This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01AR081235 (to L. K. Lepley). The authors thank the following individuals for their contribution to the fabrication and testing of our biocompatible electrode: Joel Pingel, Grant Gueller, Akhil Ramesh, Joe Letner, Jacky Tian, and Ross Brancati.

 Fabricating Epimysial EMG Electrodes for Preclinical Rodent Models 

<ol>
	<li>Grandjean, P. A., Mortimer, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+properties+of+monopolar+and+bipolar+epimysial+electrodes.">Recruitment properties of monopolar and bipolar epimysial electrodes.</a> Ann. Biomed. Eng. 14 (1), 53-66 (1986).</li><li>Memberg, W. D., Stage, T. G., Kirsch, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fully+implanted+intramuscular+bipolar+myoelectric+signal+recording+electrode.">A fully implanted intramuscular bipolar myoelectric signal recording electrode.</a> Neuromodulation J. Int. Neuromodulation Soc. 17 (8), 794-799 (2014).</li><li>Loeb, G. E., Gans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electromyography for Experimentalists. , (1986).</li><li>Boehler, C., Carli, S., Fadiga, L., Stieglitz, T., Asplund, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial:+guidelines+for+standardized+performance+tests+for+electrodes+intended+for+neural+interfaces+and+bioelectronics.">Tutorial: guidelines for standardized performance tests for electrodes intended for neural interfaces and bioelectronics.</a> Nat. Protoc. 15 (11), 3557-3578 (2020).</li><li>Patel, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+linear+8.4+&#181;m+diameter+16+channel+carbon+fiber+electrode+arrays+for+single+unit+recordings.">Insertion of linear 8.4 &#181;m diameter 16 channel carbon fiber electrode arrays for single unit recordings.</a> J. Neural Eng. 12 (4), 046009 (2015).</li><li>Richie, J. M., 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=Open-source+toolkit:+benchtop+carbon+fiber+microelectrode+array+for+nerve+recording.">Open-source toolkit: benchtop carbon fiber microelectrode array for nerve recording.</a> J. Vis. Exp. (176), e63099 (2021).</li><li>Sarolic, A., Skalic, I., Deftu, A., Sapunar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+measurement+of+bipolar+stimulation+electrodes+immersed+in+medium.">Impedance measurement of bipolar stimulation electrodes immersed in medium.</a> , 1-2 (2018).</li><li>Pritchett-Corning, K. R., Mulder, G. B., Luo, Y., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+New+Surgeon.">Principles of rodent surgery for the New Surgeon.</a> J. Vis. Exp.: JoVE. (47), e2586 (2011).</li><li>Zealear, D., Li, Y., Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+system+for+chronic+in+vivo+electromyography.">An implantable system for chronic in vivo electromyography.</a> J. Vis. Exp. JoVE. (158), e60345 (2020).</li><li>Butterfield, T. A., Leonard, T. R., Herzog, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+serial+sarcomere+number+adaptations+in+knee+extensor+muscles+of+rats+is+contraction+type+dependent.">Differential serial sarcomere number adaptations in knee extensor muscles of rats is contraction type dependent.</a> J. Appl. Physiol. Bethesda Md 1985. 99 (4), 1352-1358 (2005).</li><li>Farago, E., MacIsaac, D., Suk, M., Chan, A. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+techniques+for+surface+electromyography+signal+quality+analysis.">A review of techniques for surface electromyography signal quality analysis.</a> IEEE Rev. Biomed. Eng. 16, 472-486 (2023).</li><li>Raez, M. B. I., Hussain, M. S., Mohd-Yasin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+of+EMG+signal+analysis:+detection,+processing,+classification+and+applications.">Techniques of EMG signal analysis: detection, processing, classification and applications.</a> Biol. Proced. Online. 8, 11-35 (2006).</li><li>Tankisi, H., 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=Standards+of+instrumentation+of+EMG.">Standards of instrumentation of EMG.</a> Clin. Neurophysiol. 131 (1), 243-258 (2020).</li><li>Kumar, A., Accorsi, A., Younghwa, R., Mahasweta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J. Vis. Exp. JoVE. (99), e52793 (2015).</li><li>Wang, C., Yue, F., Kuang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+histology+characterization+using+H&amp;E+staining+and+muscle+fiber+type+classification+using+immunofluorescence+staining.">Muscle histology characterization using H&amp;E staining and muscle fiber type classification using immunofluorescence staining.</a> Bio-Protoc. 7 (10), e2279 (2017).</li><li>Kreifeldt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+versus+noise+characteristics+of+filtered+EMG+used+as+a+control+source.">Signal versus noise characteristics of filtered EMG used as a control source.</a> IEEE Trans. Biomed. Eng. BME-18 (1), 16-22 (1971).</li><li>Farina, D., Yoshida, K., Stieglitz, T., Koch, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+thin-film+electrode+for+intramuscular+electromyographic+recordings.">Multichannel thin-film electrode for intramuscular electromyographic recordings.</a> J. Appl. Physiol. 104 (3), 821-827 (2008).</li><li>Muceli, 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=Decoding+motor+neuron+activity+from+epimysial+thin-film+electrode+recordings+following+targeted+muscle+reinnervation.">Decoding motor neuron activity from epimysial thin-film electrode recordings following targeted muscle reinnervation.</a> J. Neural Eng. 16 (1), 016010 (2018).</li><li>Guo, L., Guvanasen, G., Tuthill, C., Nichols, T., Deweerth, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterization of a Stretchable Multielectrode Array for Epimysial Recording. , 694 (2011).</li><li>Zwarts, M. J., Stegeman, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+surface+EMG:+basic+aspects+and+clinical+utility.">Multichannel surface EMG: basic aspects and clinical utility.</a> Muscle Nerve. 28 (1), 1-17 (2003).</li><li>Koch, K. P., Leinenbach, C., Stieglitz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+test+of+robust+spherical+epimysial+electrodes+for+lower+limb+stimulation.">Fabrication and test of robust spherical epimysial electrodes for lower limb stimulation.</a> Aalb. Den. , (2000).</li><li>Uhlir, J. P., Triolo, R. J., Davis, J. A., Bieri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+epimysial+stimulating+electrodes+in+the+lower+extremities+of+individuals+with+spinal+cord+injury.">Performance of epimysial stimulating electrodes in the lower extremities of individuals with spinal cord injury.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 12 (2), 279-287 (2004).</li><li>Deer, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+appropriate+use+of+neurostimulation:+new+and+evolving+neurostimulation+therapies+and+applicable+treatment+for+chronic+pain+and+selected+disease+states.">The appropriate use of neurostimulation: new and evolving neurostimulation therapies and applicable treatment for chronic pain and selected disease states.</a> Neuromodulation Technol. Neural Interface. 17 (6), 599-615 (2014).</li><li>Ortiz-Catalan, M., Br&#229;nemark, R., H&#229;kansson, B., Delbeke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+viability+of+implantable+electrodes+for+the+natural+control+of+artificial+limbs:+Review+and+discussion.">On the viability of implantable electrodes for the natural control of artificial limbs: Review and discussion.</a> Biomed. Eng. OnLine. 11, 33 (2012).</li><li>Sando, I. 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=Regenerative+peripheral+nerve+interface+for+prostheses+control:+electrode+comparison.">Regenerative peripheral nerve interface for prostheses control: electrode comparison.</a> J. Reconstr. Microsurg. 32 (3), 194-199 (2016).</li></ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Our objective was to streamline the EMG fabrication process, enabling broader adoption and implementation of epimysial electrode designs, thus promoting accessibility, and advancing neuromuscular research. To this end, we present a user-friendly guide for sourcing, fabricating, and testing low-cost epimysial electrodes in-house. In hopes of supporting other research groups, we also provide supplemental 3D printing templates to facilitate the production of in-house epimysial electrodes for their research endeavors.
<p...

Electromyography (EMG) is a powerful tool for studying the electrical activity of muscle. EMG recordings can be especially useful in preclinical animal models to assess the effectiveness of interventions to treat neuromuscular dysfunction. In these models, implantable biocompatible electrodes are commonly used to assess the neurophysiological interface between motor neurons and muscle fibers. These implantable electrodes can provide localized measurements of muscle excitation and can be diverse in terms of their configuration, shape, and material, with the optimal design ultimately dictated by the location and intended use.
Despite their suitability for assessing muscle excitation in preclinical models, the use of epimysial electrodes can be limited by cost. As a result, many investigators use custom-fabricated epimysial electrodes that are produced in-house. Although resources exist detailing the fundamental considerations of electrode fabrication, testing, and use1,2, there is a need for an updated instructional guide detailing the sourcing, fabrication, and validation of epimysial electrodes using modern methods. Informed by the foundational works of Loeb and Gans3 and others in electrode theory, we present modern instructions on the sourcing and fabrication of low-cost epimysial electrodes and test their performance in a series of ex vivo and in vivo experiments. The aim is to offer a user-friendly guide for others in the scientific community to source, fabricate, and test in-house low-cost epimysial electrodes for animal use, enabling the broader quantification of muscle excitation in preclinical models.
In this protocol, we provide an instructional guide to the sourcing, fabrication, and testing of epimysial electrodes for animal use in the modern electrophysiology laboratory. Electrode parameters chosen for fabrication, such as the shape, dimensions, contact surface area, interelectrode distance, lead length, etc., were selected to suit our experimental needs and were comparable to a commercially available industry standard epimysial electrode (see the Table of Materials). We encourage other groups to modify these parameters to suit their needs in addition to selecting a reliable industry standard electrode that matches their use case.
In an effort to give readers a relatively quick sense of electrode performance, we also provide an example of an ex vivo testing protocol with the option of measuring electrode impedance. Additionally, we give an example assessment of electrode performance in vivo. The ex vivo experiment compared the custom-fabricated electrode to an industry standard in a saline bath to mimic stable physiological conditions. Impedance was also assessed ex vivo via electrochemical impedance spectroscopy (EIS). The in vivo experiment consisted of the surgical implantation of the custom-fabricated electrode into the vastus lateralis (VL) muscle of a 16-week-old female Long Evans rat (HsdBlu: LE, Envigo) to measure the EMG signal during conditions known to elicit a high or low signal (uphill, downhill walking). To assess the reliability of the custom-fabricated electrode, EMG signaling was acquired during level walking following full surgical recovery and prior to sacrifice (14 days and 56 days post implantation, respectively). Hematoxylin-eosin (H&#38;E) staining was conducted on the instrumented muscle to assess the biocompatibility of the custom-fabricated electrode.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electrode Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>Contact surface</td>
			<td>Prince and Izant PT90/IR10 1.25 mm x 5 mm foil</td>
			<td>Catalog #1040055&#160;</td>
			<td>2 per electrode 
			$7.50 per foil 
			$15.00 per electrode</td>
		</tr>
		<tr>
			<td>PFA coated stainless-steel electrode lead wire&#160;</td>
			<td>A-M Systems Multi-Stranded PFA-Coated Stainless Steel Wire 50.8 &#181;m strand diameter&#160;</td>
			<td>Catalog #793500</td>
			<td>Dependent on desired lead length (e.g., 9 inch lead wires x2) 
			$128 per 25 ft spool 
			$5.12 per foot 
			$0.42 per inch (x18) 
			$7.68 per electrode</td>
		</tr>
		<tr>
			<td>Folding jig&#160;</td>
			<td>3D printed 
			(see .gcode file)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Sealant for electrode body</td>
			<td>Nusil Med-1137 liquid silicone</td>
			<td>Catalog #MED-1137</td>
			<td>1 gram 
			$344.66 per 2 oz. (59.15 mL) 
			$5.83 per electrode</td>
		</tr>
		<tr>
			<td>Silicone base</td>
			<td>Implantech Alliedsil Silicone Sheeting-Reinforced, Long Term Implantable (8&#8221; x 6&#8221;) .007 thick</td>
			<td>Catalog #701-07&#160;</td>
			<td>10mm x 5mm sheet 
			$225.00 per 8 x 6 inch 
			$0.36 per electrode (10 mm x 5 mm)</td>
		</tr>
		<tr>
			<td>&#160;Thinner for sealant mixture</td>
			<td>Toluene 99.5% ACS Reagent 500mL or Xylene ACS 99.5%</td>
			<td>Catalog #179418-500 ML</td>
			<td>0.75 mL 
			$25.53 per 500 mL 
			$0.38 per electrode</td>
		</tr>
		<tr>
			<td>Template for perforating silicone base</td>
			<td>Cutting jig &#8211; 3D printed 
			(see CAD file)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Custom-fabricated electrode: $29.25</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Industry standard electrode (EP105 EMG Patch Electrode, 2 contacts, single-sided, 7mm x 4mm, MicroProbe for Life Science): $305.00</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Additional Fabrication Materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;&#160;</td>
			<td>&#160; &#160;&#160;</td>
			<td>&#160; &#160;&#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>3D printing software&#160;</td>
			<td>Solidworks (Solidworks, 2022)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Tig welder&#160;</td>
			<td>Micro-Tig Welder (CD1000SPM, Single Pulse Research and Light Production Resistance Spot Welder, Sunstone)</td>
			<td>SKU&#160;301010</td>
			<td>$3,500</td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Ultrasonic bath (CPX Series Ultrasonic Bath, Fisherbrand).&#160;</td>
			<td>15-337-403</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Ex Vivo Testing Materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>Data acquisition platform and software</td>
			<td>DigitalLynx 4sX Base Cheetah version 6.0 (Neuralynx Inc.)&#160;</td>
			<td>NA</td>
			<td>EMG acquisition hardware and software</td>
		</tr>
		<tr>
			<td>Electrode interface board (EIB)</td>
			<td>EIB, EIB16-QC, Neuralynx Inc.</td>
			<td>31-0603-0007</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>5 MHz Function Generator, B&#38;K Precision&#160;</td>
			<td>&#160;4005DDS220V</td>
			<td>$387.46</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>PGSTAT1 potentiostat (EcoChemie, Utrecht, Netherlands)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Stainless steel screw</td>
			<td>Fine Science Tools</td>
			<td>19010-00</td>
			<td>$98</td>
		</tr>
		<tr>
			<td>Ex Vivo Testing Materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>Rodent treadmill&#160;</td>
			<td>Exer 3/6 Open Treadmill, Columbus Instruments</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Excel Formula&#174; Pourable Dental Material, St. George Technology Inc.</td>
			<td>#24211</td>
			<td>$125.60</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Keyence BZ-X800, Keyence Corporation, Osaka, Japan&#160;</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>Optitrack Color Camera, Optitrack, NaturalPoint Inc.</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Peak detection algorithm</td>
			<td>&#8220;SciPy.signal.find_peaks - SciPy v1.8.1 Manual&#8221;, 2022</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Python software</td>
			<td>Python Software Foundation. Python Language Reference, version 3.9. Available at http://www.python.org</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>HsdBlu: LE, Envigo</td>
			<td>140</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Statistical sotware</td>
			<td>GraphPad Prism version 10.0.0 (GraphPad Software, Boston, Massachusetts USA)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

development of a low-cost epimysial electromyography electrode: a simplified workflow for fabrication and testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. 
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. 
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

Ex vivo performance 
ICCs revealed high levels of agreement between the custom-fabricated and industry standard electrodes across all waveforms (sine [ICC = 0.993], square [ICC = 0.995], triangle [ICC = 0.958]; p &#60; .001). Bland-Altman plots also revealed a high degree of signal agreement between electrodes. Bland Altman plots and Pearson correlations are summarized in Figure 3 with strong positive correlations between the custom-fabricated and industry stan...

Read this Scientific Article Protocol about Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies at JoVE.com

Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

development-low-cost-epimysial-electromyography-electrode-simplified

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Neuroscience

343 Views.  University of Michigan, Ann Arbor. Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process. 

Video: Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Modeling Human Cerebellar Development In Vitro in 2D Structure

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, disruption in cerebellar development has been implicated in many neurodevelopmental disorders, including autism, attention deficit-hyperactivity disorder (ADHD), and schizophrenia. Investigations of cerebellar development in humans have previously only been possible through post-mortem studies or neuroimaging, yet these methods are not sufficient for understanding the molecular and cellular changes occurring in vivo during early development, which is when many neurodevelopmental disorders originate. The emergence of techniques to generate human-induced pluripotent stem cells (iPSCs) from somatic cells and the ability to further re-differentiate iPSCs into neurons have paved the way for in vitro modeling of early brain development. The present study provides simplified steps toward generating cerebellar cells for applications that require a 2-dimensional (2D) monolayer structure. Cerebellar cells representing early developmental stages are derived from human iPSCs via the following steps: first, embryoid bodies are made in 3-dimensional (3D) culture, then they are treated with FGF2 and insulin to promote cerebellar fate specification, and finally, they are terminally differentiated as a monolayer on poly-l-ornithine (PLO)/laminin-coated substrates. At 35 days of differentiation, iPSC-derived cerebellar cell cultures express cerebellar markers including ATOH1, PTF1&#945;, PAX6, and KIRREL2, suggesting that this protocol generates glutamatergic and GABAergic cerebellar neuronal precursors, as well as Purkinje cell progenitors. Moreover, the differentiated cells show distinct neuronal morphology and are positive for immunofluorescence markers of neuronal identity such as TUBB3. These cells express axonal guidance molecules, including semaphorin-4C, plexin-B2, and neuropilin-1, and could serve as a model for investigating the molecular mechanisms of neurite outgrowth and synaptic connectivity. This method generates human cerebellar neurons useful for downstream applications, including gene expression, physiological, and morphological studies requiring 2D monolayer formats.

Modeling Human Cerebellar Development In Vitro in 2D Structure

The present protocol explains the generation of a 2D monolayer of cerebellar cells from induced pluripotent stem cells for investigating the early stages of cerebellar development.

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...