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

Summary

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.

Abstract

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 vitro^9,5. Last, and perhaps most importantly, a small applied volume (˜50μl) 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 approach^1,8.

Protocol

1. Subcutaneous administration of muscarinic acetylcholine receptor (mAChR) antagonists

1. 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.
2. Draw 50μl 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.
3. Anesthetize mice with ketamine-xylazine cocktail (120 mg/kg....

Discussion

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.......

Materials

List of materials used in this article

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