Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Summary

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Abstract

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP¹ and Plp-GFP mice², respectively.

Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact³.

Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings³. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins⁴. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative⁵. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species⁶.

In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation^7,8 has proven useful for studies of NMJ development, physiology and pathology⁹. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Protocol

1. Triangularis Sterni Explant (Figure 1)

Timing: 15 min.

1. Prepare surgical instruments: 2 pairs of forceps, 1 pair of scissors, 1 pair of spring scissors, 1 tissue culture dish (10-cm). Pre-bubbled (minimum for 15 min) cooled (4 °C) Ringer's solution with 5% CO₂/ 95% O₂. Fill 15-cm tissue culture dish with ice and cover it with metal plate. Prepare dissection microscope and place the 15-cm dish with ice under the dissection scope in order to cool the explant during dissection.
2. Lethally anesthetize the mouse by isoflurane overdose (or any other approved method of sacrifice). Sp....

Results

An example of a triangularis sterni explant ready for imaging dish is shown in Figure 1G. This explant is particularly suitable for imaging NMJs ex vivo as the triangularis muscle consists only of a few layers of muscle fibers. This allows obtaining high resolution images from explants derived from transgenic mouse lines that highlight either SCs (Plp-GFP²) and/or axons (Thy1-XFP¹). Critical factors for high quality imaging include: (i) avoiding touching t.......

Discussion

The SC bleaching method presented here is simple, fast and versatile: (i) It enables revealing single SC morphology and dynamics by confocal microscopy based on a single transgene - which is a significant advantage when combining the approach e.g. with disease models that require additional alleles. (ii) The method is rapid, as from dissection to fixation it can be performed in half a day. (iii) The number of applications is broad, as it can be combined with other methods, such as cell ablations, imaging organel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70% (vol/vol) ethanol solution	Roth	T913.1	in spray bottle
isoflurane	Forene, Abbott		any other approved anaesthetic can be used for lethal anaesthesia
transgenic mice (Plp-GFP)			to be obtained from cited sources
dissection microscope with cold-light illumination	Olympus SZ51		equipped with Schott KL 1500 LCD
forceps #2	Fine Science Tools	11233-20
forceps #5	Fine Science Tools	11295-00
scissors, large medical	Fine Science Tools	14108-09
scissors, small angled spring	Fine Science Tools	15033-09
in-line heater	Warner Instruments	SF-28
heating ring for 3.5-cm dishes	Warner Instruments	64-0110 DH-35
two channel temperature control system	Warner Instruments	TC-344B
superfusion pump system			custom-built
15-cm tissue culture dish	Sarstedt	83.1803.003	filled with ice and covered by metal plate
10-cm tissue culture dish	Sarstedt	83.1802.003	with oxygenated Ringer's solution
3.5-cm tissue culture dish	Sarstedt	83.1800.003	filled with Sylgard polymer
Sylgard polymer	Dow Corning	Sylgard 184 Silicone Elastomer Kit
confocal microscope	Olympus	FV1000	with argon laser
50-ml reaction tube	Sarstedt	62.547.254 PP
4% PFA in PBS	Sigma	P6148
cannula 0.5 mm x 25 mm	Neolab	E-1510
syringe 1 ml	Neolab	E-1496
synaptophysin antibody	Invitrogen	18-0130	rabbit
secondary antibody	Invitrogen	A-11012	Alexa 594
24-well plate	Sarstedt	83.1836
0.01 M PBS	Sigma	P4417
0.1 M glycine in PBS	Roth	3790.1
coverslips	Neolab	E-4132
slides	Neolab	E-4121
Vectashield	Vector labs	H-1000
minutien pins ×10	Fine Science Tools	26002-20	shortened to < 4 mm
α-Bungarotoxin coupled to Alexa 594	Invitrogen (Molecular Probes)	B-13423
Blocking Solution
0.01 M PBS
0.2% Triton-X100	Roth	3051.3
10% Normal Goat Serum	Abcam	ab7481
1% bovine serum albumin	Sigma	A7030
Ringer bubbled with 95% O2 /5% CO2
125 mM NaCl	Sigma	S7653
2.5 mM KCl	Sigma	P9333
1.25 mM NaH2PO4	Sigma	S8282
26 mM NaHCO3	Sigma	S6297
2 mM CaCl2	Sigma	21114
1 mM MgCl2	Sigma	63020
20 mM glucose	Sigma	G7528
Table 1. Specific reagents and equipments.