The authors would like to thank Dr. Martin Brandh&#248;j Skov from NMD Pharma for his valuable advice on rocuronium dosing and Arash Karimi from the Biomedical Engineering Department of Stony Brook University for his assistance in calculations. This study was supported in part by funding from NIH to WDA (R01AG067758 and R01AG078129).

All protocols were approved and performed in accordance with the regulations set forth by the Institutional Animal Care and Use Committee at the University of Missouri.
1. Animal preparation and anesthesia administration
<ol>
	<li>Put on appropriate personal protection equipment.</li>
	<li>Prior to the procedure, measure the rat's weight to determine the appropriate dose for weight-based medications and ventilator settings.</li>
	<li>Induce anesthesia with 3%-5% inhaled isoflurane. Once an adequate level of anesthesia is established, position the rat in a prone position and maintain anesthesia with 1%-3% inhaled isoflurane.</li>
	<li>Verify the adequacy of the depth of anesthesia by gently pressing the hindlimb footpad with forceps to observe the absence of a withdrawal response.</li>
	<li>Maintain body temperature at 37 °C.</li>
	<li>Apply veterinarian-approved petroleum-based ointment to the eyes to prevent dryness. Monitor the depth of anesthesia by observing the respiratory rate and assessing for withdrawal responses when applying pressure to the footpad using forceps.</li>
	<li>Shave the hindlimb to be assessed using clippers. After adequate hair removal, using adhesive tape, position the limb to be studied with the ankle fixed at approximately 90° dorsiflexion, the knee in extension, and the hip in abduction.</li>
	<li>Continuously monitor the respirations and heart rate of the rat during the entire experiment. 
	NOTE: An increase in heart rate is commonly used as an indicator of inadequate anesthetic depth.</li>
	<li>Administer isoflurane to euthanize the rat, with a dose of 5% or greater, until breathing has ceased for at least 3 min. Confirm the euthanasia by decapitation.</li>
</ol>
2. Electrode placement and setup
NOTE: NMJ transmission of the sciatic nerve and gastrocnemius muscle are assessed using the electromyography (EMG) system. Refer to the Table of Materials.
<ol>
	<li>Insert a pair of insulated 28 G monopolar needles for stimulating the sciatic nerve with the cathode into the region of the proximal hind limb, while the anode is more proximally within the subcutaneous tissue overlaying the sacrum.</li>
	<li>Ensure the stimulating electrodes are not placed in immediate proximity to the sciatic nerve or excessively deep to avoid direct harm to the sciatic nerve or other adjacent structures.</li>
	<li>Place a disposable ground electrode on the contralateral hindlimb or tail.</li>
	<li>Carefully place a 27-gauge, 25 mm needle electrode specifically designed for single fiber EMG, featuring a recording surface made of platinum-iridium material into the right gastrocnemius muscle. Ensure that the SFEMG electrodes are autoclaved prior to each use to maintain sterility.</li>
	<li>Insert the SFEMG needle electrode in parallel with the gastrocnemius muscle fibers to capture single-fiber action potentials (SFAPs).</li>
	<li>Avoid muscle damage while inserting and maneuvering the SFEMG needle within the muscle.</li>
</ol>
3. Stimulated single-fiber electromyography (SFEMG) procedure
<ol>
	<li>Apply constant current stimulation to the right sciatic nerve at 10 Hz frequency using an intensity range of 0.3-10 mA and a pulse duration of 0.1 ms.</li>
	<li>Configure the filter settings within a low frequency filter of 1 kHz and high frequency filter of 10 kHz. Adjust the Gain from 200 µV to 1000 µV per division to facilitate the visualization of potentials. Set the sweep speed at 500 µs per division.</li>
	<li>Adjust the stimulus intensity to trigger and isolate SFAPs for recording and subsequent analysis.
	<ol>
		<li>To identify and analyze a response as an SFAP, ensure that the specific criteria mentioned below are met: Ensure the rise time from the baseline peak to the negative phase is less than 500 µs, the minimum amplitude (baseline peak to negative) is at least 200 µV, and the response consistently exhibit an all-or-none behavior (stable size and shape between responses). 
		NOTE: It is essential that the recurring responses exhibit consistent upward phases without any notches or inflection points. Note that the last criterion is crucial for distinguishing a quality signal from the summation of multiple signals.</li>
	</ol>
	</li>
	<li>Calculate Jitter, or variability of SFAP latency (time between stimulation and rising phase of negative peak of SFAP) between consecutive discharges, following at least 50 stimulations (50-100 stimulations), and assess and quantify blocking. 
	NOTE: Blocking can be assessed as the presence or absence at each synapse or as the percentage of stimulations that fail to trigger single fiber action potential generation. Jitter is calculated using the following equation7 (Jitter is typically automatically calculated by clinic electromyography systems): 
	<img alt="MCD or Jitter formula, measuring network delay variation, mathematical equation for data analysis." src="/files/ftp_upload/66452/66452eq01.jpg" style="margin: auto;" data-altupdatedat="1747911167000" data-alt="Equation 1"> 
	MCD = Mean value of Consecutive Difference 
	IPI = Interpotential Interval</li>
	<li>Repeat the process for additional SFAP responses. On average, assess 10 synapses from each animal to calculate jitter and subsequently determine the average values per animal.</li>
	<li>Exclude SFAPs with jitter of less than 4 µs from the analyses to prevent the inclusion of potentials that might have been evoked by direct muscle stimulation11.</li>
	<li>When recording potentials displaying intermittent blocking, increase the stimulus intensity to verify that SFAP failure is not attributable to submaximal stimulation.</li>
</ol>

SFEMG in Rodents: Assessing NMJ Function in Health and Disease

<ol>
	<li>Stalberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+velocity+in+human+muscle+fibers+in+situ.">Propagation velocity in human muscle fibers in situ.</a> Acta Physiol Scand Suppl. 287, 1-112 (1966).</li><li>St&#229;lberg, E., Trontelj, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+normal+and+abnormal+neuromuscular+transmission+with+single+fibre+electromyography.">The study of normal and abnormal neuromuscular transmission with single fibre electromyography.</a> J Neurosci Methods. 74 (2), 145-154 (1997).</li><li>Sanders, D. B., 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=Guidelines+for+single+fiber+EMG.">Guidelines for single fiber EMG.</a> Clin Neurophysiol. 130 (8), 1417-1439 (2019).</li><li>Chugh, D., 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=Neuromuscular+junction+transmission+failure+is+a+late+phenotype+in+aging+mice.">Neuromuscular junction transmission failure is a late phenotype in aging mice.</a> Neurobiol Aging. 86, 182-190 (2020).</li><li>Padilla, C. 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=Profiling+age-related+muscle+weakness+and+wasting:+Neuromuscular+junction+transmission+as+a+driver+of+age-related+physical+decline.">Profiling age-related muscle weakness and wasting: Neuromuscular junction transmission as a driver of age-related physical decline.</a> GeroScience. 43 (3), 1265-1281 (2021).</li><li>Selvan, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-fiber+EMG:+A+review.">Single-fiber EMG: A review.</a> Ann Indian Acad Neurol. 14 (1), 64-67 (2011).</li><li>Sanders, D. B., Kouyoumdjian, J. A., St&#229;lberg, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+fiber+electromyography+and+measuring+jitter+with+concentric+needle+electrodes.">Single fiber electromyography and measuring jitter with concentric needle electrodes.</a> Muscle Nerve. 66 (2), 118-130 (2022).</li><li>Juel, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+fiber+electromyography.">Single fiber electromyography.</a> Handb Clin Neurol. 160, 303-310 (2019).</li><li>Chung, T., 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=Evidence+for+dying-back+axonal+degeneration+in+age-associated+skeletal+muscle+decline.">Evidence for dying-back axonal degeneration in age-associated skeletal muscle decline.</a> Muscle Nerve. 55 (6), 894-901 (2017).</li><li>Iyer, C. C., 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=Follistatin-induced+muscle+hypertrophy+in+aged+mice+improves+neuromuscular+junction+innervation+and+function.">Follistatin-induced muscle hypertrophy in aged mice improves neuromuscular junction innervation and function.</a> Neurobiol Aging. 104, 32-41 (2021).</li><li>Meekins, G. D., Carter, G. T., Emery, M. J., Weiss, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+degeneration+in+the+trembler-j+mouse+demonstrated+by+stimulated+single-fiber+electromyography.">Axonal degeneration in the trembler-j mouse demonstrated by stimulated single-fiber electromyography.</a> Muscle Nerve. 36 (1), 81-86 (2007).</li><li>Gooch, C. L., Mosier, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+single+fiber+electromyography+in+the+mouse:+Techniques+and+normative+data.">Stimulated single fiber electromyography in the mouse: Techniques and normative data.</a> Muscle Nerve. 24 (7), 941-945 (2001).</li><li>Chung, T., Tian, Y., Walston, J., Hoke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+single-fiber+jitter+level+is+associated+with+reduction+in+motor+function+with+aging.">Increased single-fiber jitter level is associated with reduction in motor function with aging.</a> Am J Phys Med Rehabil. 97 (8), 551-556 (2018).</li><li>Sokolow, S., 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=Impaired+neuromuscular+transmission+and+skeletal+muscle+fiber+necrosis+in+mice+lacking+na/ca+exchanger+3.">Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking na/ca exchanger 3.</a> J Clin Investig. 113 (2), 265-273 (2004).</li><li>A&#241;or, S., 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=Evaluation+of+jitter+by+stimulated+single-fiber+electromyography+in+normal+dogs.">Evaluation of jitter by stimulated single-fiber electromyography in normal dogs.</a> J Vet Intern Med. 17 (4), 545-550 (2003).</li><li>Mizrachi, T., 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=NMO-IgG+and+AQP4+peptide+can+induce+aggravation+of+eamg+and+immune-mediated+muscle+weakness.">NMO-IgG and AQP4 peptide can induce aggravation of eamg and immune-mediated muscle weakness.</a> J Immunol Res. 2018, 5389282 (2018).</li><li>Lin, T. S., Cheng, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+single-fiber+electromyography+in+the+rat.">Stimulated single-fiber electromyography in the rat.</a> Muscle Nerve. 21 (4), 482-489 (1998).</li><li>Finley, D. B., Wang, X., Graff, J. E., Herr, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+fiber+electromyographic+jitter+and+detection+of+acute+changes+in+neuromuscular+function+in+young+and+adult+rats.">Single fiber electromyographic jitter and detection of acute changes in neuromuscular function in young and adult rats.</a> J Pharmacol Toxicol Methods. 59 (2), 108-119 (2009).</li><li>Khan, Z. H., Hajipour, A., Zebardast, J., Alomairi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+relaxants+in+anesthesia+practice:+A+narrative+review.">Muscle relaxants in anesthesia practice: A narrative review.</a> Arch Anesthesiol Crit Care. 4 (4), 547-552 (2018).</li><li>Padua, L., Caliandro, P., St&#229;lberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+the+measurement+of+motor+conduction+velocity+using+a+single+fibre+emg+electrode.">A novel approach to the measurement of motor conduction velocity using a single fibre emg electrode.</a> Clin Neurophysiol. 118 (9), 1985-1990 (2007).</li><li>Khoo, A., Hay Mar, H., Borghi, M. V., Catania, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiologic+evaluation+of+myasthenia+gravis+and+its+mimics:+Real-world+experience+with+single-fiber+electromyography.">Electrophysiologic evaluation of myasthenia gravis and its mimics: Real-world experience with single-fiber electromyography.</a> Hosp Pract. 50 (5), 373-378 (2022).</li><li>Nannan, 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=A+role+of+lamin+a/c+in+preventing+neuromuscular+junction+decline+in+mice.">A role of lamin a/c in preventing neuromuscular junction decline in mice.</a> J Neurosci. 40 (38), 7203 (2020).</li><li>Alley, D. E., 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=Grip+strength+cutpoints+for+the+identification+of+clinically+relevant+weakness.">Grip strength cutpoints for the identification of clinically relevant weakness.</a> J Gerontol A Biol Sci Med Sci. 69 (5), 559-566 (2014).</li><li>Juel, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Neurophysiology+of+Neuromuscular+Junction+Disease.">Clinical Neurophysiology of Neuromuscular Junction Disease.</a> Handbook of Clinical Neurology. 161, 291-303 (2019).</li><li>Rich, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+neuromuscular+transmission+in+health+and+disease.">The control of neuromuscular transmission in health and disease.</a> Neuroscientist. 12 (2), 134-142 (2006).</li><li>Juel, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+neuromuscular+junction+disorders+in+the+electromyography+laboratory.">Evaluation of neuromuscular junction disorders in the electromyography laboratory.</a> Neurol Clin. 30 (2), 621-639 (2012).</li><li>Arnold, W. D., Clark, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+junction+transmission+failure+in+aging+and+sarcopenia:+The+nexus+of+the+neurological+and+muscular+systems.">Neuromuscular junction transmission failure in aging and sarcopenia: The nexus of the neurological and muscular systems.</a> Ageing Res Rev. 89, 101966 (2023).</li><li>Kokubun, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+values+for+concentric+needle+single+fiber+EMG.">Reference values for concentric needle single fiber EMG.</a> Rinsho Shinkeigaku. 52 (11), 1246-1248 (2012).</li><li>Testelmans, D., 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=Rocuronium+exacerbates+mechanical+ventilation-induced+diaphragm+dysfunction+in+rats.">Rocuronium exacerbates mechanical ventilation-induced diaphragm dysfunction in rats.</a> Crit Care Med. 34 (12), 3018-3023 (2006).</li><li>Suzuki, K., 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=Intravenous+infusion+of+rocuronium+bromide+prolongs+emergence+from+propofol+anesthesia+in+rats.">Intravenous infusion of rocuronium bromide prolongs emergence from propofol anesthesia in rats.</a> PLoS One. 16 (2), e0246858 (2021).</li><li>St&#229;lberg, E., 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=Reference+values+for+jitter+recorded+by+concentric+needle+electrodes+in+healthy+controls:+A+multicenter+study.">Reference values for jitter recorded by concentric needle electrodes in healthy controls: A multicenter study.</a> Muscle Nerve. 53 (3), 351-362 (2016).</li></ol>

W. David Arnold received research funding from NMD Pharma and Avidity Biosciences and is consulting for NMD Pharma, Avidity Biosciences, Dyne Therapeutics, Novartis, Design Therapeutics, and Catalyst Pharmaceuticals.

SFEMG is commonly used for diagnostic testing in patients with suspected autoimmune, acquired, and genetic forms of NMJ disease. SFEMG is considered the most sensitive test for the diagnosis of the NMJ disorder, myasthenia gravis20,21. Repetitive nerve stimulation (RNS) is another method that is more commonly used in clinical diagnostic testing and involves stimulating a peripheral nerve with a train of stimuli and quantifying the summated compound muscle action potential response of the innervated muscle5,22. RNS has consistently shown to have significantly lower sensitivity as compared with SFEMG23,24. The advantage of SFEMG lies in the ability to selectively evaluate individual muscle fibers and quantify not only failure of NMJ transmission (blocking) but also variability (jitter)2,25,26. While an established and sensitive test in the clinical setting, SFEMG has only been infrequently applied in preclinical research4,5,9,10,11,12,13,14,15,16. Animal models of neuromuscular diseases play a pivotal role in determining the underlying mechanisms of human diseases and developing therapeutic interventions. The capacity to apply outcome measures and biomarkers, such as SFEMG, across different species can streamline and expedite the transition of promising preclinical discoveries into human clinical studies27. For instance, a multicenter study in Japan focused on establishing reference values for SFEMG using concentric needles in healthy human subjects. The study indicated that the recommended cut-off values for individual MCD were 58.8 &#181;s for stimulated SFEMG in the extensor digitorum communis (EDC) muscle28. It is noteworthy that the Gastrocnemius muscle is not commonly subjected to SFEMG, with EDC being the closest muscle studied in this context. Consequently, the jitter values we obtained from rats were observed to be lower than those reported in clinical studies. These discrepancies suggest that the distribution of the safety factor varies depending on the muscle and the species under consideration.
In this report, we present a stepwise approach to applying stimulated SFEMG in rodents including steps for animal preparation, electrode placement, single fiber action potential recording, and analyses. Additionally, we present representative data in an induced model of NMJ failure (rocuronium administration) to help clearly demonstrate the concepts of increased jitter and blocking as well as the all-or-none concept of a single fiber action potential in SFEMG recording.Previously, in an experimental study, a rat without the blocking agent showed a minor MCD change (around 10 &#181;s) with no blocking in the first 3000 s. In contrast, a rat injected with 0.08 mg/Kg vecuronium exhibited increased jitter after 200 s, leading to blocking around 500 s, with an initial MCD of about 50 &#181;s, consistent with our results17. Establishing the correct dosage of rocuronium in the rats was essential to attain the targeted level of neuromuscular blockade while mitigating potential complications. While various studies employed dosages ranging from 0.1 mg/kg to 5 mg/kg for inducing muscle paralysis17,29,30, we opted for a dosage of 0.05 mg/kg. This choice aligns with the recognition that NMJ impairment initiates before the onset of muscle paralysis.
The acquisition of single fiber action potential responses using SFEMG involves several crucial steps. Notably, obtaining selective recordings requires practice to perform several tasks concurrently, including adequately delivering nerve stimulation while acquiring adequate responses. During recordings, the SFEMG recording electrode is carefully inserted and adjusted with small, steady movements to minimize muscle trauma and improve yield. Once an action potential has been identified, small adjustments of stimulation and recording electrode position should be made to optimize single fiber action potential amplitude and &#34;sharpness&#34; (short rise time). Once a potential is identified and amplitude and rise time are optimized, movements of electrodes must be minimized to ensure stability of the all-or-none responses. Steady positioning of electrodes is crucial as even minor shifts in the recording needle position can result in fluctuations in action potential characteristics, including shape, amplitude, and latency. It is important to ensure that the action potentials are stable and in compliance with all-or-none appearance. Variability of responses can be related to electrode movement or superimposition of multiple single fiber action potentials from different NMJs. Together, these factors play key roles in achieving reproducibility and stable measurement of the action potential2,26. Moreover, excluding SFAPs with jitter less than 4 &#181;s from the analyses is a prudent approach to ensure the reliability of results. This stringent criterion helps avoid the inclusion of potentials that could potentially be artifacts induced by direct muscle stimulation, thereby enhancing the accuracy of the assessment11. Another important factor in stimulation jitter studies is subliminal stimulation, particularly in pathological conditions, where many spikes have apparently increased jitter. It is essential to rely on the operator to ensure that stimulus intensity exceeds the threshold for all such spikes, as this determination cannot be made retrospectively31.
The stimulated SFEMG technique outlined in this manuscript utilizes a specialized SFEMG electrode rather than a standard concentric electromyography needle electrode. Our SFEMG electrode features a recording surface of 0.0005 mm2, in contrast to the approximately 0.019 mm2 recording surface of a standard pediatric-sized concentric electromyography needle electrode. This distinction is crucial, as larger concentric needle electrodes, with less selectivity due to their larger recording surfaces, may be suitable for voluntary SFEMG to capture apparent single fiber action potential pairs. However, in stimulation SFEMG, where multiple axons or motor units are often stimulated simultaneously, employing larger concentric needle electrodes becomes more challenging7.
A few limitations should be considered. It is also important to point out that SFEMG cannot ascertain the mechanism underlying a defect in neuromuscular transmission as may be achieved with intracellular recordings performed ex vivo27. Another essential consideration regarding SFEMG is its limitation in selectively recording from specific muscle fiber types. Consequently, SFEMG recordings are likely to represent a variable mixture of different muscle fiber types5.

Single fiber electromyography (SFEMG) was initially developed by St&#229;lberg and Ekstedt in the 1960s to identify and analyze action potentials from individual muscle fibers, primarily to study muscle fatigue1. SFEMG is the most sensitive clinical technique for the assessment of neuromuscular junction (NMJ) transmission2. SFEMG is conducted by selectively recording single fiber action potentials (SFAPs)3. NMJ transmission can be compromised due to factors like aging4,5 and various neuromuscular disorders such as myasthenia gravis and amyotrophic lateral sclerosis6. Furthermore, conditions such as ischemia, fluctuations in temperature, and the use of neuromuscular blocking agents can result in deficiencies in NMJ transmission, manifested by increased NMJ transmission variability and occurrences of NMJ failure2.
There are two approaches to recording SFEMG: stimulated and voluntary SFEMG. Voluntary SFEMG involves recording SFAPs from two NMJs supplied by the same motor axon using a concentric needle electrode inserted into the muscle being tested during voluntary activation7. Accordingly, voluntary SFEMG requires cooperation from the subject and can only assess low-threshold motor units (those activated during weak contractions)3. Stimulated SFEMG uses a pair of stimulating electrodes to stimulate motor axons while recording SFAPs with an SFEMG needle electrode inserted into the muscle being tested7.
In both voluntary and stimulated SFEMG, jitter and blocking are the two parameters used to assess and quantify NMJ transmission8. Jitter describes the variability in timing (latency) between consecutive SFAPs. During voluntary SFEMG, jitter is quantified by assessing the latency differences between a pair of SFAPs (supplied by the same motor axon) during 50 to 100 consecutive discharges. During stimulated SFEMG, jitter is quantified by assessing the latency differences between the stimulation timing and the onset of the SFAP during 50 to 100 consecutive discharges. Blocking indicates failure of NMJ transmission to trigger an SFAP response, and it can be quantified as the presence or absence of each pair of SFAPs during voluntary SFEMG or for each NMJ during stimulated SFEMG2,7.
While an established and sensitive test in the clinical setting, SFEMG has only been infrequently applied in preclinical research4,5,9,10,11,12,13,14,15,16,17,18. In this report, we outline the approach to performing and analyzing SFEMG recordings in preclinical rodent models. Furthermore, we present representative data that highlights representative findings on SFEMG that indicate impairment of NMJ transmission following administration of a non-depolarizing neuromuscular blocking agent, rocuronium.

<table><tbody><tr><td>&amp;nbsp;27 G Reusable Single Fiber Needle Electrode</td><td>Technomed</td><td>202860-000</td><td>singlefiber recording electrode</td></tr><tr><td>2 mL Glass Syringe</td><td>Kent Scientific Corporation</td><td>SOMNO-2ML</td><td></td></tr><tr><td>Detachable Cable</td><td>Technomed</td><td>202845-0000</td><td>to connect the recorder electrode to the electrodiagnostic machine</td></tr><tr><td>Disposable 2&quot; x 2&quot; disc electrode with leads</td><td>Cadwell</td><td>302290-000</td><td>ground electrode</td></tr><tr><td>disposable monopolar needles 28 G</td><td>Technomed</td><td>202270-000</td><td>cathode and anode stimulating electrodes</td></tr><tr><td>EMG needle cable (Amp/stim switch box)</td><td>Cadwell</td><td>190266-200</td><td>to connect monopolar electrodes to electrodiagnostic stimulator</td></tr><tr><td>Helping Hands alligator clip with iron base</td><td>Radio Shack</td><td>64-079</td><td>Maintaining recording electrode placement&amp;nbsp;</td></tr><tr><td>Isoflurane (250 mL bottle)</td><td>Piramal Healthcare</td><td>NA</td><td></td></tr><tr><td>monoject curved tip irrigating syringe</td><td>Covidien</td><td>81412012</td><td>utilized for application of electrode gel</td></tr><tr><td>PhysioSuite Physiological Monitoring System with RightTemp Homeothermic Warming</td><td>Kent Scientific Corporation</td><td>PS-RT</td><td>Includes infrared warming pad, rectal probe, and pad temperature probe</td></tr><tr><td>Pro trimmer Pet Grooming Kit</td><td>Oster</td><td>078577-010-003</td><td>clippers for hair removal</td></tr><tr><td>Rat Endotracheal Tubes (16 G)</td><td>Kent Scientific Corporation</td><td></td><td></td></tr><tr><td>Rocoronium Bromide</td><td>Sigma</td><td>PHR2397-500MG</td><td>neuromuscular blocker agent</td></tr><tr><td>Sierra Summit EMG system</td><td>Cadwell Industries, Inc., Kennewick, WA</td><td>NA</td><td>portable electrodiagnostic system</td></tr><tr><td>SomnoSuite Low-Flow Digital Anesthesia System</td><td>Kent Scientific Corporation</td><td>SOMNO</td><td>Includes anti-spill, anti-vapor bottle top adapter; Y adapter tubing; charcoal scavenging filter</td></tr><tr><td>Veterinarian petroleum-based ophthalmic ointment&amp;nbsp;</td><td>Puralube</td><td>26870</td><td>applied during anesthesia to avoid corneal injury</td></tr></tbody></table>

stimulated single fiber electromyography (sfemg) for assessing neuromuscular junction transmission in rodent models

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

To demonstrate increased jitter and blocking in the context of NMJ transmission failure, stimulated SFEMG was performed with and without intravenous administration of rocuronium. Rocuronium is an intermediate-acting, non-depolarizing neuromuscular blocking agent widely used in clinical settings to induce muscle paralysis during surgeries or medical procedures. It operates by competitively binding to nicotinic acetylcholine receptors at the NMJ19. Prior to the administration of rocuronium, the adult wild-type rat (Sprague Dawley) was prepared and anesthetized as described in section 1. Due to paralysis induced by rocuronium, invasive ventilatory support was used to obtain representative data (contrasting to the typical approach for anesthesia during SFEMG recordings described above in section 1). An endotracheal tube was placed, and ventilation was maintained at 60 respirations/min using a pressure-controlled mode with a pressure of 17 mmHg and positive end-expiratory pressure of 3 cm H2O while delivering 2%-3% isoflurane. Once ventilatory support and anesthesia were achieved, single fiber action potentials were recorded with and without treatment with rocuronium (bolus of 0.05 mg/kg rocuronium bromide intravenous). Figure 1 demonstrates three representative single-fiber action potential recordings from the same NMJ in an adult rat. Figure 1A demonstrates the typical morphology of a single fiber action potential (after a single stimulation) with a rise time of less than 500 µs from the baseline to the negative peak and a minimum amplitude (peak to peak) of at least 200 µV. Figure 1B demonstrates features of increased jitter and intermittent blocking following injection of rocuronium. Figure 1C demonstrates the normalization of jitter and blocking from the same synapse after the resolution of the NMJ block. For viewing simplicity, Figure 1B,C only show responses following 9 consecutive stimulations. Moreover, for better comprehension, Figure 2 shows an additional example of three superimposed single fiber action potentials observed after 10 consecutive stimulations, revealing heightened jitter and blocking.
To quantify the impact of rocuronium on NMJ transmission using stimulated SFEMG, recordings were made from 5 NMJs without any treatment and from 5 NMJ following delivery of rocuronium and were analyzed for jitter and blocking (Figure 3). Following rocuronium, jitter (Figure 3A) and blocking (Figure 3B) were significantly increased.
<img alt="Single fiber action potential graphs showing latency and amplitude changes; jitter analysis." class="xfigimg" src="/files/ftp_upload/66452/66452fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Three representative single-fiber electromyography recordings from the same neuromuscular junction (NMJ). (A) Example of a single fiber action potential following a single nerve stimulation showing criteria for amplitude (baseline to negative peak amplitude of at least 200 µV) and rise time (baseline to negative peak less than 500 µs) criteria for acceptance as single fiber potential. (B) Example of superimposed single fiber action potentials following 9 consecutive stimulations showing increased jitter and blocking indicating impaired NMJ transmission following rocuronium administration (jitter: 68.35 µs). (C) Example of superimposed single fiber action potentials following 9 consecutive stimulations showing reduced jitter and no blocking, indicating normalizing NMJ transmission after resolution of rocuronium treatment (jitter: 21.7 µs). Representative SFEMG traces reveal single muscle fiber action potentials recorded from a healthy synapse with no blocking and normal jitter in superimposed view. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66452/66452fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Single fiber action potential graph showing latency vs amplitude with blocked stimulation analysis." class="xfigimg" src="/files/ftp_upload/66452/66452fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Three superimposed single fiber action potentials (SFAPs) recorded after consecutive stimulations during rocuronium administration. Three superimposed single fiber action potentials (SFAPs) were recorded after 10 consecutive stimulations during rocuronium administration, indicating increased jitter and blocking. The jitter values for A, B, and C were 24.3 µs, 46.9 µs, and 88.7 µs, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66452/66452fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="NMJ jitter and transmission blocking bar graphs showing statistical differences with NMJ blocker use." class="xfigimg" src="/files/ftp_upload/66452/66452fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Single Fiber Electromyography assessment of neuromuscular junction transmission (NMJ) following treatment with a non-depolarizing NMJ blocking agent. (A) During rocuronium administration (0.05 mg/kg), SFEMG showed increased variability of transmission (jitter) versus healthy condition (untreated: 12.9 µs, 95% CI [7.2-16.9 µs] versus rocuronium: 40.70 µs, 95% CI [34.7-70.7 µs]) (Mann Whitney test: p = 0.0079). (B) Percentage of stimulations with NMJ blocking from each synapse on SFEMG was increased versus healthy condition (untreated: 0%, 95% CI [0-0%] versus rocuronium: 31%, 95% CI [14.0-59.0%]) (Mann Whitney test: p= 0.0079). All measurements (jitter and percentages of stimulations with NMJ blocking) were obtained from a single rat, n = 5 synapses untreated and n = 5 synapses following rocuronium treatment. Data shown as a median ± 95% confidence interval. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66452/66452fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Stimulated Single Fiber Electromyography SFEMG for Assessing Neuromuscular Junction Transmission in Rodent Models at JoVE.com

Author Spotlight: Translational Applications of Stimulated SFEMG in Rodent Models

In this study, we demonstrate a refined single fiber electromyography (SFEMG) protocol to allow in vivo measurement of neuromuscular junction (NMJ) transmission in rodent models. A step-by-step approach to the SFEMG technique is described to allow quantification of NMJ transmission variability and failure in rat gastrocnemius muscle.

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. ...

stimulated-single-fiber-electromyography-sfemg-for-assessing

Nanyang Technological University

Research

JoVE Journal

Neuroscience

578 Views.  University of Missouri. In dit rapport presenteren we een stapsgewijze aanpak om EMG met één vezel bij knaagdieren te stimuleren. Bovendien tonen we in een rattenmodel van door brocorion geïnduceerde neurovasculaire junctietransmissie falen aan van het al dan niet verschijnen van actiepotentialen van één vezel en geven we representatieve voorbeelden van verhoogde jitter en blokkering. EMG met één vezel is een gevestigde klinische diagnostische test, maar de toepassing ervan i...

Video: Auteur in de schijnwerpers: translationele toepassingen van gestimuleerde SFEMG in knaagdiermodellen

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Medicine

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biology

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

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 ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Bioengineering

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

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.

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.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

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

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

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.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

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

Source: Castells-Nobau, A., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
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: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

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