The authors would like to acknowledge Dr. Claire Warriner for contributing to the development of this method. Mark Agrios and Sajishnu Savya assisted with preparing figures. This research was supported by a Searle Scholar Award, a Sloan Research Fellowship, a Simons Collaboration on the Global Brain Pilot Award, a Whitehall Research Grant Award, The Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, NIH grant DP2 NS120847 (A.M.), and NIH grant 2T32MH067564 (A.K.).

Fabrication and Surgical Implantation of Electromyographic Electrodes in Mice

<ol><li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+large+scale+neural+activity+with+cellular+resolution+in+awake+mobile+mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Imaging large scale neural activity with cellular resolution in awake mobile mice.</a> Neuron. 56 (1), 43-57 (2007).</li>
<li>Guo, Z. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+of+cortical+activity+underlying+a+tactile+decision+in+mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Flow of cortical activity underlying a tactile decision in mice.</a> Neuron. 81 (1), 179-194 (2014).</li>
<li>Guo, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortex+commands+the+performance+of+skilled+movement%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Cortex commands the performance of skilled movement.</a> eLife. 4, e10774(2015).</li>
<li>Morandell, K., Huber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+forelimb+motor+cortex+areas+in+goal+directed+action+in+mice%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">The role of forelimb motor cortex areas in goal directed action in mice.</a> Sci Rep. 7 (1), 15759(2017).</li>
<li>Galiñanes, G. L., Bonardi, C., Huber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directional+reaching+for+water+as+a+cortex-dependent+behavioral+framework+for+mice%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Directional reaching for water as a cortex-dependent behavioral framework for mice.</a> Cell Rep. 22 (10), 2767-2783 (2018).</li>
<li>Evarts, E. V., Tanji, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reflex+and+intended+responses+in+motor+cortex+pyramidal+tract+neurons+of+monkey%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Reflex and intended responses in motor cortex pyramidal tract neurons of monkey.</a> J Neurophysiol. 39 (5), 1069-1080 (1976).</li>
<li>Hounsgaard, J., Hultborn, H., Jespersen, B., Kiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bistability+of+alpha-motoneurones+in+the+decerebrate+cat+and+in+the+acute+spinal+cat+after+intravenous+5-hydroxytryptophan%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Bistability of alpha-motoneurones in the decerebrate cat and in the acute spinal cat after intravenous 5-hydroxytryptophan.</a> J Physiol. 405, 345-367 (1988).</li>
<li>Murphy, P. R., Hammond, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+cutaneous+afferents+in+the+control+of+gamma-motoneurones+during+locomotion+in+the+decerebrate+cat%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The role of cutaneous afferents in the control of gamma-motoneurones during locomotion in the decerebrate cat.</a> J Physiol. 434, 529-547 (1991).</li>
<li>Manuel, M., Chardon, M., Tysseling, V., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaling+of+motor+output%2C+from+mouse+to+humans%5BTitle%5D)+AND+%22Physiol+Bethesda+Md%22%5BJournal%5D)">Scaling of motor output, from mouse to humans.</a> Physiol Bethesda Md. 34 (1), 5-13 (2019).</li>
<li>Sauerbrei, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+pattern+generation+during+dexterous+movement+is+input-driven%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cortical pattern generation during dexterous movement is input-driven.</a> Nature. 577 (7790), 386-391 (2020).</li>
<li>Barrett, J. M., Raineri Tapies, M. G., Shepherd, G. M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manual+dexterity+of+mice+during+food-handling+involves+the+thumb+and+a+set+of+fast+basic+movements%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Manual dexterity of mice during food-handling involves the thumb and a set of fast basic movements.</a> PLoS One. 15 (1), e0226774(2020).</li>
<li>Serradj, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Task-specific+modulation+of+corticospinal+neuron+activity+during+motor+learning+in+mice%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Task-specific modulation of corticospinal neuron activity during motor learning in mice.</a> Nat Commun. 14, 2708(2023).</li>
<li>Scholle, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+surface+EMG+characteristics+from+rat+triceps+brachii+muscle+during+treadmill+locomotion+indicate+selective+recruitment+of+functionally+distinct+muscle+regions%5BTitle%5D)+AND+%22Exp+Brain+Res%22%5BJournal%5D)">Spatiotemporal surface EMG characteristics from rat triceps brachii muscle during treadmill locomotion indicate selective recruitment of functionally distinct muscle regions.</a> Exp Brain Res. 138 (1), 26-36 (2001).</li>
<li>Scholle, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinematic+and+electromyographic+tools+for+characterizing+movement+disorders+in+mice%5BTitle%5D)+AND+%22Mov+Disord+off+J+Mov+Disord+Soc%22%5BJournal%5D)">Kinematic and electromyographic tools for characterizing movement disorders in mice.</a> Mov Disord off J Mov Disord Soc. 25 (3), 265-274 (2010).</li>
<li>Pearson, K. G., Acharya, H., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+electrode+configuration+for+recording+electromyographic+activity+in+behaving+mice%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A new electrode configuration for recording electromyographic activity in behaving mice.</a> J Neurosci Methods. 148 (1), 36-42 (2005).</li>
<li>Akay, T., Acharya, H. J., Fouad, K., Pearson, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+and+electromyographic+characterization+of+mice+lacking+EphA4+receptors%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Behavioral and electromyographic characterization of mice lacking EphA4 receptors.</a> J Neurophysiol. 96 (2), 642-651 (2006).</li>
<li>Akay, T., Tourtellotte, W. G., Arber, S., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Degradation+of+mouse+locomotor+pattern+in+the+absence+of+proprioceptive+sensory+feedback%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Degradation of mouse locomotor pattern in the absence of proprioceptive sensory feedback.</a> Proc Natl Acad Sci USA. 111 (47), 16877-16882 (2014).</li>
<li>Miri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behaviorally+selective+engagement+of+short-latency+effector+pathways+by+motor+cortex%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Behaviorally selective engagement of short-latency effector pathways by motor cortex.</a> Neuron. 95 (3), 683-696 (2017).</li>
<li>Osborne, J., Dudman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RIVETS%3A+A+mechanical+system+for+in+vivo+and+in+vitro+electrophysiology+and+imaging%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">RIVETS: A mechanical system for in vivo and in vitro electrophysiology and imaging.</a> PloS One. 9 (2), e89007(2014).</li>
<li>Santuz, A., Laflamme, O. D., Akay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+brain+integrates+proprioceptive+information+to+ensure+robust+locomotion%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The brain integrates proprioceptive information to ensure robust locomotion.</a> J Physiol. 600 (24), 5267-5294 (2022).</li>
<li>Warriner, C. L., Fageiry, S., Saxena, S., Costa, R. M., Miri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+cortical+influence+relies+on+task-specific+activity+covariation%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Motor cortical influence relies on task-specific activity covariation.</a> Cell Rep. 40, 111427(2022).</li>
</ol>

This protocol enables stable muscle activity recordings from head-fixed mice performing a variety of behaviors for several weeks. Recently, this method has been employed to examine neural control of limb musculature during behaviors such as treadmill locomotion18,20, a joystick pulling task18, and a co-contraction task21.&#160;While the protocol described here is specific for mouse elbow and wrist muscles, it is easily modified to record from different muscles or a different number of muscles by changing the length and/or total number of electrode pairs. The method described here was adapted from those used previously to record forelimb and hindlimb muscle activity in mice without head restraint15,16,17.
Electrode fabrication requires significant practice to master. Daily practice for 1-2 h is recommended while learning. Stripping the electrodes is the most challenging step due to the precise level of force required to cut the insulation without damaging the underlying wire. This level of force is dependent on the sharpness of the blade, so frequently replacing the scalpel blade can help ensure reproducibility during learning. Soldering the wires to the brass blades of the connector can also be difficult because stainless steel does not readily solder. Applying a liberal amount of stainless steel-compatible flux&#160;helps promote the connection.
The main challenge during the implantation surgery is tying the distal knot without disturbing the implanted wire or proximal knot. The proximal knot must be large enough to resist slipping into muscle at the insertion site - thus, avoid tying the knot too tight in step 2 of electrode set fabrication. If the proximal knot migrates after implantation, use carbon fiber-tipped forceps to reposition it carefully. Tighten the distal knot slowly while maintaining a firm grip on the wire with forceps to avoid pulling the entire electrode through. This step is critical to ensure the longevity of implanted electrodes: too much tension placed on the electrode can cause it to break when the animal moves, while a loose electrode can shift during recovery and lose contact with its associated muscle as the tissue heals.
Animals recover remarkably well from the surgery, though there are potential complications to note. First, mice will chew on their sutures and electrodes if given the chance. While the Elizabethan collar prevents this, it also prevents the animal from grooming itself. Some mice develop a mucus-like build-up around their eyes. Occasional male mice, particularly older ones, experience urethra blockages that can be distressing to the animal. Allowing the animal to groom itself for 20 min each day before inspecting sutures should give the animal enough time to prevent these issues.
There are important limitations of this method to note. First, these custom electrodes generally cannot resolve single motor unit activity. Furthermore, the electrical signal is not guaranteed to emanate exclusively from a specific muscle (i.e., biceps), as it is difficult to rule out crosstalk from activity in nearby synergist muscles. Therefore, in publications, researchers commonly refer to the recorded muscles by their synergy group (i.e., elbow flexor). It is recommended to perform post-mortem dissections after each experiment to verify the position of each electrode, as they could shift in the tissue during recovery.
Researchers interested in single motor unit activity should consider trying newly developed EMG electrodes by the Center for Advanced Motor Bioengineering Research (CAMBER) at Emory University. These electrodes are still being developed, but CAMBER will provide the latest electrode design. The main drawback of these electrodes is longevity: the hand-fabricated electrodes described in this protocol generally allow recordings for several weeks, whereas CAMBER electrodes work best for short-term experiments. Researchers selecting an EMG recording method can contact CAMBER directly to determine if their electrodes will be suitable for a given experiment.

In recent decades, mice have become an attractive model organism for studying the mammalian motor system. Common experimental approaches involve head-fixed mice performing motor tasks alongside the monitoring and/or perturbation of neural activity1,2,3,4,5. Motor system studies in larger species (such as cats and primates) have traditionally relied upon electromyography (EMG) to measure motor output directly during such experiments6,7,8. However, recording muscle activity in mice is challenging because their musculature is too small for commercially available EMG electrodes used in large mammal experiments9. Many researchers opt to track limb kinematics through video4,10,11 and/or behavioral performance2,4,12 to probe motor output indirectly, but these methods lack the resolution to detect the millisecond-timescale influence of neural activity and perturbation thereof on muscles. Thus, recording EMG is desirable for researchers interested in direct neural control of muscles.
EMG involves measuring the voltage between two points, typically separated by a short distance roughly parallel to the fibers of the muscle being recorded. EMG electrodes come in surface (or &#34;patch&#34;) and intramuscular (or &#34;needle&#34;) varieties. Surface electrodes are placed atop the skin or overlaid on the muscle tissue and secured with adhesive or suturing. As such, surface electrodes are less invasive than intramuscular electrodes and are most popular with humans, cats, and primates due to their relative ease of use. Surface electrodes have also been used successfully with rats and mice13,14; however, they must be hand-fabricated and surgically implanted under the skin due to the tendency of rodents to try to remove foreign objects while grooming. Intramuscular EMG electrodes, on the other hand, are surgically implanted within the muscle tissue. Because they are engulfed in muscle tissue, they provide high spatial resolution and remain fixed in position indefinitely. Thus, implanted intramuscular EMG electrodes are ideal over surface electrodes for long-term experiments using rodents. To record intramuscular EMG reliably in mice, researchers have developed a method for hand fabricating and implanting EMG electrodes in muscles as small as those in an adult mouse&#39;s forearm. These electrodes enable chronic muscle recording during motor behavior in rodents over several weeks.
The protocol described here is the result of a decade-long refinement of established methods15,16,17,18, which has yielded a procedure for hand fabricating, implanting, and recording from wire EMG electrodes chronically implanted in flexor/extensor muscle pairs of the elbow and wrist in behaving mice. The first section describes the hand fabrication of an electrode set with four electrode pairs and an 8-pin connector&#160;for the head stage interface. The next section details the surgical implantation of the electrodes intramuscularly in upper and lower arm muscles in the same surgery as headplate implantation. Finally, representative recordings from mice performing a variety of behaviors are discussed. Overall, this method is a cost-effective and customizable way to include muscle activity measurements in head-fixed behavior experiments that is ideal for labs with some electrode fabrication experience.

<table><tbody><tr><td>#11 Scalpel Blades</td><td>World Precision Instruments</td><td>504170</td><td>For EMG electrode fabrication</td></tr><tr><td>#3 Scalpel Handle</td><td>Fine Science Tools</td><td>10003-12</td><td>For EMG electrode fabrication</td></tr><tr><td>1 mL Sub-Q Syringe (100 pack)&amp;nbsp;</td><td>Becton Dickinson</td><td>309597</td><td>For administering injectable drugs</td></tr><tr><td>12-pin connector</td><td>Newark</td><td>33AC2371</td><td>12-pin connector with brass fittings; for EMG electrode fabrication</td></tr><tr><td>18 G Needles</td><td>Exel International</td><td>26419</td><td>For EMG electrode fabrication</td></tr><tr><td>27 G Needles&amp;nbsp;</td><td>Exel International</td><td>26426</td><td>For EMG electrode fabrication</td></tr><tr><td>3 M Transpore Surgical Tape</td><td>3M</td><td>1527-0</td><td>For taping animal&#039;s limbs out during surgery</td></tr><tr><td>6-0 silk sutures</td><td>Henry Schein</td><td>101-2636</td><td>These sutures work well with delicate skin around the wrists&amp;nbsp;</td></tr><tr><td>C&amp;amp;B Metabond Complete Kit</td><td>Pearson Dental</td><td>P16-0126</td><td>Dental cement to affix connector to headplate</td></tr><tr><td>C57BL6/J Mice&amp;nbsp;</td><td>Jackson Laboratories</td><td>#000664</td><td>Wild type mice&amp;nbsp;</td></tr><tr><td>Carbofib 5-CF Tweezers (2)</td><td>Aven tools&amp;nbsp;</td><td>18762</td><td>Carbon fiber tipped forceps, used to manipulate delicate parts of electrode (stripped or inserted sections)&amp;nbsp;</td></tr><tr><td>Carprodyl (Carprofen) 50 mg/mL Injection</td><td>Ceva Animal Health, LLC</td><td>G43010B</td><td>Injectable analgesic for pain management during and after surgery</td></tr><tr><td>Castroviejo Micro Needle Holder</td><td>Fine science tools</td><td>12060-01</td><td>For suturing</td></tr><tr><td>Castroviejo Needle Holder (large)</td><td>Fine science tools</td><td>12565-14</td><td>For inserting needle into muscle&amp;nbsp;</td></tr><tr><td>Delicate Bone Scraper</td><td>Fine science tools</td><td>10075-16</td><td>To separate skin from underlying tissue&amp;nbsp;</td></tr><tr><td>Dietgel 76A Dietary Supplement</td><td>Clear H2O</td><td>72-07-5022</td><td>For post-operative care</td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine science tools</td><td>11251-35</td><td>To remove fascia overlying muscle&amp;nbsp;</td></tr><tr><td>Elizabethan collar for mouse</td><td>Kent Scientific Corporation</td><td>EC201V-10</td><td>For post-operative care</td></tr><tr><td>Enrofloxacin 2.27%</td><td>Covetrus</td><td>#074743</td><td>Injectable antibiotic for use during and after surgery</td></tr><tr><td>Epoxy gel</td><td>Devcon</td><td>14265</td><td>For EMG electrode fabrication</td></tr><tr><td>Hopkins Bulldog Clamp (4)</td><td>Stoelting</td><td>10-000-481</td><td>Tissue clamps for headplate implantation</td></tr><tr><td>Isoflurane Solution</td><td>Covetrus</td><td>11695067771</td><td>Inhalable anesthesia</td></tr><tr><td>Lidocaine Hydrochloride Injectable - 2%</td><td>Covetrus</td><td>#002468</td><td>Topical analgesic for pain management during surgery</td></tr><tr><td>Medical Grade Oxygen</td><td>Airgas</td><td>OX USP200</td><td>For administering isoflurane during surgery</td></tr><tr><td>MetriCide 1 Gallon</td><td>Metrex</td><td>10-1400</td><td>Glutaraldehyde solution for cold-sterilization of headplate and electrodes&amp;nbsp;</td></tr><tr><td>MetriTest Strips 1.5%</td><td>Metrex</td><td>10-303</td><td>Test strips for monitoring glutaraldehyde solution (recommended)</td></tr><tr><td>Model 900LS Small Animal Stereotaxic Instrument</td><td>Kopf Instruments&amp;nbsp;</td><td>900LS</td><td>Stereotax with lazy susan feature that allows platform rotation during surgery&amp;nbsp;</td></tr><tr><td>PFA-coated 0.0055&quot; braided stainless steel wire&amp;nbsp;</td><td>A-M systems&amp;nbsp;</td><td>793200</td><td>For EMG electrode fabrication</td></tr><tr><td>Povidone-iodine prep pads</td><td>Dynarex</td><td>1108</td><td>For cleaning skin&amp;nbsp;</td></tr><tr><td>Puralube Vet Ointment&amp;nbsp;</td><td>Dechra</td><td>37327</td><td>Eye ointment for surgery&amp;nbsp;</td></tr><tr><td>Sterile alcohol prep pads</td><td>Dynarex</td><td>1113</td><td>For cleaning skin&amp;nbsp;</td></tr><tr><td>Straight fine #5 forceps</td><td>Fine science tools</td><td>11295-10</td><td>For curling wire after insertion&amp;nbsp;</td></tr><tr><td>Straight fine scissors</td><td>Fine science tools</td><td>14060-11</td><td>For cutting wire&amp;nbsp;</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine science tools</td><td>91500-09</td><td>For making incisions, trimming fat and fascia, and suturing&amp;nbsp;</td></tr><tr><td>Technik Tweezers 7B-SA (2)</td><td>Aven tools</td><td>18074USA</td><td>Curved blunt forceps, for general use during surgery</td></tr><tr><td>Triple Antibiotic Ointment</td><td>Walgreens</td><td>975863</td><td>Topical antibiotic for surgery</td></tr><tr><td>V-1 Tabletop Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901806</td><td>Contains all necessary equipment for anesthesia induction and scavenging including vaporizer, induction chamber, moveable plastic nose cone, and tubing&amp;nbsp;</td></tr></tbody></table>

recording forelimb muscle activity in head&#45;fixed mice with chronically implanted emg electrodes

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with unprecedented precision in head-fixed mice performing a variety of tasks. The small size of the mouse makes the measurement of motor output difficult, as the traditional method of electromyographic (EMG) recording of muscle activity was designed for larger animals like cats and primates. Pending commercially available EMG electrodes for mice, the current gold-standard method for recording muscle activity in mice is to make electrode sets in-house. This article describes a refinement of established procedures for hand fabrication of an electrode set, implantation of electrodes in the same surgery as headplate implantation, fixation of a connector on the headplate, and post-operative recovery care. Following recovery, millisecond-resolution EMG recordings can be obtained during head-fixed behavior for several weeks without noticeable changes in signal quality. These recordings enable precise measurement of forelimb muscle activity alongside in vivo neural recording and/or perturbation to probe mechanisms of motor control in mice.

Figure 2, Figure 3, and Figure 4 show normalized muscle activity recorded from the forelimb muscles of mice performing different behaviors: treadmill walking without head-fixation (Figure 2), climbing a rotating wheel under head-fixation (Figure 3), and reaching for water droplets under head-fixation (Figure 4). Figure 2 shows 1.5 s of treadmill locomotion with an approximate step cycle estimated from the time between two elbow flexor activations. Figure 3 shows 5 s of EMG data from an animal that had the wrist extensor electrode fail 6 weeks after implantation. In Figure 3A, all four electrodes produce a clean EMG signal that aligns with the turning of the wheel (which indicates climbing). Figure 3B shows the signal from the same electrodes after failure: the wrist extensor electrode produces a noisy signal that does not change with the animal&#39;s movement. Figure 4 shows 1 s of EMG from the four forelimb muscle groups during a task in which the mouse transitioned from immobility to reaching for a water droplet.
In Figure 2, Figure 3, and Figure 4, the voltage signals were amplified and bandpass filtered (250-20,000 Hz) using a differential amplifier. Raw voltage was then subsampled to 1 kHz and z-scored for comparison across datasets. Note again that while electrodes were implanted in the four muscles specified in the protocol (biceps, triceps, ECR, and PL), it is not guaranteed that adjacent synergistic muscles did not influence EMG signal; therefore, each recording is assigned to its synergy group (elbow flexor, etc.) for accuracy. Verifying isolated recordings from single muscles would require simultaneous recordings in multiple synergists to assay for crosstalk between muscle recordings, which may be prohibitively difficult, especially in the lower arm of mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66584/66584fig01.jpg" /> 
Figure 1: Schematics of the electrode set fabrication. (A) Diagram of a single electrode pair. Gray areas indicate where to strip. (B) Diagram of the connector assembly with a single completed electrode pair inserted into the connector. The diagram in (B) is not to scale. <a href="https://www.jove.com/files/ftp_upload/66584/66584fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66584/66584fig02.jpg" /> 
Figure 2: Representative EMG recording from four muscles of a freely moving (not head-fixed) mouse walking on a treadmill. The total duration is 1.5 s. The step cycle was estimated from the time between sequential elbow extensor activations. <a href="https://www.jove.com/files/ftp_upload/66584/66584fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66584/66584fig03.jpg" /> 
Figure 3: Representative EMG recording from four muscles of a head-fixed mouse performing a naturalistic climbing behavior. The 5th row shows the position of the climbing wheel read out by a rotary encoder; changes in this value indicate that the wheel is turning and the animal is actively climbing. The total duration is 5 s. (A) Recording 36 days after implantation during climbing. (B) Recording 72 days after implantation in the same mouse after the wrist extensor electrode failed. <a href="https://www.jove.com/files/ftp_upload/66584/66584fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66584/66584fig04.jpg" /> 
Figure 4: Representative EMG recording from four muscles of a head-fixed mouse transitioning from immobility to performing a reaching movement. The total duration is 1 s. <a href="https://www.jove.com/files/ftp_upload/66584/66584fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Author Spotlight: Investigating Mouse Motor Cortex Interactions from Muscle Activity to Neural Dynamics at JoVE.com

Author Spotlight: Investigating Mouse Motor Cortex Interactions from Muscle Activity to Neural Dynamics

This protocol describes the hand fabrication and surgical implantation of electromyographic (EMG) electrodes in the forelimb muscles of mice to record muscle activity during head-fixed behavior experiments.

Recording Forelimb Muscle Activity in Head&#45;Fixed Mice with Chronically Implanted EMG Electrodes

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with ...

recording-forelimb-muscle-activity-head-fixed-mice-with-chronically

Research

JoVE Journal

Neuroscience

2.2K Views.  Northwestern University. This protocol describes the hand fabrication and surgical implantation of electromyographic (EMG) electrodes in the forelimb muscles of mice to record muscle activity during head-fixed behavior experiments. 

Video: Author Spotlight: Investigating Mouse Motor Cortex Interactions from Muscle Activity to Neural Dynamics

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

Behavior

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 ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Medicine

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

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

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

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

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

Computer-based Multitaper Spectrogram Program for Electroencephalographic Data

Current web resources provide limited, user friendly tools to compute spectrograms for visualizing and quantifying electroencephalographic (EEG) data. This paper describes a Windows-based, open source code for creating EEG multitaper spectrograms. The compiled program is accessible to Windows users without software licensing. For Macintosh users, the program is limited to those with a MATLAB software license. The program is illustrated via EEG spectrograms that vary as a function of states of sleep and wakefulness, and opiate-induced alterations in those states. The EEGs of C57BL/6J mice were wirelessly recorded for 4 h after intraperitoneal injection of saline (vehicle control) and antinociceptive doses of morphine, buprenorphine, and fentanyl. Spectrograms showed that buprenorphine and morphine caused similar changes in EEG power at 1&#8722;3 Hz and 8&#8722;9 Hz. Spectrograms after administration of fentanyl revealed maximal average power bands at 3 Hz and 7 Hz. The spectrograms unmasked differential opiate effects on EEG frequency and power. These computer-based methods are generalizable across drug classes and can be readily modified to quantify and display a wide range of rhythmic biological signals.

This protocol provides an open source, compiled MATLAB program that generates multitaper spectrograms for electroencephalographic data.

Current web resources provide limited, user friendly tools to compute spectrograms for visualizing and quantifying electroencephalographic (EEG) data. ...

Micro-Drive Implant for Recording Daily Neural Activity in Juvenile Mice

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of particular importance for studying mouse models of human neuropsychiatric disorders. However, such methods often require large cranial implants, which cannot be used in mice at early developmental time points. As such, virtually no studies of in vivo physiology have been performed in freely behaving infant or juvenile mice, despite the fact that a better understanding of neurological development in this critical window would likely provide unique insights into age-dependent developmental disorders such as autism or schizophrenia. Here, a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy are described that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in mice as they age from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond, a time window roughly corresponding to the human ages of 2 years old through to adulthood. The number of recording electrodes and final recording sites can be easily modified and expanded, thus allowing flexible experimental control of the in vivo monitoring of behavior- or disease-relevant brain regions across development.

Author Spotlight: A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice  

Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond.

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Angled Silicon Multielectrode Recordings for Deep Brainstem Structures in Mice

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

Author Spotlight: Investigating Anesthesia-Induced Sleep Pathways and Neuronal Excitability in Mice

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

Source: Pollari, E. et al., <a href="https://app.jove.com/v/57741">In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates an in vivo technique for measuring compound muscle action potential (CMAP) in mice. It outlines the steps for setting up electrodes, stimulating forelimb neurons that innervate muscle fibers, and recording muscle responses. The technique assesses neuronal demyelination and functional neuron loss.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Recording Brain Electrical Activity in Behaving Mice Using Multi-Shank Linear Silicon Probes

Source: Sauer, J., et al. <a href="https://app.jove.com/v/57714">Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice.</a> J. Vis. Exp. (2018).
This video outlines a protocol for implanting a multi-shank linear silicon probe into the mouse brain to measure local field potentials. The procedure involves securing the probe to a stereotaxic unit, positioning it accurately over the target brain region, inserting it into the brain, and securing it with dental cement. After implantation, the assembly is protected with a casing. Following recovery, the setup allows for recording local field potentials, providing insights into neuronal activity.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Recording Forelimb Muscle Activity in Head-Fixed Mice with Chronically Implanted EMG Electrodes

Summary

Explore More Videos

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

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

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

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

Computer-based Multitaper Spectrogram Program for Electroencephalographic Data

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Measuring Compound Muscle Action Potentials in Mouse Forelimb Muscles In Vivo

Recording Brain Electrical Activity in Behaving Mice Using Multi-Shank Linear Silicon Probes