This work was supported by Muscular Dystrophy Association, NIH (NS062320).

1. Subcutaneous administration of muscarinic acetylcholine receptor (mAChR) antagonists
<ol>
 <li>Prepare under aseptic conditions the appropriate dose of mAChR antagonist, (cf., Table) by dissolving the drug in sterile physiological saline in 1.5mL reaction tube. The following antagonists were used: atropine, Methoctramine, 4-DAMP, AFDX-116, AFDX-384, MT 7. </li>
 <li>Draw 50&mu;l of solution into a1cc insulin syringe and use a separate syringe for each mouse. Also prepare syringes containing physiological saline onl...

<ol>
	<li>Wright, M. C., Son, Y. J. <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+is+not+required+for+terminal+sprouting+and+compensatory+reinnervation+of+neuromuscular+synapses:+re-evaluation+of+CNTF+null+mice.">Ciliary neurotrophic factor is not required for terminal sprouting and compensatory reinnervation of neuromuscular synapses: re-evaluation of CNTF null mice.</a> Exp Neurol. 205, 437-448 (2007).</li><li>Gurney, M. E., Yamamoto, H., Kwon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+motor+neuron+sprouting+in+vivo+by+ciliary+neurotrophic+factor+and+basic+fibroblast+growth+factor.">Induction of motor neuron sprouting in vivo by ciliary neurotrophic factor and basic fibroblast growth factor.</a> J Neurosci. 12, 3241-3247 (1992).</li><li>Caroni, P., Aigner, L., Schneider, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+neuronal+determinants+locally+regulate+extrasynaptic+and+synaptic+growth+at+the+adult+neuromuscular+junction.">Intrinsic neuronal determinants locally regulate extrasynaptic and synaptic growth at the adult neuromuscular junction.</a> J Cell Biol. 136, 679-692 (1997).</li><li>Witzemann, V., Brenner, H. R., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+factors+regulate+AChR+subunit+mRNAs+at+rat+neuromuscular+synapses.">Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.</a> J Cell Biol. 114, 125-141 (1991).</li><li>Angaut-Petit, D., Molgo, J., Connold, A. L., Faille, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+levator+auris+longus+muscle+of+the+mouse:+a+convenient+preparation+for+studies+of+short-+and+long-term+presynaptic+effects+of+drugs+or+toxins.">The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.</a> Neurosci Lett. 82, 83-88 (1987).</li><li>Lanuza, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-+and+postsynaptic+maturation+of+the+neuromuscular+junction+during+neonatal+synapse+elimination+depends+on+protein+kinase.">Pre- and postsynaptic maturation of the neuromuscular junction during neonatal synapse elimination depends on protein kinase.</a> C. J Neurosci Res. 67, 607-617 (2002).</li><li>Garcia, N., Santafe, M. M., Tomas, M., Lanuza, M. A., Tomas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effects+of+beta-amyloid25-35+peptide+aggregates+on+transmitter+release+in+neuromuscular+synapses.">Short-term effects of beta-amyloid25-35 peptide aggregates on transmitter release in neuromuscular synapses.</a> J Neuropathol Exp Neurol. 67, 250-259 (2008).</li><li>Wright, M. C., Cho, W. J., Son, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+motor+nerve+terminal+sprouting+induced+by+ciliary+neurotrophic+factor+vs.+botulinum+toxin.">Distinct patterns of motor nerve terminal sprouting induced by ciliary neurotrophic factor vs. botulinum toxin.</a> J Comp Neurol. 504, 1-16 (2007).</li><li>Wright, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+muscarinic+acetylcholine+receptor+subtypes+contribute+to+stability+and+growth,+but+not+compensatory+plasticity,+of+neuromuscular+synapses.">Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses.</a> J Neurosci. 29, 14942-14955 (2009).</li><li>Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+inhibits+chloride+channels+in+mature+mammalian+skeletal+muscle+by+activating+P2Y1+receptors.">Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors.</a> J Physiol. 587, 5739-5752 (2009).</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 (2009).</li><li>Dorje, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonist+binding+profiles+of+five+cloned+human+muscarinic+receptor+subtypes.">Antagonist binding profiles of five cloned human muscarinic receptor subtypes.</a> J Pharmacol Exp Ther. 256, 727-733 (1991).</li><li>Caulfield, M. P., Birdsall, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Union+of+Pharmacology.+XVII.+Classification+of+muscarinic+acetylcholine+receptors.">International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors.</a> Pharmacol Rev. 50, 279-290 (1998).</li></ol>

The method presented here permits investigation of previously unrecognized roles of subtype-specific mAChR signaling in the stability and maintenance of mammalian NMJs. This method will also be useful to test the effects of neurotrophic factors and pharmacological agents. For example, our laboratory found that Ciliary Neurotrophic Factor (CNTF) elicited sprouting from nearly all LAL nerve terminals in adult mice1. This result contrasted with prior studies of CNTF-treated hind limb muscles, which reported moder...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>Hospira</td>
			<td>NDC0409-2051-05</td>
			<td>Dose: 120mg/kg</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>Lloyd Laboratories</td>
			<td>LA33806</td>
			<td>Dose: 8mg/kg</td>
		</tr>
		<tr>
			<td>atropine</td>
			<td>Sigma-Aldrich </td>
			<td>A0132</td>
			<td>(&gt;98% purity); Dose: 0.2mg/kg &#x2013; 20mg/kg</td>
		</tr>
		<tr>
			<td>atropine</td>
			<td>Voigt Global Distribution</td>
			<td>AT105</td>
			<td>Pharmaceutical grade</td>
		</tr>
		<tr>
			<td>Methoctramine</td>
			<td>Sigma-Aldrich</td>
			<td>M105</td>
			<td>Dose: 100 - 400M</td>
		</tr>
		<tr>
			<td>4-DAMP</td>
			<td>Sigma-Aldrich</td>
			<td>D142</td>
			<td>Dose: 2.5mg/kg</td>
		</tr>
		<tr>
			<td>AFDX-116</td>
			<td>Tocris Bioscience</td>
			<td>1105</td>
			<td>250M</td>
		</tr>
		<tr>
			<td>AFDX-384</td>
			<td>Tocris Bioscience</td>
			<td>1345</td>
			<td>50M - 500M</td>
		</tr>
		<tr>
			<td>MT 7</td>
			<td>Peptides International</td>
			<td>PMT-4340-s</td>
			<td>0.1M - 1M</td>
		</tr>
		<tr>
			<td>1X Phosphate Buffered Saline, pH 7.4</td>
			<td>Invitrogen</td>
			<td>10010049</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>T353-500</td>
			<td>Make 10% solution first by dissolving 10g/100mL de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>Sodium pentobarbitol</td>
			<td>Virbac Animal Health</td>
			<td>NDC-051311-050-01</td>
			<td>Dose: 390mg/kg</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Dow Corning</td>
			<td>Part # 184</td>
			<td>Follow instructions that come with kit, can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs</td>
		</tr>
		<tr>
			<td>0.1M Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G-7126</td>
			<td>Add 0.185g to 25mL of 2% BSA/PBS</td>
		</tr>
		<tr>
			<td>2% Bovine serum albumin (2% BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A3059-100g</td>
			<td>Dissolve 2g BSA into 100mL of 1X PBS</td>
		</tr>
		<tr>
			<td>0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer)</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-100mL</td>
			<td>Dissolve 0.2ml/100mL 2% BSA/PBS</td>
		</tr>
		<tr>
			<td>&alpha;-bungarotoxin</td>
			<td>Invitrogen</td>
			<td>T1175</td>
			<td>Use at concentration of 1:200</td>
		</tr>
		<tr>
			<td>SMI-312</td>
			<td>Sternberger Monoclonals</td>
			<td>SMI312</td>
			<td>Use at concentration of 1:1000</td>
		</tr>
		<tr>
			<td>SV2</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>SV2-Supernatant</td>
			<td>Use at concentration of 1:10</td>
		</tr>
		<tr>
			<td>S100</td>
			<td>Dako</td>
			<td>Z0311</td>
			<td>Use at concentration of 1:400</td>
		</tr>
		<tr>
			<td>FITC- goat anti-mouse IgG1</td>
			<td>Roche</td>
			<td>03117731001</td>
			<td>Use at concentration of 1:200, but if background is high, try 1:400</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 647 conjugated goat anti-rabbit</td>
			<td>Invitrogen</td>
			<td>A21244</td>
			<td>Use at concentration of 1:200</td>
		</tr>
		<tr>
			<td>Vectashield fluorescent mounting media</td>
			<td>Vector laboratories</td>
			<td>H-1000</td>
			<td>This is not a hard-set media, you will need to secure the cover slip with clear nail polish.</td>
		</tr>
		<tr>
			<td>Small Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15002-08</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K Views.  Arcadia University. We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.