasive stimulation and evaluation of NMJ function18,19. Through genetic engineering, light-sensitive proteins can be integrated into the genome of hiPSCs. Integrating channelrhodopsin-2 (ChR2), an ion channel that opens in response to blue light, into the membrane of excitable cells such as neurons allows for non-contact spatiotemporal control over cell activation20,21,22. hiPSCs carrying ChR2 can be differentiated into optogenetic motoneurons sensitive to blue light, removing the need for typical invasive electrodes that stimulate neurons and avoiding unwanted stimulation of the muscle cells by electrodes23. This system uses optogenetic motoneurons to stimulate contractions in non-optogenetic skeletal muscle cells. Combining video acquisition and controlled blue light illumination allows for the co-cultured tissues to be simultaneously stimulated and recorded for NMJ function.
MG is caused by autoantibodies targeting nicotinic acetylcholine receptors (AChR), which results in decreased NMJ function and muscle weakness24. It is diagnosed based on presented symptoms, electrodiagnosis, and detection of autoantibodies via serological blood tests. However, not all autoantibodies involved in MG have been identified, and some seronegative patients are diagnosed with MG but with no recognized antibodies25,26. Our system allows for repeated functional assessment of the NMJ before and after the addition of serum from MG patients, providing invaluable insight into the functional and biochemical changes caused by the MG antibodies18. Our protocol illustrates how to produce 3D in vitro models of functional human NMJ that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. We demonstrate the versatility of the system in two platforms, a microfluidic device, and a larger open-well bioreactor platform.

<table><tbody><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</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>&lt;strong&gt;Media and Supplements&lt;/strong&gt;</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>&lt;strong&gt;Plasticware&lt;/strong&gt;</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 &amp;micro;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&amp;ndash;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&amp;deg; 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>&lt;strong&gt;Optics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pluriStrainer 400 &amp;micro;m, 25 pack, sterile</td><td>PluriSelect</td><td>43-50400-03</td><td></td></tr><tr><td>pluriStrainer 500 &amp;micro;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>&lt;strong&gt;Molds&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Female Threaded Hex Standoffs,&amp;nbsp; 3 1/2&quot; 10-32, Partially Threaded 1/2&quot;</td><td>McMaster</td><td>91920A046</td><td></td></tr><tr><td>Low-Profile&amp;nbsp;C-Clamp</td><td>McMaster</td><td>1705A12</td><td></td></tr><tr><td>&lt;strong&gt;Growth Factors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adenosine 3&amp;prime;,5&amp;prime;-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&amp;amp;D Systems</td><td>2634/10</td><td></td></tr><tr><td>Human CNTF, research grade, 5 &amp;micro;g</td><td>Miltenyl Biotec</td><td>130-096-336</td><td></td></tr><tr><td>Human Vitronectin Protein, CF</td><td>R&amp;amp;D Systems</td><td>2349-VN-100</td><td></td></tr><tr><td>Human Vitronectin Protein, CF</td><td>R&amp;amp;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&amp;amp;D Systems</td><td>6624-AG-050</td><td></td></tr><tr><td>Recombinant Human GDNF Protein, CF 50ug</td><td>R&amp;amp;D Systems</td><td>212-GD-050/CF</td><td></td></tr><tr><td>Recombinant Human Neurotrophin 3&amp;nbsp;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&amp;amp;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>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;alpha;-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>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;nbsp;(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>&lt;strong&gt;Software&lt;/strong&gt;</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>

engineering and characterization of an optogenetic model of the human neuromuscular junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

Neuromuscular junctions were generated by co-culturing optogenetic hiPSC-derived motoneurons with non-optogenetic skeletal muscle tissue. Human primary skeletal myoblasts (SkM) were seeded into the platforms and differentiated into multinucleated myotubes using the 2-week protocol. The optogenetic motoneurons were differentiated separately, but in parallel with the myotube differentiation, and then seeded into the platform (Figure 1). The tissues began contracting in response to blue light s...

Watch this Scientific Journal Video about Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction at JoVE.com

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

engineering-characterization-an-optogenetic-model-human-neuromuscular

Research

JoVE Journal

Bioengineering

2.3K Views.  Columbia University. Dit protocol beschrijft hoe functionele 3D in vitro menselijke neuromusculaire juncties kunnen worden ontworpen die kunnen worden gebruikt om neuromusculaire pathologieën te onderzoeken en potentiële therapeutica te testen. Deze techniek maakt gebruik van gelijktijdige optogenetische stimulatie en videoverwerking. Het is gekwantificeerd veranderingen in neuromusculaire junctiefunctie tijdens de ontwikkeling en als gevolg van externe factoren, zoals neurotoxinen en serum van myasthenia gravi...

Video: Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

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.

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

Developmental Biology

Measuring Neuromuscular Junction Functionality

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

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

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

Neuroscience

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

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 ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

SFEMG in Rodents: Assessing NMJ Function in Health and Disease

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

Author Spotlight: Translational Applications of Stimulated SFEMG in Rodent Models

In this study, we demonstrate a refined single fiber electromyography (SFEMG) protocol to allow in vivo measurement of neuromuscular junction (NMJ) transmission in rodent models. A step-by-step approach to the SFEMG technique is described to allow quantification of NMJ transmission variability and failure in rat gastrocnemius muscle.

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. ...

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

Author Spotlight: Regenerative Roles of Muscle Proteins at Neuromuscular Junction Post-Nerve Injury

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://www.jove.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Drosophila Neuromuscular Junction NMJ Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Larva

Generating Neuromuscular Junctions Using a Microfluidic Device

Source: Stoklund Dittlau, K. et al., <a href="https://app.jove.com/v/62959">Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
All reagents and equipment used in ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Samenvatting

Meer video's verkennen