Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

Podsumowanie

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Streszczenie

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

Wprowadzenie

The mammal neuromuscular junction (NMJ) is a large cholinergic tripartite synapse made up of the motor neuron nerve ending, the postsynaptic membrane on the skeletal muscle fiber, and the terminal Schwann cells^1,2,3. This synapse exhibits high morphological and functional plasticity^4,5,6,7,8, even during adulthood when NMJs can undergo dynamic structural modifications. For example, some researchers have shown that ....

Protokół

All animal procedures were performed according to the guidelines of the National Law N° 18611 for Care of Animals Used for Experimental Purposes. The protocol was approved by the Institutional Ethical Committee (CEUA IIBCE, Protocol Number 004/09/2015).

1. Muscle dissection (Day 1)

NOTE: Before starting, make 40 mL of 0.5% paraformaldehyde (PFA), pH 7.4 in Dulbecco´s phosphate saline (DPBS). Optionally, make 20 mL of 4% PFA. Prepare 5 mL aliquots and freeze at -20 °C. On the day of dissection, defrost a 4% aliquot and add 35 mL of DPBS to obtain 40 mL of 0.5% PFA.

1. Isolation of E....

Wyniki

This protocol offers a straightforward method to isolate and immunostain muscle fibers from two different types of muscles (fast- and slow-twitch muscles, see Figure 1). Using the correct markers and / or probes, NMJ components can be detected and evaluated since a quantitative point of view to assess some of the morphological changes that can occur as consequence of illness progression or a specific drug treatment. In the present study, presynaptic and postsynaptic components of the NMJ wer.......

Dyskusje

In this article, we present a detailed protocol for the dissection of two rat skeletal muscles (one slow-twitch and the other fast-twitch), fiber muscle isolation and immunofluorescence detection of pre and postsynaptic markers to quantitatively assess NMJ changes as well as assembly/disassembly processes. This kind of protocol can be useful in rodent models^41,42 to evaluate NMJ during physiological or pathological processes as exemplified here in a model of.......

Materiały

Name	Company	Catalog Number	Comments
Stereomicroscope with cool light illumination	Nikon	SMZ-10A
Rocking platform	Biometra (WT 16)	042-500
Cover glasses (24 x 32 mm)	Deltalab	D102432
Premium (Plus) microscope slides	PORLAB	PC-201-16
Tweezers	F.S.T	11253-20
Uniband LA-4C Scissors 125mm	E.M.S	77910-26
Disponsable surgical blades #10	Sakira Medical	1567
Disponsable sterile syringe (1 ml)	Sakira Medical	1569
Super PAP pen	E.M.S	71310
100 μl or 200 μl pipette	Finnpipette	9400130
Confocal microscope	Zeiss	LSM 800 - AiryScan
NTac:SD-TgN(SOD1G93A)L26H rats	Taconic	2148-M
1X PBS (Dulbecco)	Gibco	21600-010
Paraformaldehyde	Sigma	158127
Triton X-100	Sigma	T8787
Glycine	Amresco	167
BSA	Bio Basic INC.	9048-46-8
Glycerol	Mallinckrodt	5092
Tris	Amresco	497
Purified anti-Neurofilament H (NF-H), Phosphorylated Antibody	BioLegend	801601	Previously Covance # SMI 31P
Purified anti-Neurofilament H (NF-H), Nonphosphorylated Antibody	BioLegend	801701	Previously Covance # SMI-32P
Alexa Fluor 488 goat anti-Mouse IgG (H+L)	Thermo Scientific	A11029
α-Bungarotoxin, biotin-XX conjugate	Invitrogen	B1196
Streptavidin, Alexa Fluor 555 conjugate	Invitrogen	S32355
Diaminophenylindole (DAPI)	Sigma	D8417