We thank Dr. Ying Xiong for discussions and comments on the manuscript. This work is supported by a grant from the National Science Foundation of China (NSFC) to C. M. (31500839).

1. Dissection of larvae
NOTE: The dissecting solution hemolymph-like saline (HL3.1)18 and the fixing solution 4% paraformaldehyde (PFA)19,20are used at room temperature because the microtubules depolymerize when the temperature is too low.
<ol>
	<li>Pick out a wandering 3rd instar larva with long blunt forceps. Wash it with HL3.1 and place it on the dissection dish under the stereomicroscope. 
	NOTE: Wandering 3rd instar larva is identified by branched anterior spiracle and crawls around the tube at approximately 96 h (25 &#176;C)21.</li>
	<li>Position the larva with the dorsal or ventral side up. Identify the dorsal side by the two long tracheas and the ventral by the abdominal denticle belts.
	<ol>
		<li>Depending on the tissue to observe, opt for dorsal or ventral dissection. This will allow for a more precise and detailed view of the specific area of interest. For NMJ and muscle cell observation, cut open the larval dorsal and ventral regions, respectively.</li>
	</ol>
	</li>
	<li>Pin the mouth hooks and the tail. Adjust the pins to keep the larva in an extended state. Add a drop of HL3.1 to the larva to prevent it from drying. 
	NOTE: Ca2+ -free HL 3.1 can be used to minimize contraction of the muscles during dissection.</li>
	<li>Use the dissecting scissors to make a small transverse cut close to the posterior end, but do not cut off the posterior end. Then cut along the ventral midline towards the anterior end.</li>
	<li>Insert four insect pins on the four corners of the larva. Readjust insect pins such that the larva is maximally stretched in all directions.</li>
	<li>Use the forceps to remove internal organs while not damaging the muscles.</li>
</ol>
2. Fixation
<ol>
	<li>Add 100 &#181;L of PFA (4%) to immerse carcasses for 40 min while the carcasses are still pinned on the dissecting dish. 
	CAUTION: Effective protection measures are taken to avoid direct contact with skin or inhalation, as PFA is a hazard.</li>
	<li>Dismount the pins and transfer the sample to a 2 mL microcentrifuge tube. Rinse off PFA with 1x phosphate buffered saline containing 0.2% Triton X-100 (0.2% PBST) to fill 2 mL microcentrifuge tube for 10 min on the decoloring shaker at 15 rpm. Repeat the wash process 5x.</li>
</ol>
3. Immunocytochemistry
<ol>
	<li>Immerse the carcasses in blocking agent (5% goat serum in 0.2% PBST) and block for 40 min at room temperature.</li>
	<li>Remove the blocking agent and replace it with 200 &#181;L of the primary antibody (e.g., anti-&#945;-tubulin, 1:1000; anti-Futsch, 1:50) diluted with 0.2% PBST at 4 &#176;C overnight. Visualize muscle microtubules by immunostaining with monoclonal anti-&#945;-tubulin. Anti-Futsch can indirectly reflect the microtubule morphology in NMJ.</li>
	<li>After incubation of the primary antibody, wash larvae with 0.2% PBST for 10 min. Repeat 5x.</li>
	<li>Incubate the larvae with 200 &#181;L of secondary antibody (e.g., goat anti-Mouse-488, 1:1000) diluted with 0.2% PBST at room temperature for 1.5 h in the dark.</li>
	<li>Next, add a nuclear dye such as TO-PRO(R) 3 iodide (T3605) at a concentration of 1:1000 into the incubating tube for 30 min when staining the microtubules in the dark20,22.</li>
	<li>Rinse off the secondary antibody and the nuclear dye with 0.2% PBST for 10 min. Repeat 5x in the dark.</li>
</ol>
4. Mounting
<ol>
	<li>Place the larval carcass in 0.2% PBST on a glass slide and adjust it under the stereomicroscope. Make sure the inner surface of the larval carcass is facing up and all larval carcasses are arranged as desired.</li>
	<li>Absorb excess PBST solution with a wipe and gently add a drop of antifade mounting medium.</li>
	<li>Place a coverslip on the slide to cover the dissected larvae slowly and gently to avoid bubbles.</li>
	<li>Apply fingernail polish around the coverslip. Place the slide in the dark space to reduce fluorescent attenuation.</li>
</ol>
5. Image acquisition
<ol>
	<li>To acquire the images, use a laser scanning confocal microscope, select the 60x oil immersion objective (numerical aperture 1.42) or similar, and adjust the laser power and wavelength based on the experiment.</li>
	<li>To identify the NMJ, capture images in muscle 4 in segment A3 (as shown in the location in Figure 1F). Select a 488 nm laser to activate the &#945;-tubulin or Futsch and 543 nm to activate the HRP imaging track. Adjust the parameters to a frame size of 800 pixels x 800 pixels, a digital zoom of 2.0, and an imaging interval of 0.8 &#181;m in NMJs (Figure 2).</li>
	<li>To identify the microtubules in muscle, capture images of muscle 2 in segment A3-A5 (as shown in the location in Figure 1G) as it has fewer tracheal branches. Choose a 488 nm laser for activating &#945;-tubulin and a 635 nm laser for activating the T3605 imaging track. Adjust the parameters to a frame size of 1024 pixels x 1024 pixels, a digital zoom of 3.0, and an imaging interval of 0.4 &#181;m in muscle cells (Figure 3).</li>
</ol>

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+katanin+in+severing+microtubules+in+the+neuron.">An essential role for katanin in severing microtubules in the neuron.</a> The Journal of cell biology. 145 (2), 305-315 (1999).</li><li>Hu, Z., 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=Fidgetin+regulates+cultured+astrocyte+migration+by+severing+tyrosinated+microtubules+at+the+leading+edge.">Fidgetin regulates cultured astrocyte migration by severing tyrosinated microtubules at the leading edge.</a> Molecular biology of the cell. 28 (4), 545-553 (2017).</li><li>Feizabadi, M. S., Castillon, V. 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S., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+embryonic+neuromuscular+synapse+of+Drosophila+melanogaster.">Development of the embryonic neuromuscular synapse of Drosophila melanogaster.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 13 (1), 144-166 (1993).</li><li>Xiong, 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=HDAC6+mutations+rescue+human+tau-induced+microtubule+defects+in+Drosophila.">HDAC6 mutations rescue human tau-induced microtubule defects in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (12), 4604-4609 (2013).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Mao, C. 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=Microtubule-severing+protein+Katanin+regulates+neuromuscular+junction+development+and+dendritic+elaboration+in+Drosophila.">Microtubule-severing protein Katanin regulates neuromuscular junction development and dendritic elaboration in Drosophila.</a> Development. 141 (5), 1064-1074 (2014).</li><li>Jin, S., 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=Drosophila+Tubulin-specific+chaperone+E+functions+at+neuromuscular+synapses+and+is+required+for+microtubule+network+formation.">Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation.</a> Development. 136 (9), 1571-1581 (2009).</li><li>Sarthi, J., Elefant, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dTip60+HAT+activity+controls+synaptic+bouton+expansion+at+the+Drosophila+neuromuscular+junction.">dTip60 HAT activity controls synaptic bouton expansion at the Drosophila neuromuscular junction.</a> PloS one. 6 (10), 26202 (2011).</li><li>Weingarten, M. 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The authors have nothing to disclose.

Here a protocol is described for the dissection and immunostaining of Drosophila larval neuromuscular junctions and muscle cells. There are several essential points to consider. Firstly, avoiding injury to the observed muscles is crucial during the dissection process. It may be worth fixing the fillet before removing internal organs to prevent direct contact between the forceps and the muscles. To avoid muscle damage or separation from the larval epidermis, it is important to ensure that the speed of the shaker ...

Microtubules (MTs), as one of the structural components of the cytoskeleton, play an important role in diverse biological processes, including cell division, cell growth and motility, intracellular transport, and the maintenance of cell shape. Microtubule dynamics and function are modulated by interactions with other proteins, such as MAP1, MAP2, Tau, Katanin, and Kinesin1,2,3,4,5.
In neurons, microtubules are essential for the development and maintenance of axons and dendrites. Abnormalities in microtubules lead to dysfunction and even the death of neurons. For instance, in the brain of Alzheimer&#39;s patients, Tau protein hyperphosphorylation reduces the stability of the microtubule network, causing neurological irregularities6. Thus, examining microtubule networks will contribute to a comprehension of neurodevelopment and the pathogenesis of neurological diseases.
The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fiber, which is an excellent and powerful model system for studying synaptic structure and functions7. Futsch is a protein in Drosophila that is homologous to the microtubule-binding protein MAP1B found in mammals8. It is expressed only in neurons and plays a role in the development of the NMJ&#39;s synaptic buttons8,9. In wild-type, filamentous bundles that run along the center of NMJ processes are visualized by immunostaining with anti-Futsch. When reaching NMJ&#39;s end, this bundle has the ability to either form a loop consisting of microtubules or to lose its filamentous structure, resulting in a diffuse and punctate appearance10. Microtubule loops are associated with paused growth cones, which suggests the microtubule array is stable11. Therefore, we can indirectly determine the stable microtubule development in NMJ by Futsch staining. The large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. The factors affecting the stability of the microtubule network can be found by analyzing the density and shape of microtubules. Simultaneously, the microtubule network status of muscle cells can be cross-verified with the result of NMJ to obtain more comprehensive conclusions.
Many protocols have been employed for investigating the network and dynamics of microtubules. However, these researches have often focused on in vitro studies12,13,14,15,16. Alternatively, some in vivo experiments have employed electron microscopy to detect the cytoskeleton17. According to the specific binding of fluorescently labeled antibodies or chemical dyes to proteins or DNA, the methods presented here allow the detection of microtubule networks in NMJ at the level of individual neurons in vivo, with results corroborated by observations in muscle cells. This protocol is simple, stable, and repeatable when combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examinations and genetic screenings for the role of microtubule network regulatory proteins in the nervous system in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor Plus 405 phalloidin</td>
			<td>invitrogen</td>
			<td>A30104</td>
			<td>dilute 1:200</td>
		</tr>
		<tr>
			<td>Enhanced Antifade Mounting Medium</td>
			<td>Beyotime</td>
			<td>P0128M</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW confocal microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>Thermo Fisher</td>
			<td>A-11001</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Laser confocal microscope LSM 710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>66vision</td>
			<td>54138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Futsch antibody</td>
			<td>Developmental Studies Hybridoma Bank&#160;&#160;</td>
			<td>22C10</td>
			<td>dilute 1:50</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5168</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Wako</td>
			<td>168-20955</td>
			<td>Final concentration: 4% in PB Buffer</td>
		</tr>
		<tr>
			<td>Stainless Steel Minutien Pins</td>
			<td>Entomoravia</td>
			<td></td>
			<td>0.1mm Diam</td>
		</tr>
		<tr>
			<td>Stereomicroscope SMZ161</td>
			<td>Motic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereoscopic fluorescence microscope BX41</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-conjugated goat anti-HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>dilute 1:100</td>
		</tr>
		<tr>
			<td>TO-PRO(R) 3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Transfer decoloring shaker TS-8</td>
			<td>Kylin-Bell lab instruments</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>BioFroxx</td>
			<td>1139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>dumont</td>
			<td>500342</td>
			<td></td>
		</tr>
	</tbody>
</table>

using drosophila larval neuromuscular junction and muscle cells to visualize microtubule network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

We demonstrated a step-by-step procedure for visualizing the microtubule network in both neuromuscular junctions (NMJs) and muscle cells. Following dissection according to the schematic diagram (Figure 1A-E), immunostaining is performed, and images are subsequently observed and collected under a laser confocal microscope or a stereoscopic fluorescence microscope (Figure 1F,G).
Both pre-and post-synapt...

Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Currently, microtubule dynamics are observed indirectly through microtubule-binding proteins such as EB1. Since alphabeta tubulin heterodimers are abundant in the cytoplasm, fold directly, neighboring tubulin heterodimers are lacking the dynamics of microtubule networks in vivo. Our protocol visualizes microtubules in neuromuscular synapses and muscle cells.When combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examinations and the genetic screening for the role of microtubule network regulatory proteins in the nervous system in vivo. Our research focuses on the relationship between the cytoskeleton and the neurodevelopment, as well as its implications for neurological diseases. We hope to discover the mechanism of neurodevelopment and the parthenogenesis for neurological diseases by highlighting the effect of cytoskeletal regulatory proteins on neurons.

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1.4K Views.  Hubei University. Currently, microtubule dynamics are observed indirectly through microtubule-binding proteins such as EB1. Since alphabeta tubulin heterodimers are abundant in the cytoplasm, fold directly, neighboring tubulin heterodimers are lacking the dynamics of microtubule networks in vivo. Our protocol visualizes microtubules in neuromuscular synapses and muscle cells.When combined with the powerful genetic tools available in Drosophila melanogaster, enabling a diverse range of phenotypic examina...

Video: Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...