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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+large+scale+neural+activity+with+cellular+resolution+in+awake+mobile+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+of+cortical+activity+underlying+a+tactile+decision+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortex+commands+the+performance+of+skilled+movement.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+forelimb+motor+cortex+areas+in+goal+directed+action+in+mice.">The role of forelimb motor cortex areas in goal directed action in mice.</a> Sci Rep. 7 (1), 15759 (2017).</li><li>Gali&#241;anes, G. L., Bonardi, C., Huber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+reaching+for+water+as+a+cortex-dependent+behavioral+framework+for+mice.">Directional reaching for water as a cortex-dependent behavioral framework for mice.</a> Cell Rep. 22 (10), 2767-2783 (2018).</li><li>Evarts, E. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cutaneous+afferents+in+the+control+of+gamma-motoneurones+during+locomotion+in+the+decerebrate+cat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+of+motor+output,+from+mouse+to+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+pattern+generation+during+dexterous+movement+is+input-driven.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+dexterity+of+mice+during+food-handling+involves+the+thumb+and+a+set+of+fast+basic+movements.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Task-specific+modulation+of+corticospinal+neuron+activity+during+motor+learning+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+surface+EMG+characteristics+from+rat+triceps+brachii+muscle+during+treadmill+locomotion+indicate+selective+recruitment+of+functionally+distinct+muscle+regions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+and+electromyographic+tools+for+characterizing+movement+disorders+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+electrode+configuration+for+recording+electromyographic+activity+in+behaving+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electromyographic+characterization+of+mice+lacking+EphA4+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+mouse+locomotor+pattern+in+the+absence+of+proprioceptive+sensory+feedback.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behaviorally+selective+engagement+of+short-latency+effector+pathways+by+motor+cortex.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIVETS:+A+mechanical+system+for+in+vivo+and+in+vitro+electrophysiology+and+imaging.">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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)&#160;</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&#160;</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&#39;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&#160;</td>
		</tr>
		<tr>
			<td>C&#38;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&#160;</td>
			<td>Jackson Laboratories</td>
			<td>#000664</td>
			<td>Wild type mice&#160;</td>
		</tr>
		<tr>
			<td>Carbofib 5-CF Tweezers (2)</td>
			<td>Aven tools&#160;</td>
			<td>18762</td>
			<td>Carbon fiber tipped forceps, used to manipulate delicate parts of electrode (stripped or inserted sections)&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Delicate Bone Scraper</td>
			<td>Fine science tools</td>
			<td>10075-16</td>
			<td>To separate skin from underlying tissue&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>900LS</td>
			<td>Stereotax with lazy susan feature that allows platform rotation during surgery&#160;</td>
		</tr>
		<tr>
			<td>PFA-coated 0.0055&#34; braided stainless steel wire&#160;</td>
			<td>A-M systems&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment&#160;</td>
			<td>Dechra</td>
			<td>37327</td>
			<td>Eye ointment for surgery&#160;</td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pads</td>
			<td>Dynarex</td>
			<td>1113</td>
			<td>For cleaning skin&#160;</td>
		</tr>
		<tr>
			<td>Straight fine #5 forceps</td>
			<td>Fine science tools</td>
			<td>11295-10</td>
			<td>For curling wire after insertion&#160;</td>
		</tr>
		<tr>
			<td>Straight fine scissors</td>
			<td>Fine science tools</td>
			<td>14060-11</td>
			<td>For cutting wire&#160;</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&#160;</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&#160;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.0K 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

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

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

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