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&#39;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 &#176;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&#176; 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 &#181;V to 1000 &#181;V per division to facilitate the visualization of potentials. Set the sweep speed at 500 &#181;s 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 &#181;s, the minimum amplitude (baseline peak to negative) is at least 200 &#181;V, 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="Equation 1" src="/files/ftp_upload/66452/66452eq01.jpg" style="margin: auto;" /> 
	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 &#181;s 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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;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&#34; x 2&#34; 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&#160;</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&#160;</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 adul...

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. ...

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Research

JoVE Journal

Neuroscience

493 Views.  University of Missouri. 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. 

Video: Author Spotlight: Translational Applications of Stimulated SFEMG in Rodent Models

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 ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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.

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 ...

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 ...

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 ...

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 ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...