The authors thank Nikky Corthout and Sebastian Munck from LiMoNe, Research Group Molecular Neurobiology (VIB-KU Leuven) for their advice on live-cell calcium transient fluorescence recordings. This research was supported by the Fulbright Commission to Belgium and Luxembourg, KU Leuven (C1 and &#34;Opening the Future&#34; Fund), the VIB, the Agency for Innovation by Science and Technology (IWT; SBO-iPSCAF), the &#34;Fund for Scientific Research Flanders&#34; (FWO-Vlaanderen), Target ALS, the ALS Liga Belgi&#235; (A Cure for ALS), the Belgian Government (Interuniversity Attraction Poles Program P7/16 initiated by the Belgian Federal Science Policy Office), the Thierry Latran Foundation and the &#34;Association Belge contre les Maladies neuro-Musculaires&#34; (ABMM). T.V. and J.B. are supported by Ph.D. fellowships awarded by FWO-Vlaanderen.

L.V.D.B. has a patent on the use of HDAC inhibitors in Charcot-Marie-Tooth disease (US-2013227717-A1), is a scientific co-founder of Augustine Therapeutics, and a member of its scientific advisory board. The other authors declare no competing interests.

The protocol describes a relatively easy-to-use method, which generates human motor units with functional NMJs in commercially available microfluidic devices in less than 30 days. It is described how the NMJs can be assessed morphologically through standard techniques such as ICC and SEM and functionally through live-cell calcium recordings.
A large advantage of this protocol is the use of stem cell technology. This allows for full adaptability in which NMJs can be evaluated in both health and disease, independently of the donor profile. The model has proven already successful and beneficial in ALS research, where we identified impairments in neurite outgrowth, regrowth, and NMJ numbers as novel phenotypes due to mutations in the FUS gene18. With this model, it is possible to expand the research to include sporadic forms of ALS, where the etiology is unknown, by using iPSCs from sporadic ALS patients. This provides an advantage over traditional animal models, which rely on transgenic overexpression of mutated genes to recapitulate human disease23,24. In addition, our fully human system allows for potential recapitulation of human-specific physiology and disease. Previous studies demonstrated the differences between rodent and human NMJ morphology25, which suggests that caution must be implemented when using rodents to address human NMJ pathology. Although this system is a relative simple in vitro setup, which lacks the complexity of an in vivo model, it was possible to demonstrate that the NMJ morphology displayed in the microfluidic devices resembled NMJs of human amputates25. Furthermore, this model allows for NMJ evaluation during NMJ formation and maturation, potentially revealing early disease phenotypes, which are absent, unidentifiable, or overlooked in human post-mortem samples.
MABs provide a valid option to generate myotubes, although their limited survival of 10 days is a disadvantage of the system. The myotube survival relies on their attachment to the surface, which is likely compromised by spontaneous contractions of the myofibers. After more than 10 days, most myotubes will have detached, rendering the NMJ culture unusable. Ideally, the myotubes would be generated from iPSCs as well. However, current protocols have proven difficult to reproduce26 due to variability in fusion index27,28,29,30.
By using commercially available microfluidic devices, we generated a standardized system, which is fully accessible. Other NMJ models exist31,32,33,34,35,36,37,38,39,40,41,42. However, they typically rely on single compartments, which lack the compartmentalization and fluidic isolation between cell types, or on custom-made culture vessels, which lowers the availability and potentially also the reproducibility. The microfluidic devices used for this protocol can be purchased with microgrooves of various lengths, which allows for further analysis such as axonal transport43,44 or axotomy18,45,46 investigations. The fluidic isolation between compartments further enables compartmentalized drug treatment of either motor neurons or myotubes, which can be favorable in therapy development. More companies specializing in microfluidics have emerged, which has opened up for a large selection of device design and features, further promoting the accessibility for in vitro research.
In conclusion, we have developed a protocol providing a reliable, versatile and easy method to culture human motor units with functional NMJs.

NMJs facilitate the communication between lower spinal motor neurons and skeletal muscle fibers through the release of neurotransmitters1. In motor neuron disorders like ALS and SMA, the NMJs degenerate, which causes a disruption in the communication with the muscles2,3,4,5,6,7. This results in patients gradually losing their muscle function, which causes them to be wheelchair-bound and eventually dependent on respiratory life-support due to progressive atrophy of vital muscle groups like the diaphragm. The exact underlying mechanisms responsible for this profound loss of NMJs in these disorders are unknown. Many studies have been done on transgenic animal models, which has given us some insights into the pathogenesis of NMJ degeneration5,6,8,9,10,11. However, to fully understand the pathology and counteract the denervation, it is important to have a human system, which allows full accessibility.
Here, the protocol describes a relatively simple way to generate human NMJs through the co-culturing of hiPSC-derived motor neurons and human primary MAB-derived myotubes using commercially available microfluidic devices. The use of microfluidics to polarize and fluidically isolate the somas and axons of neurons has been known since the first description of the &#39;Campenot&#39; chambers12 in the late 1970s. Since then, more microfluidic designs have been fabricated, including commercial options. The devices used in this protocol contain two compartments, and each compartment consists of two wells connected with a channel13. The two compartments are mirrored and connected with several microgrooves. These microgrooves have a size that facilitates neurite growth while maintaining fluidic isolation between the two compartments through a capillary hydrostatic pressure13,14. Using this system, it is possible to culture motor neurons in one compartment and muscle cells in the other one, each in their specific culture medium, while still facilitating a physical connection through neurites passing through the microgrooves and engaging with the muscle cells. This model provides a fully accessible and adaptable in vitro system of a human motor unit, which can be used to study early NMJ pathology in diseases like ALS and SMA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#945;-bungarotoxin (Btx) Alexa fluor 555</td>
			<td>Thermo Fisher Scientific</td>
			<td>B35451</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>CHEM-Lab NV</td>
			<td>CL00.0116.1000</td>
			<td>Coating component. H226, H314. P280</td>
		</tr>
		<tr>
			<td>Aclar 33C sheet (SEM sheet)</td>
			<td>Electron Microscopy Sciences</td>
			<td>50425-25</td>
			<td>Thickness: 7.8 mil</td>
		</tr>
		<tr>
			<td>Agrin (recombinant human protein)</td>
			<td>R&#38;D systems</td>
			<td>6624-AG-050</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Alexa fluor IgG (H+L) 488 donkey-anti rabbit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Alexa fluor IgG (H+L) 555 donkey-anti goat</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21432</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Alexa fluor IgG (H+L) 555 donkey-anti mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31570</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Alexa fluor IgG (H+L) 647 donkey-anti mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31571</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4403</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>&#946;III-tubulin (Tubulin)</td>
			<td>Abcam</td>
			<td>ab7751</td>
			<td>Antibody (1:500)</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>31350010</td>
			<td>Media component. H317. P280.</td>
		</tr>
		<tr>
			<td>B-27 without vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587-010</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>BDNF (brain-derived neurotrophic factor)</td>
			<td>Peprotech</td>
			<td>450-02B</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>bFGF (recombinant human basic fibroblast growth factor)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Choline acetyltransferase (ChAT)</td>
			<td>Millipore</td>
			<td>ab144P</td>
			<td>Antibody (1:500)</td>
		</tr>
		<tr>
			<td>Collagen from calfskin</td>
			<td>Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td>Coating component</td>
		</tr>
		<tr>
			<td>CNTF (ciliary neurotrophic factor)</td>
			<td>Peprotech</td>
			<td>450-13B</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>DAPI Nucblue Live Cell Stain ReadyProbes reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td>Immunocytochemistry component</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Tocris Bioscience</td>
			<td>2634</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Desmin</td>
			<td>Abcam</td>
			<td>Ab15200</td>
			<td>Antibody (1:200)</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650-100ML</td>
			<td>Cryopreservation component. H315, H319, H335. P280.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190250</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>20.821.296</td>
			<td>Sterilization. H225. P280</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Fluo-4 AM live cell dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>Calcium imaging dye</td>
		</tr>
		<tr>
			<td>Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td>Immunocytochemistry component</td>
		</tr>
		<tr>
			<td>GDNF (glial cell line-derived neurotrophic factor)</td>
			<td>Peprotech</td>
			<td>450-10B</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Agar Scientific</td>
			<td>R1020</td>
			<td>Fixation component. EUH071, H301, H314, H317, H330, H334, H410. P280.</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16050122</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Human alkaline phosphatase</td>
			<td>R&#38;D systems</td>
			<td>MAB1448</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td></td>
			<td>ICC analysis</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Insulin transferrin selenium</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Islet-1</td>
			<td>Millipore</td>
			<td>ab4326</td>
			<td>Antibody (1:400)</td>
		</tr>
		<tr>
			<td>Knockout serum replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828-028</td>
			<td>Cryopreservation component</td>
		</tr>
		<tr>
			<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>Coating component and media supplement</td>
		</tr>
		<tr>
			<td>Leica SP8 DMI8 confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>ICC confocal microscopy</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Myogenin (MyoG)</td>
			<td>Abcam</td>
			<td>Ab124800</td>
			<td>Antibody (1:500)</td>
		</tr>
		<tr>
			<td>Myosin heavy chain (MyHC)</td>
			<td>In-house, SCIL</td>
			<td></td>
			<td>Antibody (1:20)</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Coating and media component</td>
		</tr>
		<tr>
			<td>Neurofilament heavy chain (NEFH)</td>
			<td>Abcam</td>
			<td>AB8135</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Live-cell calcium imaging microscopy</td>
		</tr>
		<tr>
			<td>NIS-Elements AR 4.30.02 software</td>
			<td>Nikon</td>
			<td></td>
			<td>Live-cell calcium imaging analysis</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Sigma</td>
			<td>D9663-10ML</td>
			<td>Immunocytochemistry component</td>
		</tr>
		<tr>
			<td>Olig2</td>
			<td>IBL</td>
			<td>18953</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>Storing equipment</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>Fixation component. H302, H312, H315, H317, H319, H332, H335, H341, H350. P280.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (5000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070063</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Petri dish (3 cm)</td>
			<td>nunc</td>
			<td>153066</td>
			<td>Diameter: 3 cm</td>
		</tr>
		<tr>
			<td>Petri dish (10 cm)</td>
			<td>Sarstedt</td>
			<td>833.902</td>
			<td>Diameter: 10 cm</td>
		</tr>
		<tr>
			<td>Plate (6-well)</td>
			<td>Cellstar Greiner bio-one</td>
			<td>657160</td>
			<td>Culture plate</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3000MP</td>
			<td>Fluo-4 dye solvent</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Coating component</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>CHEM-Lab NV</td>
			<td>CL00.1133.1000</td>
			<td>Calcium imaging reagent</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Media supplement. H302, H315, H360FD, H410. P280.</td>
		</tr>
		<tr>
			<td>RevitaCell supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2644501</td>
			<td>ROCK inhibitor solution</td>
		</tr>
		<tr>
			<td>Smoothened agonist</td>
			<td>Merch Millipore</td>
			<td>566660</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Sodium cacodylate buffer</td>
			<td>Sigma</td>
			<td>C0250</td>
			<td>Fixation component. H301, H331, H350, H410. P280.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Synaptophysin (SYP)</td>
			<td>Cell Signaling</td>
			<td>5461S</td>
			<td>Antibody (1:1000)</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Sigma</td>
			<td>CLS3276</td>
			<td>Culture plate</td>
		</tr>
		<tr>
			<td>Titin</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>9D10</td>
			<td>Antibody (1:300)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250ML</td>
			<td>Immunocytochemistry component. H302, H315, H318, H319, H410, H411. P280</td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605010</td>
			<td>MAB dissociation solution</td>
		</tr>
		<tr>
			<td>Tubocyrarine hydrochloride pentahydrate</td>
			<td>Sigma</td>
			<td>T2379-100G</td>
			<td>Acetylcholine receptor blocker. H301. P280.</td>
		</tr>
		<tr>
			<td>XonaChips pre-assembled microfluidic device</td>
			<td>Xona Microfluidics</td>
			<td>XC150</td>
			<td>Microgroove length: 150 &#956;m</td>
		</tr>
		<tr>
			<td>Xona Silicone microfluidics device</td>
			<td>Xona Microfluidics</td>
			<td>SND75</td>
			<td>Microgroove length: 75 &#956;m</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices at JoVE.com

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

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 ...

generation-human-motor-units-with-functional-neuromuscular-junctions

Research

JoVE Journal

Neuroscience

4.5K Views.  KU Leuven - University of Leuven. 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. 

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

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

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

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

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

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...

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 ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.