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 only for each mouse in the control group. Keep syringes on ice until ready to inject. </li>
 <li>Anesthetize mice with ketamine-xylazine cocktail (120 mg/kg ketamine, 8 mg/kg xylazine) Check for appropriate anesthetic depth by observing lack of response to toe pinch.</li>
 <li>After proper sedation, shave hair covering the rostral region of the head to the caudal region of the neck, wipe remaining hair away with 70% ethanol. </li>
 <li>Place mouse under stereomicroscope, and insert needle parallel to LAL muscle while pulling up lightly on the needle of the syringe (Fig. 1A,B). This ensures that the needle is inserted into overlying connective tissue and does not directly damage the LAL muscle itself. Insert the needle so that the tip covers the caudal aspect of the LAL muscle.</li>
 <li>Very slowly begin to inject solution over the right LAL muscle while withdrawing the syringe; the total injection time should be approximately 1 minute. If injected properly, the solution should form a "dome" that is roughly 1 cm in diameter, in the overlying skin that will remain for at least one hour. A uniformly injected solution will cover the entire rostral and caudal regions of the right LAL muscle. </li>
 <li>Repeat the procedure every 12 hours for 1 to 7 days. For other studies1, we used the same protocol to inject growth factors every 12 hours for up to 21 days. No abnormal responses were observed from mice administered with anesthesia 2x a day for up to 21 days.</li>
</ol>
2. Dissection of LAL muscles
<ol>
 <li>Euthanize mice with an overdose of sodium pentobarbital ( 390 mg/kg). Death is assured by assessing lack of heartbeat after thoracotomy is performed.</li>
 <li>Pin down the mouse in a dissection dish, dorsal side up with 1 pin in each paw and 1 pin through the nose. </li>
 <li>Make the first incision through the skin only, using small spring scissors to cut along the left LAL from the region just proximal to the ear to the region around the left shoulder blade.</li>
 <li>Carefully remove the skin in this region, but avoid cutting too close to the right ear as this is one of the points of attachment for the right LAL muscle. </li>
 <li>Locate the centrally positioned adipose tissue strip of lipid along the midline, on the superficial surface of the head, where the right and left LAL muscles meet. Using small spring scissors, cut 1cm to the right of the fat tissue (toward the left ear ) from the proximal edge of the left LAL muscle until reaching the shoulder (Fig. 1C). </li>
 <li>Once the incision is made, peel back the right LAL muscle with small forceps to expose the ventral side of the muscle. Trim the connective tissue and fascia while carefully pulling up on the right LAL muscle with forceps (Fig. 1C). </li>
 <li>Cut around the caudal end of the right LAL at the base of the ear. Continue cutting, moving towards the rostral end of the right ear, keeping the right LAL turned over (Fig. 1C).. It is better to include more tissue at this step than risk damaging the LAL itself. If the muscle begins to dry out, pipette 1X PBS over the muscle.</li>
 <li>Place dissected right LAL in 30mm culture Sylgard dish containing 1X PBS, dorsal side up. Pin down four corners of muscle with small insect pins. </li>
 <li>Using small forceps and spring scissors, clean connective tissue from dorsal and ventral surfaces of the right LAL muscle. Turn muscle over and re-pin in order to clean off ventral surface. 2-3 muscles may be placed in the same 30mm dish.</li>
</ol>
3. Triple immunostaining of LAL NMJs: Day 1
<ol>
 <li>Before beginning immunostaining, prepare all needed reagents. 1X PBS, 2% bovine serum albumin BSA)/PBS, 0.1M Glycine in 2%BSA/PBS, 0.2% Triton X100 in 2% BSA/PBS, and 4% paraformaldehyde (PFA) in 1X PBS will be needed. Pre-cool methanol in -20&deg;C freezer. </li>
 <li>Leave LAL muscles pinned in the dish dorsal side up during this process. Discard 1X PBS solution and add enough 4% (PFA) to cover the muscle. Place dish on shaker set at speed 2 or 3 for 20 minutes. Unless otherwise noted, the dish should be placed on a shaker for the following steps. </li>
 <li>Discard 4% PFA and wash the muscle with 1X PBS 3 times, 10' each. </li>
 <li>Discard last 1X PBS wash and add 0.1M Glycine in 2% BSA/PBS solution for 30'. </li>
 <li>Discard 0.1M Glycine solution and add 1X PBS containing rhodamine conjugated &alpha;-bungarotoxin at a concentration of 1:200 for 15'.</li>
 <li>Discard &alpha;-bungarotoxin solution and wash muscle in 1X PBS, 3 times 10' each. </li>
 <li>Permeabilize tissue by adding pre-cooled methanol and place the dish in the -20&deg;C freezer for precisely 5 minutes, no shaking needed. </li>
 <li>Remove methanol and wash 3 times 10' each with 1X PBS. Discard last wash and block tissue with 0.2% triton X-100 in 2% BSA/PBS for 1 hour. The washing and blocking steps are performed at room temperature. </li>
 <li>Incubate the muscles overnight, shaking, at 4&deg;C in a cocktail of primary antibodies diluted in the blocking solution (cf., Table). </li>
</ol>
4. Triple immunostaining of LAL NMJs: Day 2
<ol>
<li>Wash muscles in 1X PBS 3 times, 10' each at room temperature. </li>
<li>Add cocktail of secondary antibodies in blocking solution for 1 hour at room temperature (cf., Table). The muscles must be protected from light from this step onward to prevent photo-bleaching of Alexa-Fluor 647 fluorescence.</li>
<li>Wash muscles in 1X PBS 3 times, 10' each. </li>
<li>Clean off any remaining connective tissue in 1X PBS, cut the lateral borders of the LAL muscles and mount on slides dorsal side up, using glass cover-slips and mounting media. Use clear nail polish to secure cover slip in place. </li>
<li>Protect slides from light and store at -20&deg;C until ready for analysis. </li>
</ol>
5. Confocal imaging of LAL NMJs
<ol>
 <li>High-resolution confocal images are obtained with a Leica Plan Apo 63X oil objective (1.4NA) on a Leica TCS 4D confocal microscope. </li>
 <li>Fluorescein and Alexa 647 signals were simultaneously scanned with laser lines 488 nm (Argon, 488 nm, 20 mW) and 633 nm (HeNe, 633 nm, 10 mW), respectively. Sequentially, Rhodamine signal is then scanned with the laser line 561 nm (DPSS, 561, 20 mW). Optical sections were collected at 0.3 &mu;m intervals, and the number of images in the z-plane varied from 60-80. The pinhole is adjusted to Airy 1 (107.97 um). The image format 1024 x 1024 (X,Y), with a zoom factor of 1, at 400Hz speed results in an image size of 234.32 &mu;m x 234.32 &mu;m and pixel size of 229.05 nm x 229.05 nm.</li>
 <li>Leica TCS-NT acquisition software is then used to reconstruct z-series images into maximum intensity projections. </li>
 <li>Multipanel images are adjusted for brightness and contrast using Adobe Photoshop.</li>
 <li>For a typical LAL muscle, one should be able to analyze approximately 75-100 en face NMJs. Synaptic stability is determined by features such as spatial alignment of pre-, peri- and postsynaptic elements (i.e., nerve, Schwann cells and muscle), and measurements of synaptic area (i.e., synapse size).</li>
</ol>
Representative results: 
At the adult mammalian NMJ, a single motor axon elaborates fine branches that form highly differentiated arbors of a nerve terminal (Fig. 2; Green) on a single muscle fiber, precisely apposed to postsynaptic clusters of nicotinic AChRs (Fig. 2; Red). Perisynaptically, terminal Schwann cells tightly cover all the branches of presynaptic nerve terminals (Fig. 2; Blue). The structural and functional integrity of this tripartite organization is severely perturbed by daily application of subtype-specific mAChR inhibitors. In the example presented here (Fig. 3), 4-DAMP, a mAChR antagonist with high affinity for M1, M3, M4 and M5 mAChR subtypes , evokes selective elimination of nerve terminals from numerous NMJs throughout the muscle surface (Fig. 3B, C). In addition, terminal Schwann cells are abnormally quiescent8 as evidenced by bright S100 labeling without process extension (Fig. 3B", 3C"). Postsynaptically, muscle fibers are normal and there is no loss of nAChRs (Fig. 3B', 3C'). 
<img src="/files/ftp_upload/3124/3124fig1.jpg" alt="Figure 1"/> Figure 1. Anatomical location and organization of the LAL muscle. Location of rostral (rLAL), caudal (cLAL), and the LAL nerve (LALn) and corresponding endplate bands are shown (A, inset). The location and orientation of the needle in respect to the LAL muscle is shown for the injection procedure (B). Incision points are shown for the dissection procedure (C). 
<img src="/files/ftp_upload/3124/3124fig2.jpg" alt="Figure 2"/> Figure 2. Tripartite organization of the NMJ. High-magnification confocal views of a mouse NMJ. A single axon elaborates fine terminal branches (A), tightly covered by terminal Schwann cell and their processes (A', asterisks point to terminal Schwann cell bodies). Postsynaptically in a muscle fiber, a cluster of nicotinic AChRs is precisely apposed to the branches of nerve terminals and terminal Schwann cells.
<img src="/files/ftp_upload/3124/3124fig3.jpg" alt="Figure 3"/> Figure 3. LAL muscles treated with 4-DAMP, a mAChR antagonist. Low and high-magnification confocal views of LAL muscles treated with vehicle or 4-DAMP. In contrast to the vehicle-treated muscle (A-A'''), numerous NMJs in the 4-DAMP-treated muscle lack nerve terminals (B-B''', C-C'''). A boxed area in Figure 2B is zoomed in Figure 2C.

<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>

No conflicts of interest declared.

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. نحن تصف الإجراءات اللازمة لإدارة المتكررة من مثبطات المسكارينية يشير إلى الرافعة الأذن الطويلة (LAL) عضلات الفئران البالغين الشباب وimmunostaining لاحقة من التقاطعات في الاعصاب (NMJs) في wholemounts. عضلة LAL ومزايا فريدة للكشف عن في الجسم الحي التأثيرات الدوائية على NMJs....

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

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Medicine

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biology

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Immunology and Infection

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

A Heterotopic Mouse Model for Studying Laryngeal Transplantation

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other animal models. Although first described by Shipchandler et al. in 2009, this technique is not widely used, possibly due to the difficulties in learning the microsurgical technique and time required to master it. This paper describes the surgical steps in detail, as well as potential pitfalls to avoid, in order to encourage effective use of this technique.
In this model, the bilateral carotid arteries of the donor larynx are anastomosed to the recipient carotid artery and external jugular vein, allowing for blood flow through the graft. Blood flow can be confirmed intraoperatively by the visualization of blood filling in the graft bilateral carotid arteries, reddening of the thyroid glands of the graft, and bleeding from micro vessels in the graft. The crucial elements for success include delicate preservation of the graft vessels, making the correct size arteriotomy and venotomy, and using the appropriate number of sutures on the arterial-arterial and arterial-venous anastomoses to secure vessels without leakage and prevent occlusion. 
Anyone can become proficient in this model with sufficient training and perform the procedure in approximately 3 h. If performed successfully, this model allows for immunologic studies to be performed with ease and at low cost.

The aim of this manuscript is to describe the microsurgical steps required to perform a heterotopic laryngeal transplant in mice. The advantages of this mouse model compared to other animal models of laryngeal transplantation are its cost-effectiveness and the availability of immunologic assays and data.

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other ...

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

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

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

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

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

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