Name: The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2014-12-26T14:00:00.000+00:00
Duration: 12 min 18 s
Description: Learn to quantify neuromuscular junction structure using confocal fluorescence microscopy in mouse muscle. This method measures synapse size, fragmentation, and postsynaptic protein density for studying neuromuscular disorders.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the National Health and Medical Research Council [570930]. Imaging was carried out at the Bosch Institute Advanced Microscopy Facility. Former members of the lab, whose work is cited, are thanked for their contributions to developing these methods.

<ol><li>Schmidt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuregulin%2FErbB+regulate+neuromuscular+junction+development+by+phosphorylation+of+%CE%B1-dystrobrevin%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin.</a> J Cell Biol. 195, 1171-1184 (2011).</li>
<li>Amenta, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biglycan+is+an+extracellular+MuSK+binding+protein+important+for+synapse+stability%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Biglycan is an extracellular MuSK binding protein important for synapse stability.</a> J Neurosci. 32, 2324-2334 (2012).</li>
<li>Samuel, M. A., Valdez, G., Tapia, J. C., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrin+and+Synaptic+Laminin+Are+Required+to+Maintain+Adult+Neuromuscular+Junctions%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions.</a> PLOS ONE. 7, e46663(2012).</li>
<li>Valdez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attenuation+of+age-related+changes+in+mouse+neuromuscular+synapses+by+caloric+restriction+and+exercise%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+%28USA%29%22%5BJournal%5D)">Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise.</a> Proc Natl Acad Sci (USA). 107, 14863-14868 (2010).</li>
<li>Yampolsky, P., Pacifici, P. G., Witzemann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+muscle-driven+synaptic+remodeling+in+the+neuromuscular+junction+after+denervation%29%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Differential muscle-driven synaptic remodeling in the neuromuscular junction after denervation).</a> Eur J Neurosci. 31, 646-658 (2010).</li>
<li>Li, Y., Lee, Y., Thompson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+Aging+Mouse+Neuromuscular+Junctions+Are+Explained+by+Degeneration+and+Regeneration+of+Muscle+Fiber+Segments+at+the+Synapse%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Changes in Aging Mouse Neuromuscular Junctions Are Explained by Degeneration and Regeneration of Muscle Fiber Segments at the Synapse.</a> J Neurosci. 31, 14910-14919 (2011).</li>
<li>Zhu, H., Bhattacharyya, B. J., Lin, H., Gomez, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+IP3R1+receptors+amplify+physiological+and+pathological+synaptic+calcium+signals%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals.</a> J Neurosci. 31, 15269-15283 (2011).</li>
<li>Valdez, G., Tapia, J. C., Lichtman, J. W., Fox, M. A., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shared+resistance+to+aging+and+ALS+in+neuromuscular+junctions+of+specific+muscles%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Shared resistance to aging and ALS in neuromuscular junctions of specific muscles.</a> PLoS ONE. 7, e34640(2012).</li>
<li>Perez-Garcia, M. J., Burden, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+MuSK+Activity+Delays+Denervation+and+Improves+Motor+Function+in+ALS+Mice%5BTitle%5D)+AND+%22Cell+reports%22%5BJournal%5D)">Increasing MuSK Activity Delays Denervation and Improves Motor Function in ALS Mice.</a> Cell reports. 2, 1-6 (2012).</li>
<li>Klooster, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle-specific+kinase+myasthenia+gravis+IgG4+autoantibodies+cause+severe+neuromuscular+junction+dysfunction+in+mice%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice.</a> Brain. 135, 1081-1101 (2012).</li>
<li>Pratt, S. J., Shah, S. B., Ward, C. W., Inacio, M. P., Stains, J. P., Lovering, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+in+vivo+injury+on+the+neuromuscular+junction+in+healthy+and+dystrophic+muscles%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Effects of in vivo injury on the neuromuscular junction in healthy and dystrophic muscles.</a> J Physiol. 591, 559-570 (2013).</li>
<li>Landis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+call+for+transparent+reporting+to+optimize+the+predictive+value+of+preclinical+research%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A call for transparent reporting to optimize the predictive value of preclinical research.</a> Nature. 490, 187-191 (2012).</li>
<li>Gervásio, O. L., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+ratio+of+rapsyn+to+ACh+receptor+stabilizes+postsynaptic+receptors+at+the+mouse+neuromuscular+synapse%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Increased ratio of rapsyn to ACh receptor stabilizes postsynaptic receptors at the mouse neuromuscular synapse.</a> J Physiol. 562, 673-685 (2005).</li>
<li>Gervásio, O. L., Armson, P. F., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+increase+in+the+amount+of+rapsyn+per+acetylcholine+receptor+promotes+postsynaptic+receptor+packing+and+stability%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability.</a> Dev Biol. 305, 262-275 (2007).</li>
<li>Brockhausen, J., Cole, R. N., Gervásio, O. L., Ngo, S. T., Noakes, P. G., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+agrin+increases+postsynaptic+ACh+receptor+packing+by+elevating+rapsyn+protein+at+the+mouse+neuromuscular+synapse%5BTitle%5D)+AND+%22Dev+Neurobiol%22%5BJournal%5D)">Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse.</a> Dev Neurobiol. 68, 1153-1169 (2008).</li>
<li>Cole, R. N., Reddel, S. W., Gervásio, O. L., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-MuSK+patient+antibodies+disrupt+the+mouse+neuromuscular+junction%5BTitle%5D)+AND+%22Ann+Neurol%22%5BJournal%5D)">Anti-MuSK patient antibodies disrupt the mouse neuromuscular junction.</a> Ann Neurol. 63, 782-789 (2008).</li>
<li>Morsch, M., Reddel, S. W., Ghazanfari, N., Toyka, K. V., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+Specific+Kinase+autoantibodies+cause+synaptic+failure+through+progressive+wastage+of+postsynaptic+acetylcholine+receptors%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Muscle Specific Kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors.</a> Exp Neurol. 237, 237-286 (2012).</li>
<li>Cole, R. N., Ghazanfari, N., Ngo, S. T., Gervasio, O. L., Reddel, S. W., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient+autoantibodies+deplete+postsynaptic+Muscle+Specific+Kinase+leading+to+disassembly+of+the+ACh+receptor+scaffold+and+myasthenia+gravis+in+mice%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Patient autoantibodies deplete postsynaptic Muscle Specific Kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.</a> J Physiol. 588, 3217-3229 (2010).</li>
<li>Viegas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passive+and+active+immunization+models+of+MuSK-Ab+positive+myasthenia%3A+Electrophysiological+evidence+for+pre+and+postsynaptic+defects%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Passive and active immunization models of MuSK-Ab positive myasthenia: Electrophysiological evidence for pre and postsynaptic defects.</a> Exp Neurol. 234, 506-512 (2012).</li>
<li>Morsch, M., Reddel, S. W., Ghazanfari, N., Toyka, K. V., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyridostigmine+but+not+3%2C4-diaminopyridine+exacerbates+ACh+receptor+loss+and+myasthenia+induced+in+mice+by+Muscle+Specific+Kinase+autoantibody%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by Muscle Specific Kinase autoantibody.</a> J Physiol. 591, 2747-2762 (2013).</li>
<li>Ghazanfari, N., Morsch, M., Reddel, S. W., Liang, S. X., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+Specific+Kinase+autoantibodies+suppress+the+MuSK+pathway+and+ACh+receptor+retention+at+the+mouse+neuromuscular+junction%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Muscle Specific Kinase autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.</a> J Physiol. 592, 2881-2897 (2014).</li>
<li>Cheng, A., Morsch, M., Murata, Y., Ghazanfari, N., Reddel, S. W., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequence+of+age-associated+changes+to+the+mouse+neuromuscular+junction+and+the+protective+effects+of+voluntary+exercise%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Sequence of age-associated changes to the mouse neuromuscular junction and the protective effects of voluntary exercise.</a> PLoS One. 8, e67970(2013).</li>
<li>Schaefer, A. M., Sanes, J. R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+compensatory+subpopulation+of+motor+neurons+in+a+mouse+model+of+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis.</a> J Comp Neurol. 490, 209-219 (2005).</li>
<li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+bioscience+research+reporting%3A+the+ARRIVE+guidelines+for+reporting+animal+research%5BTitle%5D)+AND+%22PLos+Biol%22%5BJournal%5D)">Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.</a> PLos Biol. 8, e1000412(2010).</li>
<li>Shimizu, S. Routes of Administration. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aShimizu%2C+S+%22The+Laboratory+Mouse%22">The Laboratory Mouse</a>. Hedrich, H. J., Bullock, G. , Elsevier. (2004).</li>
<li>Chiasson, R. B. Laboratory anatomy of the white rat. , Brown. Dubuque, Iowa. (1988).</li>
<li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+Animal+Perfusion+Fixation+for+Rodents%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Whole Animal Perfusion Fixation for Rodents.</a> J. Vis. Exp. (65), e3564(2012).</li>
<li>Mitra, A. K., Stroud McCarthy, M. P., M, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+structure+of+the+nicotinic+acetylcholine+receptor+and+location+of+the+major+associated+43-kD+cytoskeletal+protein%2C+determined+at+22A+by+low+dose+electron+microscopy+and+x-ray+diffraction+to+12.5A%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22A by low dose electron microscopy and x-ray diffraction to 12.5A.</a> J Cell Biol. 109, 755-774 (1989).</li>
<li>Paas, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+microscopic+evidence+for+nucleation+and+growth+of+3D+acetylcholine+receptor+microcrystals+in+structured+lipid-detergent+matrices%5BTitle%5D)+AND+%22Proc.+Natl+Acad.+Sci.+%28USA%29%22%5BJournal%5D)">Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid-detergent matrices.</a> Proc. Natl Acad. Sci. (USA). 100, 11309-11314 (2003).</li>
<li>Samson, A. O., Scherf, T., Eisenstein, M., Chill, J. H., Anglister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanism+for+acetylhcoline+receptor+inhibition+by+%CE%B1-neurotoxins+and+species-specific+resistance+to+%CE%B1-bungarotoxin+revealed+by+NMR%29%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The mechanism for acetylhcoline receptor inhibition by α-neurotoxins and species-specific resistance to α-bungarotoxin revealed by NMR).</a> Neuron. 35, 319-332 (2002).</li>
<li>Ghazanfari, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+Specific+Kinase%3A+Organiser+of+synaptic+membrane+domains%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">Muscle Specific Kinase: Organiser of synaptic membrane domains.</a> Int J Biochem Cell Biol. 43, 295-298 (2011).</li>
<li>Ghazanfari, N., Morsch, M., Tse, N., Reddel, S. W., Phillips, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+the+%CE%B22-adrenoceptor+agonist%2C+albuterol%2C+in+a+mouse+model+of+anti-MuSK+myasthenia+gravis%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Effects of the β2-adrenoceptor agonist, albuterol, in a mouse model of anti-MuSK myasthenia gravis.</a> PLoS ONE. 9, e87840(2014).</li>
<li>Prakash, Y. S., Miller, S. M., Huang, M., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphology+of+diaphragm+neuromuscular+junctions+on+different+fibre+types%5BTitle%5D)+AND+%22J+Neurocytol%22%5BJournal%5D)">Morphology of diaphragm neuromuscular junctions on different fibre types.</a> J Neurocytol. 25, 88-100 (1996).</li>
<li>Salpeter, M. M., Harris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution+and+turnover+rate+of+acetylcholine+receptors+throughout+the+junction+folds+at+a+vertebrate+neuromuscular+junction%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.</a> J Cell Biol. 96, 1781-1785 (1983).</li>
<li>Soper, S. A., Nutter, H. L., Keller, R. A., Davis, L. M., Shera, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+photophysical+constants+of+several+fluorescent+dyes+pertaining+to+ultrasensitive+fluorescence+spectroscopy%5BTitle%5D)+AND+%22Photochem+Photobiol%22%5BJournal%5D)">The photophysical constants of several fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy.</a> Photochem Photobiol. 57, 972-977 (1993).</li>
<li>Panchuk-Voloshina, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alexa+dyes%2C+a+series+of+new+fluorescent+dyes+that+yield+exceptionally+bright%2C+photostable+conjugates%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.</a> J Histochem Cytochem. 47, 1179-1188 (1999).</li>
</ol>

The authors declare they have no competing financial interests.

The protocols described here have enabled us to reliably measure and quantify changes in the properties of the NMJ across a range of conditions, including normal aging and disease states. The methods described for en face NMJ images will allow researchers to compare the area of pre- and postsynaptic specializations and the area of synaptic overlap/alignment. To compare the relative intensity of pre- and postsynaptic proteins the second protocol, which uses transverse optical sections, is preferred. The third protocol spe...

The neuromuscular junction (NMJ) is the critical relay synapse that mediates communication between the nervous system and skeletal muscle. It is required for all voluntary movement. Fluorescence microscopy has long been used to study the effects of transgenes on the mouse NMJ 1-3 or to compare the effects of age, diet, exercise and disease upon rodent NMJs 4-11. Such studies have taught us much about the physiology and pathophysiology of the NMJ, but the diverse parameters reported (e.g., AChR area, endplate area, perimeter length, fragmentation indices) often make it difficult to compare the findings of these studies. There is an increasing expectation for pre-clinical researchers to be able to demonstrate reproducibility, particularly in studies with rodent models of disease 12. The protocols described here were refined through a series of studies that investigated developmental, physiological and pathophysiological changes to the NMJ. Such studies require measurement of the area of synaptic specializations at the mouse motor endplate and the relative density of packing of synaptic proteins within postsynaptic specializations13-15.
The utility of these methods is illustrated by recent studies in a mouse model of anti-MuSK myasthenia gravis. Daily injections of IgG from anti-MuSK-positive myasthenia gravis patients into adult mice caused them to become weak within 2 weeks 16. Confocal maximum-projection images of muscle sections that were double-labeled for synaptophysin (in nerve-terminals) and postsynaptic AChRs revealed a progressive decline in the area of AChR staining as the primary change. Importantly the rate of decline was sufficient to explain comparable declines in the amplitude of synaptic potentials, failure of synaptic transmission and muscle weakness 17,18. Qualitatively similar findings were reported by other research groups 10,19. The same NMJ measurement methods have since been used to assess the impact of three drugs for treating anti-MuSK myasthenia gravis in this mouse model 20,21.
Sedentary aging can lead to loss of neuromuscular connections. The protocols described here have revealed an age-associated decline in the area of nerve terminal synaptophysin at motor endplates as mice progress into old age. The same methods revealed that voluntary exercise could largely prevent the reduction in presynaptic nerve terminal area 22, consistent with previous work by other groups 4. Loss of neuromuscular connections also occurs in the SOD1G93A mouse model of amyotrophic lateral sclerosis 9,23.
The studies mentioned above demonstrate that a variety of health conditions may lead to reductions in the area of either pre- or post-synaptic specializations at the NMJ. This may result in impaired synaptic function or may herald complete loss of the neuromuscular connection. Three protocols are described that allow quantitation of the area and density of synaptic specializations. The purpose of the first protocol is to provide a practical and reproducible measure of the areas of pre- and post-synaptic specializations and their alignment at mammalian NMJs, using fluorescence microscopy. Two-dimensional maximum projection confocal images and image analysis with NIH ImageJ is used to detect changes in the area of synaptophysin staining (synaptic vesicles), postsynaptic AChRs and synaptic overlap area. Confocal imaging parameters (gain and offset level) are optimized for each NMJ so as to maximize the visual information used to discern the area of synaptic specialization. Neuromuscular failure can also result from changes in the density of postsynaptic AChR and/or other synaptic proteins. The second protocol can be applied to detect changes in the relative density of postsynaptic proteins such as MuSK, rapsyn, dystroglycan, phosphorylated Src kinase and phosphorylated AChR 18,21.
In myasthenia gravis, a reduced density of AChR within the postsynaptic membrane is the immediate cause of synaptic failure and muscle weakness. The third protocol describes a Fluorescence Resonance Energy Transfer (FRET) method to assess changes in the proximity of adjacent AChRs within postsynaptic membranes 14,15. This method detects energy transfer between neighboring AChRs labeled with fluorescent-&#945;-bungarotoxin (BGT). FRET occurs only when the fluorescent donor and acceptor probes are less than 10 nm apart. This can reveal (submicroscopic) changes in the tightness of AChR packing that may directly relate to the amplitude of synaptic potentials.
These three protocols, refined over the past decade, provide complementary measures of NMJ integrity in a consistent and reproducible way. Use of standardized protocols and parameters should facilitate comparison of the effects of genes and environmental interventions upon the mammalian NMJ.

<table><tbody><tr><td>Scanning confocal microscope</td><td>Leica</td><td>DM 2000 with&amp;nbsp; TCS SP2 system</td><td>Most scanning confocal microscopes should be suitable.&amp;nbsp;</td></tr><tr><td></td><td>Zeiss</td><td>LSM 510 Meta&amp;nbsp;</td><td></td></tr><tr><td></td><td>Leica</td><td>SPE-II</td><td></td></tr><tr><td>Alexa555-a-bungarotoxin (red-BGT)</td><td>Life technologies</td><td>B35451</td><td>Used for labelling AChRs</td></tr><tr><td>Alexa647-&amp;alpha;-bungarotoxin (far-red-BGT)</td><td>Life technologies</td><td>B35450</td><td>Far red fluorescence: barely visible through the eyepiece&amp;nbsp;</td></tr><tr><td>rabbit anti-synaptophysin</td><td>Life technologies</td><td>18-0130</td><td>Different batches of primary antibody differ in effective working dilution</td></tr><tr><td>FITC-anti-rapsyn mab1234</td><td>Milipore</td><td>FCMAB134F</td><td>Monoclonal antibody conjugated to FITC</td></tr><tr><td>FITC-donkey anti-rabbit IgG</td><td>Jackson</td><td>711-095-152</td><td>Polyclonal secondary antibodies can vary in quality according to source and batch</td></tr><tr><td>Optimal Cutting Temperature compound (O.T.C.)</td><td>ProSciTech</td><td>IA018</td><td>Cryostat embedding matrix for freezing&amp;nbsp; muscles</td></tr><tr><td>DABCO</td><td>Sigma</td><td>10981</td><td>Mounting medium that slows photobleaching of fluorophores</td></tr></tbody></table>

the neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy

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.

Measurement of Synaptic Area at the NMJ
Any estimate of area relies upon the drawing of a boundary to define the extent of synaptic specializations. In healthy young adult muscles NMJ images should display well-defined boundaries for both AChR and synaptophysin staining (Figure 2A and B). Fluorescence intensity for both AChR and synaptophysin rises sharply at the boundary between the peri-synaptic and synaptic portion of the motor endplate (<s...

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. ...

the-neuromuscular-junction-measuring-synapse-size-fragmentation

Research

JoVE Journal

Neuroscience

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Automated Quantification of Synaptic Fluorescence in C. elegans

The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Marinello, M., et al. Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy. J. Vis. Exp. (2021)This video demonstrates the preparation, immunostaining, and imaging of neuromuscular junctions in rat muscle tissue using confocal and super-resolution microscopy to visualize and analyze presynaptic and postsynaptic structures.

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

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.

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

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Learn to quantify neuromuscular junction structure using confocal fluorescence microscopy in mouse muscle. This method measures synapse size, fragmentation, and postsynaptic protein density for studying neuromuscular disorders.

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy (Video) | JoVE Journal

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

Explore More Videos