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

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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.

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

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 ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide limited information about muscle fiber membrane properties and underlying disease mechanisms. Muscle velocity recovery cycles (MVRCs) illustrate how the velocity of a muscle action potential depends on the time after a preceding action potential. MVRCs are closely related to changes in membrane potential that follow an action potential, thereby providing information about muscle fiber membrane properties. MVRCs may be recorded quickly and easily by direct stimulation and recording from multi-fiber bundles in vivo. MVRCs have been helpful in understanding disease mechanisms in several neuromuscular disorders. Studies in patients with channelopathies have demonstrated the different effects of specific ion channel mutations on muscle excitability. MVRCs have been previously tested in patients with neurogenic muscles. In this prior study, muscle relative refraction period (MRRP) was prolonged, and early supernormality (ESN) and late supernormality (LSN) were reduced in patients compared to healthy controls. Thereby, MVRCs can provide in vivo evidence of membrane depolarization in intact human muscle fibers that underlie their reduced excitability. The protocol presented here describes how to record MVRCs and analyze the recordings. MVRCs can serve as a fast, simple, and useful method for revealing disease mechanisms across a broad range of neuromuscular disorders.

Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide ...

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...