We would like to thank Manuela Budak, Ljiljana Marinkovi and Kristina Wullimann for excellent technical assistance. We thank W. Macklin for providing Plp-GFP mice. T.M. is supported by the Institute of Advanced Studies (Technische Universit&auml;t M&uuml;nchen), by the Deutsche Forschungsgemeinschaft (DFG; Sonderforschungsbereich SFB 596), by the Alexander-von-Humboldt-Foundation and by the national funding agency ("Bundesministerium f&uuml;r Bildung und Forschung") in the frame of ERA-Net NEURON "iPSoALS" and "2-photon imaging". T.M. and M.B. are supported by the Center for Integrated Protein Science (Munich). P.M. was supported by the Graduate School of Technische Universit&auml;t M&uuml;nchen (TUM-GS).

1. Triangularis Sterni Explant (Figure 1)
Timing: 15 min.
<ol>
 <li>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 &deg;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.</li>
 <li>Lethally anesthetize the mouse by isoflurane overdose (or any other approved method of sacrifice). Spray the mouse with 70% ethanol to prevent fur contamination of the explant. For SC bleaching, we used Plp-GFP mice, which can be crossed to Thy1-OFP3 or Thy1-Membow13 for simultaneous visualization of the axon.</li>
 <li>Using the pair of large scissors, make a midline incision of the skin over the sternum and two incisions parallel to the lower edge of the rib cage.</li>
 <li>Using the pair of large scissors, remove the skin, open the abdominal wall, and make incisions parallel to the rib cage all the way back to the vertebral column. Then dissect off the pectoral muscles by making incisions close to the muscle insertion at the sternum.</li>
 <li>Cut the diaphragm open just below the xiphoid cartilage and dissect off the diaphragm along its costal insertions. </li>
 <li>Holding the rib cage by the xiphoid process with a set of forceps, start cutting the ribs off the vertebral column, as close as possible to their insertions. The left and right cuts should converge above the heart towards the manubrium sterni. Try to avoid cutting major blood vessels (especially the subclavian veins). Better visibility is achieved by gently lifting the preparation while holding onto the xiphoid process with forceps.</li>
 <li>Make a transverse cut just below the manubrium sterni and transfer the explant into the 10-cm dish with cooled 5% CO2/ 95% O2-bubbled Ringer's solution. Place the 10-cm dish on the metal plate covering the 15-cm tissue culture dish filled with ice. All further steps should be done under the dissection microscope.</li>
 <li>Using small spring scissors remove the remnants of thymus, pleura, diaphragm (inside) and pectoral muscles (outside; Figure 1B-D).</li>
 <li>To fit the explant to the 3.5-cm dish, dissect off all the ribs that do not insert to the sternum. This is best done using small spring scissors and cutting the tissue in between ribs (Figure 1E).</li>
 <li>Pin the triangularis sterni nerve-muscle explant into the Sylgard coated 3.5-cm tissue culture dish using minutien pins (shortened to &lt; 4 mm; Figure 1G). Two pins should go through cartilaginous (white) parts of the sternum. Insert at least two pins through the ribs both on the left and right side of the explant aiming for the softer cartilaginous parts and avoiding the ribs under or close to the triangularis muscle. The more pins are used, the better (we typically use 8-10). As you pin down the preparation, make sure that the ribs are somewhat spread, fixing the muscle into a slightly stretched position.</li>
 <li>Optional: Visualize synaptic sites containing acetylcholine receptors by adding bungarotoxin coupled to Alexa dyes (concentration of 0.8 &mu;g/ml in 5% CO2/ 95% O2-bubbled Ringer's solution). Incubate pinned explant in Sylgard coated dish for 15-20 min at room temperature then wash several times with 5% CO2/ 95% O2-bubbled Ringer's solution.</li>
</ol>
2. Bleaching SCs and Optional Time-lapse Microscopy (Figure 2)
Timing: 30 - 45 min + optional 1 - 5 hr for time lapse.
<ol>
 <li>Transfer 3.5-cm Sylgard-coated dish to a confocal microscope equipped with an argon laser (488 nm wavelength) and water immersion objectives (4x, 0.13 NA; 20x, 0.5 NA and 100x, 1.0 NA or 60x, 0.9 NA).</li>
 <li>Optional for time-lapse microscopy: Insert 3.5-cm Sylgard-coated dish into heating ring, install superfusion system and temperature probe (Figure 2A). Make sure that none of them touch the explant, the rim of the dish or the Sylgard coating. Heat the sample to 33-35 &deg;C. For SC bleaching without time-lapse, room temperature is better, as cells show less dynamics between bleaching steps.</li>
 <li>With a 4x air objective observe innervation pattern of triangularis sterni muscle and find triangularis sterni endplate band. Switch to 20x dipping-cone objective and start looking for superficial regions within the endplate band (areas overlying ribs are good candidates). Change objective to 100x or 60x dipping-cone objective to check on individual NMJs. Ideal is an NMJ that is covered by several SCs and is located very superficially (Figure 2B). If you perform time-lapse imaging after bleaching, select up to 3 NMJs that are relatively close together to increase yield.</li>
 <li>Acquire a confocal image of the NMJ before photo-bleaching individual SCs (Figure 2B). (For full resolution imaging, e.g. on an Olympus FV1000 microscope, use a 100x, 1.0 NA objective and 2.5 zoom, which results in Nyquist-limited sampling of ~0.1 &mu;m per pixel.)</li>
 <li>"Park" the 488 nm laser beam centrally on the nucleus of a SC using maximal power for 5 seconds (alternatively an efficient scan pattern in a small region of interest can be used, such as the "tornado scan" function on an Olympus microscope; as we use a confocal microscope, the laser is focussed into the image-conjugate planes and hence, at 488 nm with an objective of 1.0 N.A., &lt; 0.5 &mu;m in diameter). Re-focus on the cell and judge bleaching result. If necessary repeat bleaching step again until GFP levels are reduced to near-background levels. Acquire an image after bleaching (Figure 2C). It is critical to make sure that the bleached region is outside of any overlap between two cells - if there is overlap, bleaching in a second cell will be obvious.</li>
 <li>Repeat step above until all but one SC are bleached (Figure 2D-E). The last "unbleached" SC is suitable for confocal time-lapse microscopy. Reconstruct single cells by subtraction of images, e.g. using Photoshop software, to delineate single SC morphology and territory (Figure 2F). Be aware that subtracting two mages adds noise (e.g. by importing them in consecutive "layers" in Photoshop and then setting the top channel to "difference" in the drop down menu at the top of the channels tab). This can be circumvented by using the "despeckle" function on the channels before subtraction and cropping away any background that obviously does not originate from the bleached cell. To optimize contrast, it can be necessary to adjust the brightness of the two channels in the "levels" window. The resulting image of the bleached cell is the superimposed on the original image of a synapse, pseudo-colored in a unique hue and made transparent to color the territory of the bleached cell in the original image (from doing this for all bleached and the last, unbleached cells the image shown in Figure 2F results).</li>
 <li>Optional: Start confocal time-lapse microscopy after bleaching all but one SCs. Take an image at fixed intervals (for example every 5 to 10 min). Use as little laser power as possible. Up to 3 NMJs can be imaged at the same session.</li>
 <li>Make a map of the time-lapsed NMJs by taking an image at 100x without zoom, then images of the region using 20x and 4x objectives (Figure 2G-I). This "map" is needed to find NMJs following immunohistochemistry of a fixed muscle.</li>
 <li>For image processing, convert individual images into maximum intensity projections and save in tiff format. Combine all z-projected time-points into a stack and align in xy, e.g. using the "stackreg" plugin10 in Fiji (a package based on the open source software ImageJ; National Institutes of Health).</li>
</ol>
3. Fixation and Immunohistochemistry (Figure 3)
Timing: Overnight.
<ol>
 <li>Transfer the explant into a 50-ml reaction tube containing at least 15 ml 4% PFA by carefully removing the pins. Post-fix the anterior thoracic wall containing the triangularis sterni muscle for 1 hr on ice. Rinse fixed tissue with 0.1 M glycine in PBS (to quench residual fixative and hence reduce background) for at least 10 min. Samples can be stored at this point and processed later.</li>
 <li>Transfer fixed explant to a 10-cm Sylgard coated tissue culture dish filled with 0.01 M PBS. Cut out a trapezoid shape with one side parallel to the sternum and the other through the bone-to-cartilage transitions - this contains the triangularis muscle on its surface. Remove lower ribs with a horizontal (i.e. trans-sternal) cut so that the caudal end of the triangularis muscle can be freely accessed (Figure 3A-D).</li>
 <li>Insert a hypodermic needle as a pin into the upper part of the trapezoid (Figure 3E). Use a hypodermic needle attached to a 1 ml syringe and forceps to remove the triangularis muscle on the surface by gently holding onto a caudal corner of the muscle and using the needle as a micro-scalpel. Make sure that cuts are made parallel to the plane of the thoracic wall. Once the caudal part of the muscle is released, insert another hypodermic needle as a pin below the muscle through the thoracic wall - this immobilizes the preparation and allows exerting a gentle pull on the muscle using forceps (Figure 3F). As you are cutting connective tissue insertions, "peel" away the muscle from the thorax. Cut last caudal attachment with spring scissors. Remove fat and blood vessel remnants using forceps. Be careful not to rip away the nerves. Note: Use separate sets of tools and dishes for working with live and fixed tissue.</li>
 <li>Start immunohistochemistry (using a standard protocol) by transferring tissue samples into blocking solution for 1 hr on a shaker at room temperature. Smallest possible volume for staining is 200 &mu;l using a 96-well plate.</li>
 <li>Apply primary antibody diluted in blocking solution, 200 &mu;l per well, overnight at 4 &deg;C on a shaker.</li>
 <li>Wash in 0.01 M PBS three times for 10 min.</li>
 <li>Apply suitable secondary antibody for 1-2 hr at room temperature diluted in blocking solution.</li>
 <li>Wash in 0.01 M PBS three times for 10 min.</li>
 <li>Mount in anti-fading medium (e.g. Vectashield) and coverslip.</li>
 <li>Place slide on magnetic metal plate, place magnet on top of the sample to flatten for a few hours or overnight at 4 &deg;C. Seal with nail polish.</li>
</ol>

<ol>
	<li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Mallon, B. S., Shick, H. E., Kidd, G. J., Macklin, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolipid+promoter+activity+distinguishes+two+populations+of+NG2-positive+cells+throughout+neonatal+cortical+development.">Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development.</a> J. Neurosci. 22, 876-885 (2002).</li><li>Brill, M. S., Lichtman, J. W., Thompson, W., Zuo, Y., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+constraints+dictate+glial+territories+at+murine+neuromuscular+junctions.">Spatial constraints dictate glial territories at murine neuromuscular junctions.</a> The Journal of cell biology. 195, 293-305 (2011).</li><li>Livet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Thompson, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+photoswitchable+GFP+in+Schwann+cells.">Transgenic mice with photoswitchable GFP in Schwann cells.</a> Society for Neuroscience. Program 241, (2008).</li><li>Williams, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+development+of+outer+retinal+synapses+in+the+absence+of+glial+contact.">In vivo development of outer retinal synapses in the absence of glial contact.</a> J. Neurosci. 30, 11951-11961 (2010).</li><li>Kerschensteiner, M., Reuter, M. S., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+motor+axon+dynamics+in+murine+triangularis+sterni+explants.">Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants.</a> Nat. Protoc. 3, 1645-1653 (2008).</li><li>McArdle, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+the+triangularis+sterni+muscle+of+the+mouse+for+investigations+of+synaptic+phenomena.">Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena.</a> J. Neurosci. Methods. 4, 109-115 (1981).</li><li>Marinkovi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+deficits+and+degeneration+can+evolve+independently+in+mouse+models+of+amyotrophic+lateral+sclerosis.">Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 4296-4301 (2012).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7, 27-41 (1998).</li><li>Brill, M. S., Lichtman, J. W., Thompson, W., Zuo, Y., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+constraints+dictate+glial+territories+at+murine+neuromuscular+junctions.">Spatial constraints dictate glial territories at murine neuromuscular junctions.</a> The Journal of cell biology. 195, 293-305 (2011).</li><li>Zuo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> J. Neurosci. 24, 10999-11009 (2004).</li></ol>

No conflicts of interest declared.

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...

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

sequential photo-bleaching to delineate single schwann cells at the neuromuscular junction

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 preparation7,8 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.

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...

Watch this Scientific Journal Video about Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction at JoVE.com

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

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.

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 ...

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JoVE Journal

Neuroscience

10.6K vistas.  Technische Universität München. Visualización de las células individuales en los tejidos densamente empaquetadas, tales como terminales de las células de Schwann (SC) en las uniones neuromusculares (NMJs), es un reto. "Sequential foto-blanqueamiento" permite delimitar SCs individuales terminal, por ejemplo en el explante de músculo triangularis esternón, una conveniente preparación nervio-músculo, donde secuencial de blanqueamiento puede ser combinado con las imágenes de lapso de tiemp...

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

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...