This work was supported by grants from the Canadian Institutes of Health Research (grant number MOP 38040) to R.K., Muscular Dystrophy Association (USA) to R.K., Families of SMA to R.K. and L.M.M, The SMA Trust to T.H.G., and The Muscular Dystrophy Campaign to T.H.G.. L.M.M is a recipient of a Multiple Sclerosis Society of Canada Postdoctoral Fellowship, and R.K. is a recipient of a University Health Research Chair from University of Ottawa.

All procedures should be performed to the animal care standards set out by the institution.
1. Dissection of the Abdominal Musculature from the Mouse
<ol>
	<li>Before starting, make up 4% paraformaldehyde (PFA). Caution: Always keep PFA within a fume hood and wear appropriate protective equipment.</li>
	<li>Euthanize mouse by an approved method. Note: The mouse shown in the video was euthanized by overdose of CO2 and cervical dislocation. This mouse is a 4-week-old wild-type mouse on a CD1/C57Bl6 hybrid background. This mouse was bred in the animal facilities at the University of Ottawa from mice that were originally purchased.</li>
	<li>Make an initial incision through the skin at the level of the hip and cut through the skin all the way around the mouse.</li>
	<li>Peel off the skin, pulling upwards until you reach the level of the forelimb.</li>
	<li>Cut through the abdominal musculature at the level of the hip and continue the incision until you reach the vertebral column.</li>
	<li>At this point, start to cut upwards through the musculature and the ribs until you reach the upper limb.</li>
	<li>Cut straight across until you reach the vertebral column on the other side.</li>
	<li>Cut downwards until you reach the point at which you started.</li>
	<li>Release the diaphragm from underneath (taking care not to damage the TVA muscle) and place it in phosphate buffered saline (PBS) in a SYLGARD-coated dissection dish with 0.2 mm dissection pins.</li>
	<li>Pin out the rib cage and associated musculature in the dish, superficial side up, making sure the muscle is fully stretched out.</li>
	<li>Pour off the 1x PBS and replace with 4% PFA.</li>
	<li>Cover and leave on a rocking platform at room temperature for 15 min.</li>
	<li>Wash 3x in 1x PBS at room temperature for 10 min.</li>
</ol>
* At this point the fixed muscles can be left in a fridge overnight before subsequent dissection.
2. Isolation of the TVA Muscle
<ol>
	<li>To proceed with isolation of the TVA muscle, under a dissection microscope, begin by cutting through the external oblique muscle at the level of the last few ribs (See Figure 1 for an annotated guide).</li>
	<li>Cut straight down beside the linea alba (taking care not to cut through the TVA below) until you reach the start of the internal oblique muscle.</li>
	<li>Then cut across to release the overlying rectus abdominis and external oblique muscles from the upper part of the TVA below.</li>
	<li>Remove the blood vessel and any overlying fat from the TVA muscle.</li>
	<li>Release the muscle from the overlying last rib.</li>
	<li>Cut around the margins of the muscle and remove to a 24-well plate containing PBS.</li>
</ol>
3. Immunofluorescent Labeling and Microscopy of the TVA Muscle
For all subsequent steps, remove liquid using a fine tipped pipette and leave the plate on a rocking platform. Unless otherwise stated, all subsequent steps are performed at room temperature.
<ol>
	<li>Incubate in PBS containing 1:1,000 fluorescently tagged bungarotoxin for 30 min to label the NMJs. This can be done in a dark room or under foil.</li>
	<li>Permeabilize muscle by adding 300 &#181;l per well of 2% Triton X-100 in PBS and leave on a rocking platform for 30 min.</li>
	<li>Incubate in blocking reagent (4% BSA, 1% Triton in PBS) for 30 min.</li>
	<li>Incubate in blocking reagent containing primary antibodies (neurofilament 1:100; synaptic vesicle protein 2, 1:250) at 4 &#176;C overnight.</li>
	<li>Wash 3x in 1x PBS.</li>
	<li>Incubate in PBS containing 1:250 secondary antibody for 2-4 hr.</li>
	<li>Wash 3x in 1x PBS.</li>
	<li>Mount muscles onto glass slides using sufficient fluorescent mounting media and coverslip.</li>
	<li>Slides are best viewed using a double band pass filter on a standard epifluorescent microscope.</li>
	<li>NMJs are best imaged using a z-series projection on a confocal microscope equipped with at least a 40X&#160;objective.</li>
</ol>

<ol>
	<li>Sanes, J. R., Lichtman, J. W. <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+vertebrate+neuromuscular+junction.">Development of the vertebrate neuromuscular junction.</a> Annu. Rev. Neurosci. 22, 389-442 (1999).</li><li>Dupuis, L., Loeffler, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+junction+destruction+during+amyotrophic+lateral+sclerosis:+insights+from+transgenic+models.">Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models.</a> Curr. Opin. Pharmacol. 9, 341-346 (2009).</li><li>Murray, L. M., Talbot, K., Gillingwater, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+neuromuscular+synaptic+vulnerability+in+motor+neurone+disease:+amyotrophic+lateral+sclerosis+and+spinal+muscular+atrophy.">Review: neuromuscular synaptic vulnerability in motor neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy.</a> Neuropathol. Appl. Neurobiol. 36, 133-156 (2010).</li><li>Murray, L. M., 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=Selective+vulnerability+of+motor+neurons+and+dissociation+of+pre-+and+post-synaptic+pathology+at+the+neuromuscular+junction+in+mouse+models+of+spinal+muscular+atrophy.">Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy.</a> Hum. Mol. Genet. 17, 949-962 (2008).</li><li>Kariya, 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=Reduced+SMN+protein+impairs+maturation+of+the+neuromuscular+junctions+in+mouse+models+of+spinal+muscular+atrophy.">Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy.</a> Hum. Mol. Genet. 17, 2552-2569 (2008).</li><li>Iguchi, 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=Loss+of+TDP-43+causes+age-dependent+progressive+motor+neuron+degeneration.">Loss of TDP-43 causes age-dependent progressive motor neuron degeneration.</a> Brain. 136, 1371-1382 (2013).</li><li>Martinez-Hernandez, R., 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=Synaptic+defects+in+type+I+spinal+muscular+atrophy+in+human+development.">Synaptic defects in type I spinal muscular atrophy in human development.</a> J. Pathol. 229, 49-61 (2013).</li><li>Fischer, L. R., Li, Y., Asress, S. A., Jones, D. P., Glass, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+SOD1+leads+to+oxidative+stress+in+peripheral+nerve+and+causes+a+progressive+distal+motor+axonopathy.">Absence of SOD1 leads to oxidative stress in peripheral nerve and causes a progressive distal motor axonopathy.</a> Exp. Neurol. 233, 163-171 (2012).</li><li>Cifuentes-Diaz, C., 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=Neurofilament+accumulation+at+the+motor+endplate+and+lack+of+axonal+sprouting+in+a+spinal+muscular+atrophy+mouse+model.">Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model.</a> Hum. Mol. Genet. 11, 1439-1447 (2002).</li><li>Fischer, L. R., 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=Amyotrophic+lateral+sclerosis+is+a+distal+axonopathy:+evidence+in+mice+and+man.">Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man.</a> Exp. Neurol.. 185, 232-240 (2004).</li><li>Diers, A., Kaczinski, M., Grohmann, K., Hubner, C., Stoltenburg-Didinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultrastructure+of+peripheral+nerve,+motor+end-plate+and+skeletal+muscle+in+patients+suffering+from+spinal+muscular+atrophy+with+respiratory+distress+type+1+(SMARD1).">The ultrastructure of peripheral nerve, motor end-plate and skeletal muscle in patients suffering from spinal muscular atrophy with respiratory distress type 1 (SMARD1).</a> Acta Neuropathol. 110, 289-297 (2005).</li><li>Ang, E. T., 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=Motor+axonal+sprouting+and+neuromuscular+junction+loss+in+an+animal+model+of+Charcot-Marie-Tooth+disease.">Motor axonal sprouting and neuromuscular junction loss in an animal model of Charcot-Marie-Tooth disease.</a> J. Neuropathol. Exp. Neurol. 69, 281-293 (2010).</li><li>Simon, C. M., Jablonka, S., Ruiz, R., Tabares, L., Sendtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+neurotrophic+factor-induced+sprouting+preserves+motor+function+in+a+mouse+model+of+mild+spinal+muscular+atrophy.">Ciliary neurotrophic factor-induced sprouting preserves motor function in a mouse model of mild spinal muscular atrophy.</a> Hum. Mol. Genet. 19, 973-986 (2010).</li><li>Murray, L. M., Gillingwater, T. H., Parson, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mouse+cranial+muscles+to+investigate+neuromuscular+pathology+in+vivo.">Using mouse cranial muscles to investigate neuromuscular pathology in vivo.</a> Neuromuscul. Disord. 20, 740-743 (2010).</li><li>Brill, M. S., Marinkovic, P., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+photo-bleaching+to+delineate+single+Schwann+cells+at+the+neuromuscular+junction.">Sequential photo-bleaching to delineate single Schwann cells at the neuromuscular junction.</a> J. Vis. Exp. (4460), (2013).</li><li>Murray, L. M., Thomson, D., Conklin, A., Wishart, T. M., Gillingwater, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+translation+elongation+factor+(eEF1A2)+expression+in+vivo+differentiates+between+Wallerian+degeneration+and+dying-back+neuronal+pathology.">Loss of translation elongation factor (eEF1A2) expression in vivo differentiates between Wallerian degeneration and dying-back neuronal pathology.</a> J. Anat. 213, 633-645 (2008).</li><li>Bowerman, M., Murray, L. M., Beauvais, A., Pinheiro, B., Kothary, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+smn+threshold+in+mice+dictates+onset+of+an+intermediate+spinal+muscular+atrophy+phenotype+associated+with+a+distinct+neuromuscular+junction+pathology.">A critical smn threshold in mice dictates onset of an intermediate spinal muscular atrophy phenotype associated with a distinct neuromuscular junction pathology.</a> Neuromuscul. Disord. 22, 263-276 (2012).</li><li>Comley, L. H., 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=ApoE+isoform-specific+regulation+of+regeneration+in+the+peripheral+nervous+system.">ApoE isoform-specific regulation of regeneration in the peripheral nervous system.</a> Hum. Mol. Genet. 20, 2406-2421 (2011).</li><li>Wright, M., Kim, A., Son, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+administration+of+muscarinic+antagonists+and+triple-immunostaining+of+the+levator+auris+longus+muscle+in+mice.">Subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice.</a> J. Vis. Exp. (55), (2011).</li><li>Ling, K. K., Gibbs, R. M., Feng, Z., Ko, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+neuromuscular+denervation+of+clinically+relevant+muscles+in+a+mouse+model+of+spinal+muscular+atrophy.">Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy.</a> Hum. Mol. Genet. 21, 185-195 (2012).</li></ol>

The authors have nothing to disclose.

In this video, we have detailed a protocol for the dissection of the TVA muscle from the mouse and for the whole-mount immunofluorescent labeling of NMJs within the muscle. We also present data showing this muscle can be used to analyze neuromuscular junction pathology in a mouse model of SMA.
Success in this technique is reliant on a number of factors. Some of the most common problems are outlined below. Firstly: poor immunohistochemical staining. There can be a number of reasons for this, one of the most common being use of different reagents to the ones listed in this protocol. A high quality electron microscopy grade PFA is very important to ensure good staining, as are the choice of antibodies listed in this protocol. In addition, in older animals (i.e. &#62; 3 months), getting good quality staining can be more difficult. This is because of increased thickness of the fascia surrounding the muscle and the increase in fat accumulation between the external oblique and tranversus abdominis. It is important to strip off the fat, before proceeding to immunofluorescence. It may also be necessary to strip off some of the fascia covering the muscle, which can become thickened. It is sometimes difficult to strip the fascia and fat from the muscle without incurring some damage to the muscle fiber and a disruption to the innervation pattern. However if this technique is performed carefully, good quality staining can be acquired from mice up to at least 1 year of age. In younger mice (i.e. less than 3 months of age) it should not be necessary to perform any teasing or separation of muscle fibers. Secondly: difficultly in finding NMJs following dissection and staining. This is often because the dissection has not extended underneath the last rib. The majority of the NMJs are located just underneath the last rib and therefore care must be taken to ensure this part of the muscle is included in the dissection. Thirdly: adherence of the EO muscle to the TVA muscle. This is often a complaint when individuals attempt to extend the dissection below the level of the internal oblique (IO) muscle. The area of the TVA muscle where the IO is also present is more difficult to analyze as it can be difficult to distinguish which muscle is which. For this reason, we routinely just dissect the most superior part of the TVA muscle. At this level, there is no adherence between the EO and TVA muscles, and therefore this should not be a significant problem.
One significant hindrance to using the TVA muscle, compared to appendicular muscles, is accessibility for either surgical manipulations or injection of substances. These types of experiments can be crucial to investigate NMJ physiology in a given muscle. Although the TVA is certainly less easily accessible than more commonly used muscles such as tibialis anterior or gastrocnemius, previous work has shown it is possible to denervate the TVA by surgical injury of the intercostal nerves18. We have recently also used this muscle for local administration of substances under general anesthetic (unpublished data). Although these experiments can represent a moderate technical challenge, this work shows that they are feasible and thus extend the usefulness of this muscle for analysis of NMJs under both pathological and physiological manipulation.
The TVA muscle is one of a number of thin flat muscles situated throughout the body that can be used for whole-mount analysis of innervation patterns. Other muscles include a group of cranial muscles innervated by motor neurons originating from the facial nucleus of the brainstem, encompassing levator auris longus, auricularis superior, and adductor auris longus, the dissections for which have been described previously14,19. Furthermore, the musculature surrounding the TVA muscle, including EO, IO, and rectus abdominis, can also be labeled and used for NMJ analysis. For comprehensive analysis of NMJ pathology in a mouse model, it is important to consider a number of muscles situated throughout the body and not to restrict analysis to a single muscle. This is exemplified in mouse models of motor neuron diseases where there is significant heterogeneity in levels of NMJ pathology between different muscles20. Such intermuscular variability is an extremely valuable tool when investigating the mechanism of motor neuron vulnerability&#160;and therefore restricting analysis to a single muscle could significantly diminish the potential of the research.

Neuromuscular junctions (NMJs) are the synaptic connection between a lower motor neuron and a skeletal muscle fiber. They are traditionally considered a tripartite synapse, made up of a neuron (presynaptic terminal), muscle fiber (post synaptic terminal), and terminal Schwann cell1. NMJs appear to be early and significant targets in pathology in a range of motor neuron diseases and mouse models2,3. Typical symptoms include denervation, where the motor endplate becomes devoid of a presynaptic innervation, swelling of the presynaptic terminal, and a reduction in the complexity of the NMJ morphology4-11. Compensatory responses can also be noted, which include terminal and nodal sprouting, where axonal processes extend from remaining synaptic terminals or internodes to reinnervated denervated endplates12,13. Due to the tight correlation between synaptic activity and NMJ morphology, a great deal of information can be gained about the functional status of motor neurons from analysis of NMJ morphology. As loss of NMJs frequently represents one of the first aspects of neuromuscular pathology4,10, quantification at the level of innervation can give important information about the progression of pathology and the potential effect of a therapeutic intervention. Furthermore, as NMJ loss represents a significant step in pathological progression, the development of therapeutics that can stabilize connections and encourage regeneration may yield significant benefit.
When analyzing NMJ morphology, muscle choice is of great importance. Some of the primary considerations might include muscle fiber type, body position, and comparative analysis to human conditions. In addition, where manipulations such as injection of substances or traumatic nerve injury are required, experimental accessibility is also important to consider. In general, it is preferable to analyze a range of muscles positioned throughout the body reflecting a range of motor unit subtypes. Often, however, muscle choice is influenced by ease of dissection. Consequently, NMJ analysis is often performed exclusively on large appendicular muscles such as gastrocnemius. To obtain good NMJ staining in such muscles, sectioning or mechanical disruption of muscle fibers is often required. As a result, the innervation pattern may become disrupted and a comprehensive and high quality analysis of innervation patterns, sprouting and denervation is often compromised. An alternative approach is to use thin flat muscles which do not require sectioning and can be stained and mounted intact, allowing a comprehensive overview of the entire innervation of the muscle. There are a number of muscles which can be used for such an analysis, including a group of cranial muscles, (encompassing levator auris longus, auricularis superior, and adductor auris longus)14, thoracic muscles (e.g. triangularis sterni)15, and abdominal (e.g. transversus abdominis (TVA)) muscles. The major hindrance in using such muscles is the technical expertise required to dissect them without damage.
In this video, we provide a protocol to dissect and perform immunofluorescent labeling of the TVA muscle from the mouse to allow a comprehensive analysis of innervation pattern and NMJ morphology. The TVA muscle is a predominantly slow twitch muscle comprising the deepest layer of abdominal musculature and is innervated by the lower intercostal nerves. Previous work has shown it to be consistently highly vulnerable to pathology in a number of mouse models of the childhood motor neuron disease spinal muscular atrophy (SMA) and in other mouse models of early onset motor neuron degeneration4,16. We therefore suggest that the TVA is a useful muscle for undertaking NMJ analysis in peripheral neuropathies.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Paraformaldehyde Aqueous Solution (16% )</td>
			<td style="width:259px;">Electron Micropscopy Sciences</td>
			<td style="width:216px;">15700</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Dumont #5 Forceps</td>
			<td style="width:259px;">Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Angled Sprung Scissors</td>
			<td style="width:259px;">Fine Science Tools</td>
			<td>15006-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Fine Scissors - ToughCut</td>
			<td style="width:259px;">Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">SYLGARD 184 Silicone Elastomer Kit</td>
			<td style="width:259px;">Dow Corning</td>
			<td style="width:216px;">dependant on local supplier</td>
			<td>Use this to make dissection dish for pinning out muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Minutien Pins</td>
			<td style="width:259px;">Fine science tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">&#945;-Bungarotoxin, Alexa Fluor 488 Conjugate</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:216px;">B-13422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Albumin from Bovine Serum</td>
			<td style="width:259px;">Sigma Aldrich</td>
			<td style="width:216px;">A4503</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">Neurofilament Primary antibody (2H3), Supernatant</td>
			<td style="width:259px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">SV2 Primary antibody (SV2), Supernatant</td>
			<td style="width:259px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse IgG (H+L)</td>
			<td style="width:259px;">Jackson ImmunoResearch</td>
			<td>115-166-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Fluorescence Mounting Medium</td>
			<td style="width:259px;">Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Slides (Superfrost Plus; White)</td>
			<td style="width:259px;">Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Coverslips</td>
			<td style="width:259px;">Fisher</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Triton X-100</td>
			<td style="width:259px;">Sigma Aldrich</td>
			<td style="width:216px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">CD1/C57Bl6 mouse</td>
			<td style="width:259px;">Jackson Labs</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The above protocol directs the isolation and staining of the TVA muscle for NMJ analysis. This allows whole-mount analysis of muscle innervation patterns as well as high-resolution analysis of NMJ morphology (Figure 2). This technique can be successfully applied to reveal NMJ pathology in mouse models of motor neuron disease, such as SMA4,17(Figure 3). In mouse models of SMA there is significant intramuscular variability in pathology, however the TVA muscle is consistently highly affected. This is evidenced by denervation of motor endplates, accumulation of neurofilaments at the presynaptic terminal and terminal sprouting (Figure 3). The results presented here thus demonstrate that the technique described above can be a powerful method to provide a comprehensive overview of innervation and analysis of NMJ pathology in mouse models.
<img alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/51162/51162fig1highres.jpg" src="/files/ftp_upload/51162/51162fig1.jpg" width="600px" /> 
Figure 1. Overview of abdominal musculature of the mouse. (A)&#160;Image show the thoracic cage with attached abdominal musculature, which has been removed from a recently euthanized mouse and pinned out in a dissection dish superficial side up (A). (B)&#160;Dissection in A has been annotated to mark the approximate boundaries of the abdominal muscles. The superficial abdominal muscles can be seen on the left side of the dissection, and include external oblique (outlined in blue) and rectus abdominis (outlined in red). On the right hand side of the dissection, the superficial muscles have been removed allowed visualization of the transversus abdominis muscle (outlined in green) and the internal oblique muscle (outlined in yellow). The aim of this protocol is to direct the dissection of the superior part of the TVA muscle, shown here as a solid green triangle. <a href="https://www.jove.com/files/ftp_upload/51162/51162fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="4in" fo:src="/files/ftp_upload/51162/51162fig2highres.jpg" src="/files/ftp_upload/51162/51162fig2.jpg" width="600px" /> 
Figure 2. Whole-mount overview of neuromuscular junctions in TVA muscle. Images showing example NMJs from the TVA muscle visualized with immunofluorescent staining for neurofilament (NF; green), synaptic vesicle protein 2 (SV2; green) and bungarotoxin (BTX; red). Images are montaged fluorescent micrographs showing the whole muscle (A) or confocal images showing groups (B) or individual (C) NMJs. Scale bar = 800 &#181;m (A), 70 &#181;m (B), 25 &#181;m (C). <a href="https://www.jove.com/files/ftp_upload/51162/51162fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/51162/51162fig3highres.jpg" src="/files/ftp_upload/51162/51162fig3.jpg" width="600px" /> 
Figure 3. Neuromuscular junction pathology in TVA muscle from a mouse model of SMA.&#160;Confocal micrographs showing NMJs from the TVA muscle visualized with immunofluorescent staining for neurofilament (NF; green), synaptic vesicle protein 2 (SV2; green) and bungarotoxin (BTX; red) from either control (Smn2B/+) or SMA mouse model (Smn2B/-). Note that whilst normal NMJ morphology can be observed in control mice, in TVA muscles from Smn2B/- mice there is evidence of full denervation (white arrowhead), partial denervation (purple arrowhead) terminal sprouting (blue arrowhead) and presynaptic swelling (yellow arrowhead). The post-synaptic endplates are also less complex reflecting an apparently less mature phenotype. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/51162/51162fig3highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis at JoVE.com

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.

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 ...

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11.4K Views.  Ottawa Hospital Research Institute. 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.

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

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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 ...

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 ...