The authors are grateful to Dr. Margaret Wardle for her exceptional training and technical assistance. This work was funded in part by Swedish Research Council grant FYF-2013-687 (IM).

This protocol is based on Mayo et al.31 (Experiment 1) and Ree et al.32 (Experiment 2). Ethical approval was granted by the Regional Ethical Review Board, Link&#246;ping, Sweden (Experiment 1) and the local ethical committee at the Department of Psychology, University of Oslo, Norway (Experiment 2).
1. Participant Screening and Preparation
<ol>
	<li>Recruit participants who lack tactile or uncorrected visual disturbances and are free of any neurological or psychiatric disorder, unless a specific patient population is being recruited.</li>
	<li>Ensure that participants are fully able to understand task instructions (e.g., fluent in the language that tasks are administered).</li>
	<li>If including more than one task (e.g. Experienced, Observed), ensure that task order is counterbalanced across participants, stratifying for gender, age, or other distinguishing factors.</li>
</ol>
2. Stimuli and Task Construction
NOTE: See Table 1 for experimental design.
<ol>
	<li>Experienced touch task (Experiments 1 and 2)
	<ol>
		<li>Create trials such that they consist of a baseline period, touch administration, and self-report ratings, all separated by jittered ITIs.
		<ol>
			<li>Baseline periods consist of a blank screen, fixation cross, or other neutral scene prior to tactile stimulation.</li>
			<li>Tactile stimulation is followed by a short (e.g. 1-2 s) ITI, then self-report ratings are obtained.</li>
			<li>A jittered inter-trial interval (ITI; e.g. 6-7 s) follows self-report ratings to allow muscle activity to return to baseline levels before the next trial begins.</li>
		</ol>
		</li>
		<li>Use either audio (Experiment 131) or visual (Experiment 232) cues to ensure that touch is delivered at the appropriate velocity.
		<ol>
			<li>To use audio cues, have cues delivered to headphones worn by the experimenter to track the pace of the stimulation using a metronome. Distinguish velocities using tones of differing pitches (or other distinguishing audio cue, e.g., a cue saying &#34;10 cm/s&#34;) that precede the stimulation cues.</li>
			<li>To use visual cues, display cues on a tablet only in view of the experimenter. Use a moving bar to track velocity of touch administration.</li>
		</ol>
		</li>
		<li>Prior to the start of the study, practice to ensure that touch is delivered at the appropriate velocity and a consistent pressure. To do so, apply brushstrokes to the scale in a similar manner as to the participant. The scale readout is used to determine if the pressure changes throughout touch administration. For instance, a pressure of 0.4 N would read as 40 g on the scale.</li>
	</ol>
	</li>
	<li>Observed touch task (Experiment 1)
	<ol>
		<li>Ensure that videos of touch administration are of similar length, regardless of velocity.
		<ol>
			<li>Include both CT-optimal (1-10 cm/s) and non-optimal (less than 1 cm/s or more than 10 cm/s) velocities.</li>
		</ol>
		</li>
		<li>Start trials with a fixation cross or other neutral condition followed by video. 
		NOTE: Videos contain touch delivered to CT-rich hairy skin (arm), CT-lacking glabrous skin (palm), and a non-social condition in which touch is delivered to a fake wooden arm (Fig. 2; see supplemental videos).
		<ol>
			<li>After a 1-2 s ITI, obtain self-report ratings.</li>
			<li>Allow another 6-7 ITI following ratings to precede the next trial to allow EMG activity to return to baseline.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Facial Electromyography
<ol>
	<li>Data acquisition and filtering guidelines (based on previous protocols27,34)
	<ol>
		<li>Use software to apply filtering steps either in real-time or offline. Typical filtering steps include a comb band stop filter to filter out potential noise from AC power (50/60 Hz), followed by smoothing and rectification. 
		NOTE: Initial basic filtering steps may be set on EMG amplifiers (e.g., a high pass filter of 10 Hz and a low pass filter of 500 Hz or 1,000 Hz).</li>
	</ol>
	</li>
	<li>Electrode application (based on previous protocols27,34)
	<ol>
		<li>Briefly describe the application process to the participant. Use neutral words (&#34;sensor&#34;) instead of potentially anxiety-evoking words (&#34;electrode&#34;)34.
		<ol>
			<li>Decide what information to tell the participants regarding the purpose of the sensors. 
			NOTE: In the current studies, participants were told sensors would measure muscle and sweat activity during the session.</li>
		</ol>
		</li>
		<li>Clean the participants&#39; skin prior to electrode application.
		<ol>
			<li>Use water to wipe clean the areas in which sensors will be applied.</li>
			<li>Use an exfoliant scrub to lightly abrade the same areas. Use caution to prevent major skin irritation, though minor irritation is likely to occur.</li>
		</ol>
		</li>
		<li>Use electrode pairs consisting of two 4 mm shielded bipolar recording electrodes plus one monopolar reference electrode.
		<ol>
			<li>Apply adhesive collars to the electrodes such that they adhere to the skin.</li>
			<li>Once collars adhere to the outer rim of the electrodes, fill sensors with a conductive electrode gel, taking care to prevent the formation of air bubbles.</li>
		</ol>
		</li>
		<li>Place electrode pairs parallel to the muscle(s) of interest and perpendicular to potential sources of noise, such as other muscles34.
		<ol>
			<li>Corrugator: Affix one electrode directly above the eyebrow along an imaginary vertical line that traverses the inner corner of the eye. Place the second electrode 1 cm lateral and slightly superior to the first, along the border of the eyebrow.</li>
			<li>Zygomatic: Place the first sensor midway along an imaginary line that connects the upper ear (where the ear meets the skull) and the corner of the mouth. Place the second electrode 1 cm medial (towards the mouth). Take care to avoid the masseter muscle.</li>
			<li>Use an 8 mm unshielded, monopolar recording electrode as a reference electrode. Place the electrode in the middle of the forehead, equidistant (above) the inner brows and (below) the hairline.</li>
			<li>Ensure that electrode wires are placed such that they do not impede vision. Use medical tape to ensure long-term adherence of the electrodes to skin and reduce noise/artifacts due to cord movement.</li>
		</ol>
		</li>
		<li>Determine the quality of electrode application with an impedance monitor. Acceptable impedance levels are below 20 k&#8486;. If electrodes need to be reapplied to reach appropriate impedance levels, use a clean pair of electrodes.</li>
	</ol>
	</li>
</ol>
4. Task Procedure
<ol>
	<li>General order
	<ol>
		<li>Following sensor application, complete task(s). If using more than one task, counterbalance order across participants.</li>
		<li>Ensure that participants are seated comfortably to minimize extraneous movement that may introduce movement artifacts34.</li>
	</ol>
	</li>
	<li>Experienced touch task
	<ol>
		<li>Seat participants in front of computer with the to-be-touched arm extended laterally, resting comfortably (e.g., on a cushion). 
		NOTE: It is recommended to apply touch to the arm that is not being used for self-reported ratings in order to minimize potential movement artifacts in the EMG signal.</li>
		<li>Occlude view of the arm from the participant either using a curtain separator31 or goggles that occlude lateral vision (Figure 132) 35.</li>
		<li>Instruct the participant to focus on how the touch makes them feel.</li>
		<li>Vary touch location to avoid CT fatigue36.</li>
		<li>Administer touch using a 75 mm goat hair brush applied to designated section(s) marked on the arm (and palm). Alternatively, apply touch using a force-controlled robot37.</li>
		<li>Use consistent touch administration direction, e.g., back-and-forth (distal-to-proximal, then proximal to distal) or single direction (proximal-to-distal only)</li>
	</ol>
	</li>
	<li>Observed touch task
	<ol>
		<li>Seat participant in front of the computer that will display the videos.</li>
		<li>Instruct the participant that they will have to rate how the video made them feel.</li>
		<li>Ensure that the participant is out of view of the experimenter34.</li>
	</ol>
	</li>
</ol>
5. Data Cleaning and Analysis
<ol>
	<li>To assess the mean EMG activation to a specific touch stimulus type, compare the response to the touch stimulus to the preceding baseline, i.e. [mean activation during 6 s touch stimulation] - [mean activation during 1 s prestimulus &#34;baseline&#34;], as suggested by Fridlund and Cacioppo34.
	<ol>
		<li>Average responses for each touch stimulus type (CT-optimal, non-optimal and, if appropriate, each location (arm/palm).</li>
		<li>Do this for each muscle (corrugator, zygomatic) and self-report rating (pleasantness, intensity) individually.</li>
	</ol>
	</li>
	<li>To obtain a more sensitive time course, compute mean EMG activation during smaller time intervals (e.g., 700 ms; see Figure 532). Subtract the same 1 s baseline from all intervals to remove baseline EMG activity. 
	NOTE: Prior to analysis, it is recommended to have data manually checked by raters blinded to touch conditions to eliminate trials with artifactual activations34.</li>
</ol>

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B., Olausson, H., Wessberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unmyelinated+Afferents+Constitute+a+Second+System+Coding+Tactile+Stimuli+of+the+Human+Hairy+Skin.">Unmyelinated Afferents Constitute a Second System Coding Tactile Stimuli of the Human Hairy Skin.</a> Journal of Neurophysiology. 81 (6), 2753-2763 (1999).</li><li>Triscoli, C., Olausson, H., Sailer, U., Ignell, H., Croy, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CT-optimized+skin+stroking+delivered+by+hand+or+robot+is+comparable.">CT-optimized skin stroking delivered by hand or robot is comparable.</a> Frontiers in Behavioral Neuroscience. 7, 208 (2013).</li><li>Croy, I., 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=Interpersonal+stroking+touch+is+targeted+to+C+tactile+afferent+activation.">Interpersonal stroking touch is targeted to C tactile afferent activation.</a> Behavioural Brain Research. 297, 37-40 (2016).</li><li>Keizer, A., de Jong, J. R., Bartlema, L., Dijkerman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+perception+of+the+arm+manipulates+the+experienced+pleasantness+of+touch.">Visual perception of the arm manipulates the experienced pleasantness of touch.</a> Developmental Cognitive Neuroscience. , (2017).</li><li>Pawling, R., Cannon, P. R., McGlone, F. P., Walker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-tactile+afferent+stimulating+touch+carries+a+positive+affective+value.">C-tactile afferent stimulating touch carries a positive affective value.</a> PloS One. 12 (3), 0173457 (2017).</li><li>Scheele, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oxytocin-induced+facilitation+of+neural+and+emotional+responses+to+social+touch+correlates+inversely+with+autism+traits.">An oxytocin-induced facilitation of neural and emotional responses to social touch correlates inversely with autism traits.</a> Neuropsychopharmacology. 39 (9), 2078-2085 (2014).</li><li>Ackerley, R., Saar, K., McGlone, F., Backlund Wasling, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+sensory+and+emotional+perception+of+touch:+differences+between+glabrous+and+hairy+skin.">Quantifying the sensory and emotional perception of touch: differences between glabrous and hairy skin.</a> Frontiers in Behavioral Neuroscience. 8 (34), (2014).</li><li>Loken, L. S., Evert, M., Wessberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleasantness+of+touch+in+human+glabrous+and+hairy+skin:+order+effects+on+affective+ratings.">Pleasantness of touch in human glabrous and hairy skin: order effects on affective ratings.</a> Brain Research. 1417, 9-15 (2011).</li></ol>

The authors have nothing to disclose

Here, we report on the use of facial electromyography (EMG) as a method to study affective responses to observed and experienced touch. Previously, many studies have focused on the use of self-report ratings to characterize the affective quality of touch. Touch that optimally activates CT afferents (e.g., 1-10 cm/s) is consistently rated as more pleasant than either faster or slower touch velocities10. In contrast, ratings of intensity seem to track with velocity, with faster touch velocities rate...

C-tactile (CT) afferents are proposed to convey the affective component of touch, which can be distinguished from the discriminative aspects of touch processed via A&#946; fibers1,2. CT-mediated affective touch is believed to play an integral role in social affiliative behaviors3, leading to the &#34;skin as a social organ&#34; hypothesis4. Physical5,6, developmental7, and psychiatric8,9 factors can influence CT-mediated touch processing. Thus, establishing an objective measure to quantify affective reactions to CT-relevant touch is critical to allow for comparisons across populations.
In recent years, much insight has been gained regarding the characteristics of CT afferents. These unmyelinated afferents demonstrate an inverted U-shaped firing frequency, with velocities of 1-10 cm/s (&#34;CT-optimal&#34;) eliciting the greatest frequency and both greater (&#34;fast non-optimal&#34;) or lesser (&#34;slow non-optimal&#34;) velocities eliciting reduced firing10. CT firing frequency correlates with self-reported ratings of touch &#34;pleasantness&#34;, producing a similar inverted U-shaped curve in pleasantness ratings10. Moreover, CT-afferents also respond most robustly to stimuli close to skin temperature11. These fibers also show distinct conduction speeds. The unmyelinated CT afferents are slower2 and thus the volley of afferent input to the cortex shows a temporal lag when compared to the speed of the faster, myelinated A&#946; fibers1,12. Affective and discriminative touch can also be distinguished on a neural level. While both types of touch activate overlapping somatosensory areas, affective touch is more likely to activate the posterior insula, while discriminative touch activates sensorimotor areas13,14,15,16. This activation pattern is consistent whether the touch is directly experienced or merely observed17, suggesting that affective touch is not just a &#34;bottom-up&#34; process driven by physical activation of CT afferents, but also involves &#34;top-down&#34; integration of multimodal sensory processing.
Situations in which CT processing is deficient or otherwise atypical has also provided insight into the functional significance of these afferents. In a unique patient group with a heritable mutation affecting the nerve growth factor &#946; gene, there is a reduction in the density of thin and unmyelinated nerve fibers, including CT afferents. Compared to healthy controls, these patients report touch at CT-optimal velocities as less pleasant5. The converse scenario is also true; patients who lack myelinated A&#946; fibers are able to retain a faint sensation of pleasant touch carried by the still intact CT afferents6. Abnormal affective touch processing is not just confined to instances of physical changes in CT-afferents. Across patient and healthy populations, those higher on the spectrum of autistic traits reported reduced pleasantness ratings of touch8. Psychiatric patients also demonstrate reduced hedonic ratings of affective touch, with a history of childhood maltreatment as one of the most consistent predictors of dysregulated affective touch awareness8. Dysregulation in the CT-based affective touch system in anorexia nervosa has also been reported9. Thus, both physical and psychological factors can influence affective touch processing, and as such, it is imperative to establish methodologies that can be applied to all individuals in an equitable and comparable manner.
Insights into normo-typical and dysregulated affective processing have the opportunity to provide a more nuanced picture of many patient groups. However, one potential limitation of affective touch research is the necessity of self-reported ratings. At times, self-report can be unreliable18 and subject to recall bias19. Inquiries of self-report can psychologically remove a participant from the current setting, limiting the ecological validity of the responses and removing them temporally from the experience20. Moreover, self-report relies on a firm understanding of language and semantics, making cross-cultural and developmentally diverse (e.g. infant and toddler-aged individuals) comparisons challenging. For instance, individuals with an autism spectrum diagnosis frequently show distinct behavioral responses to touch21, but can also have difficulties in communicating verbally22. Thus, finding non-invasive methods to measure responses to touch that circumvent a reliance on self-report may translate, at least, to a better understanding of the mechanisms of affective touch, and at most, novel insights into dysregulation of social processing in patient populations.
Facial electromyography (EMG) is a suitable candidate to objectively assess affective responses to touch. It has been used to measure valence-specific reactions to visual23, audio-visual24, olfactory25, and gustatory26 stimuli. Facial EMG is a safe and non-invasive method consisting of surface electrodes that adhere to the face27. These surface electrodes record facial muscle activity continuously in real-time with time scale sensitivity in the tens of milliseconds. Of particular interest is the corrugator supercilii (&#34;corrugator&#34;), which is activated when furrowing the brow and relaxes during a smile. As a result, corrugator activity has a linear relationship with affective valence, with increased response to negative stimuli and decreased activity in response to positive stimuli28. In addition, the zygomaticus major (&#34;zygomatic&#34;) is the muscle activated as the corners of the mouth pull up into a smile. The zygomatic displays a &#34;J-shaped&#34; activation pattern with positive stimuli eliciting the greatest response, and the most negative stimuli eliciting a greater response than neutral stimuli28. Facial EMG recordings of these muscles can even be observed when stimuli are presented outside conscious awareness or when individuals are explicitly trying to suppress their reactions29,30. Importantly, facial EMG can be used alone or in combination with self-report ratings or other physiological recordings. Thus, it is an ideal methodology to assess affective reactions to tactile stimulation31,32.
In sum, facial EMG can be combined with self-report ratings to determine how CT-optimal tactile stimulation influences facial muscle activity as a potential indicator of affective response. One can take advantage of the velocity-dependent firing frequency of CTs to apply touch at CT-optimal and non-optimal velocities, and touch can be applied both to the CT-rich arm and the putatively CT-lacking palm. Comparisons can be made across modalities to determine whether affective responses to touch require direct stimulation or can be elicited via mere observation, suggestive of shared processing across sensory modalities. Finally, upon establishing facial EMG as a suitable methodology to study affective reactions to affective touch, researchers can then explore how affective touch processing may be influenced by various interventions (e.g., drug administration; stress exposure), how it changes throughout development7, how it is influenced by the relationship of the interactants33, and whether it is dysregulated in clinical populations8.

<table><tbody><tr><td>4 mm Ag-AgCl sheilded reusable electrodes</td><td>Biopac</td><td>EL654</td><td></td></tr><tr><td>75 mm goat hair brush</td><td>IN-EX Color AB</td><td>77062</td><td>Touch application; https://www.in-exfarg.se</td></tr><tr><td>8 mm Ag-AgCl unsheilded reusable electrode</td><td>Biopac</td><td></td><td></td></tr><tr><td>Acqknowledge software</td><td>Biopac</td><td>ACK100W</td><td>Used for application of filtering steps, analysis</td></tr><tr><td>Adhesive collars</td><td>Biopac</td><td>ADD204</td><td></td></tr><tr><td>Cables</td><td>Biopac</td><td>BN-EL30-LEAD3; LEAD2</td><td>LEAD3 includes ground, LEAD2 is only bipolar recording electrodes</td></tr><tr><td>Electro-gel</td><td>Biopac</td><td>GEL100</td><td></td></tr><tr><td>EMG aplifier x 2</td><td>Biopac</td><td>BN-EMG2</td><td></td></tr><tr><td>El-Prep</td><td>Biopac</td><td>ELPREP</td><td>Facial exfoliant</td></tr><tr><td>MP160 data acqusition system</td><td>Biopac</td><td>MP160WSW</td><td></td></tr><tr><td>Presentation software</td><td>Neurobehavioral systems</td><td></td><td>Task presentation software</td></tr></tbody></table>

using facial electromyography to assess facial muscle reactions to experienced and observed affective touch in humans

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. Touch that optimally activates CT fibers is consistently rated as hedonically pleasant. Patient groups with impaired social-emotional functioning also show disordered affective touch ratings. However, relying on self-reported ratings of touch has many limitations, including recall bias and communication barriers. Here, we describe a methodological approach to study affective responses to touch via facial electromyography (EMG) that circumvents the reliance on self-report ratings. Facial EMG is an objective, quantitative, and non-invasive method to measure facial muscle activity indicative of affective responses. Responses can be assessed across healthy and patient populations without the need for verbal communication. Here, we provide two separate datasets demonstrating that CT-optimal and non-optimal touch elicit distinct facial muscle reactions. Moreover, facial EMG responses are consistent across stimulus modalities, e.g. tactile (experienced touch) and visual (observed touch). Finally, the temporal resolution of facial EMG can detect responses on timescales that supersede that of verbal reporting. Together, our data suggest that facial EMG is a suitable methodology for use in affective tactile research that can be used to supplement, or in some cases, supplant, existing measures.

CT-optimal touch elicits distinct EMG responses compared to fast non-optimal touch across modalities 
The first experiment addressed whether differential EMG reactivity could be detected in response to CT-optimal (3 cm/s) and fast non-optimal (30 cm/s) tactile stimulation that was directly experienced (Figure 3) or merely observed (Figure 2 and Figure 3)31</sup...

Watch this Scientific Journal Video about Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans at JoVE.com

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

We describe a protocol to assess facial muscle activity in response to experienced and observed tactile stimulation using facial electromyography.

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. ...

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11.0K Views.  University of Oslo. We describe a protocol to assess facial muscle activity in response to experienced and observed tactile stimulation using facial electromyography.

Video: Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

Corticospinal Excitability Modulation During Action Observation

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone else's action becomes anticipatory simulation of a complementary act. TMS was delivered to the left primary motor cortex corresponding to the hand to induce the highest level of MEP activity from the abductor digiti minimi (ADM; the muscle serving little finger abduction) as well as the first dorsal interosseus (FDI; the muscle serving index finger flexion/extension) muscles. A neuronavigation system was used to maintain the position of the TMS coil, and electromyographic (EMG) activity was recorded from the right ADM and FDI muscles. Producing original data with regard to motor resonance, the combined TMS/MEP technique has taken research on the perception-action coupling mechanism a step further. Specifically, it has answered the questions of how and when observing another person's actions produces motor facilitation in an onlooker's corresponding muscles and in what way corticospinal excitability is modulated in social contexts.

Single-pulse transcranial magnetic stimulation over the primary motor cortex, neuronavigation, and registration of electromyographic activity of hand muscles were used in this study to explore corticospinal excitability while participants were observing action sequences.

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Medicine

Conscious and Non-conscious Representations of Emotional Faces in Asperger's Syndrome

Several neuroimaging studies have suggested that the low spatial frequency content in an emotional face mainly activates the amygdala, pulvinar, and superior colliculus especially with fearful faces1-3. These regions constitute the limbic structure in non-conscious perception of emotions and modulate cortical activity either directly or indirectly2. In contrast, the conscious representation of emotions is more pronounced in the anterior cingulate, prefrontal cortex, and somatosensory cortex for directing voluntary attention to details in faces3,4. Asperger&#39;s syndrome (AS)5,6 represents an atypical mental disturbance that affects sensory, affective and communicative abilities, without interfering with normal linguistic skills and intellectual ability. Several studies have found that functional deficits in the neural circuitry important for facial emotion recognition can partly explain social communication failure in patients with AS7-9. In order to clarify the interplay between conscious and non-conscious representations of emotional faces in AS, an EEG experimental protocol is designed with two tasks involving emotionality evaluation of either photograph or line-drawing faces. A pilot study is introduced for selecting face stimuli that minimize the differences in reaction times and scores assigned to facial emotions between the pretested patients with AS and IQ/gender-matched healthy controls. Information from the pretested patients was used to develop the scoring system used for the emotionality evaluation. Research into facial emotions and visual stimuli with different spatial frequency contents has reached discrepant findings depending on the demographic characteristics of participants and task demands2. The experimental protocol is intended to clarify deficits in patients with AS in processing emotional faces when compared with healthy controls by controlling for factors unrelated to recognition of facial emotions, such as task difficulty, IQ and gender.

An EEG experimental protocol is designed to clarify the interplay between conscious and non-conscious representations of emotional faces in patients with Asperger&#39;s syndrome. The technique suggests that patients with Asperger&#39;s syndrome have deficits in non-conscious representation of emotional faces, but have comparable performance in conscious representation with healthy controls.

Several neuroimaging studies have suggested that the low spatial frequency content in an emotional face mainly activates the amygdala, pulvinar, and ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

Real-Time Mouse Eye Squint Detection for Migraine Pain Tracking Using DeepLabCut

An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.

Author Spotlight: Deciphering Electrical Networks Behind Complex Brain Activities and Disorders

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head ...

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of providing precise and controlled stimulation. This article introduces a technique for recording somatosensory event-related potentials (ERP) that uses a novel mechanical stimulation method involving skin deformation using a robotic device. Controlled deformation of the facial skin is used to modulate kinesthetic inputs through excitation of cutaneous mechanoreceptors. By combining somatosensory stimulation with electroencephalographic recording, somatosensory evoked responses can be successfully measured at the level of the cortex. Somatosensory stimulation can be combined with the stimulation of other sensory modalities to assess multisensory interactions. For speech, orofacial stimulation is combined with speech sound stimulation to assess the contribution of multi-sensory processing including the effects of timing differences. The ability to precisely control orofacial somatosensory stimulation during speech perception and speech production with ERP recording is an important tool that provides new insight into the neural organization and neural representations for speech.

This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of ...

Neuroscience

Capturing Dynamic Finger Gesturing with High-resolution Surface Electromyography and Computer Vision

Finger gestures are a critical element in human communication, and as such, finger gesture recognition is widely studied as a human-computer interface for state-of-the-art prosthetics and optimized rehabilitation. Surface electromyography (sEMG), in conjunction with deep learning methods, is considered a promising method in this domain. However, current methods often rely on cumbersome recording setups and the identification of static hand positions, limiting their effectiveness in real-world applications. The protocol we report here presents an advanced approach combining a wearable surface EMG and finger&#160;tracking system to capture comprehensive data during dynamic hand movements. The method records muscle activity from soft printed electrode arrays (16 electrodes) placed on the forearm as subjects perform gestures in different hand positions and during movement. Visual instructions prompt subjects to perform specific gestures while EMG and finger positions are recorded. The integration of synchronized EMG recordings and finger tracking data enables comprehensive analysis of muscle activity patterns and corresponding gestures. The reported approach demonstrates the potential of combining EMG and visual tracking technologies as an important resource for developing intuitive and responsive gesture recognition systems with applications in prosthetics, rehabilitation, and interactive technologies. This protocol aims to guide researchers and practitioners, fostering further innovation and application of gesture recognition in dynamic and real-world scenarios.

The paper presents a comprehensive protocol for simultaneously recording hand-electromyography (EMG) and visual finger tracking during natural finger gesturing. The visual data is designed to serve as the ground truth for the development of accurate EMG-based computational models for finger gesture recognition.

Finger gestures are a critical element in human communication, and as such, finger gesture recognition is widely studied as a human-computer interface for ...

Bioengineering

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral manipulations, there remain significant gaps in the understanding of the neural processes involved in reach planning, execution, and control. The novel approach described here combines a two-dimensional reaching task with transcranial magnetic stimulation (TMS) and concurrent electromyography (EMG) recording from multiple muscles. This method allows for the noninvasive detection of corticospinal activity at precise time points during the unfolding of reaching movements. The example task code includes a delayed response reaching task with two possible targets displayed &#177; 45&#176; off the midline. Single pulse TMS is delivered on the majority of task trials, either at the onset of the preparatory cue (baseline) or 100 ms prior to the imperative cue (delay). This sample design is suitable for investigating changes in corticospinal excitability during reach preparation. The sample code also includes a visuomotor perturbation (i.e., cursor rotation of &#177; 20&#176;) to investigate the effects of adaptation on corticospinal excitability during reach preparation. The task parameters and TMS delivery can be adjusted to address specific hypotheses about the state of the motor system during reaching behavior. In the initial implementation, motor evoked potentials (MEPs) were successfully elicited on 83% of TMS trials, and reach trajectories were recorded on all trials.

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...

SFEMG in Rodents: Assessing NMJ Function in Health and Disease

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

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

Author Spotlight: Translational Applications of Stimulated SFEMG in Rodent Models

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

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

Using TMS to Measure Motor Excitability During Action Observation

Source: Laboratories of Jonas T. Kaplan and Sarah I. Gimbel&#8212;University of Southern California
Transcranial Magnetic Stimulation (TMS) is a non-invasive brain stimulation technique that involves passing current through an insulated coil placed against the scalp. A brief magnetic field is created by current in the coil, and because of the physical process of induction, this leads to a current in the nearby neural tissue. Depending on the duration, frequency, and magnitude of these magnetic pulses, the underlying neural circuitry can be affected in many different ways. Here, we demonstrate the technique of single-pulse TMS, in which one brief magnetic pulse is used to stimulate the neocortex.
One observable effect of TMS is that it can produce muscle twitches when applied over the motor cortex. Due to the somatotopic organization of the motor cortex, different muscles can be targeted depending on the precise placement of the coil. The electrical signals that cause these muscle twitches, called motor evoked potentials, or MEPs, can be recorded and quantified by electrodes placed on the skin over the targeted muscle. The amplitude of MEPs can be interpreted to reflect the underlying excitability of the motor cortex; for example, when the motor cortex is activated, observed MEPs are larger. 
In this experiment, based on a study originally performed by Fadiga and colleagues1 and since replicated by many others,2 we use single-pulse TMS to test the excitability of motor cortex during action observation. It is known that motor cortex can be activated not only when we move, but when we watch others perform movements. A common interpretation of this phenomenon is that it reflects a simulation process that may play a role in the understanding of others' actions. Here we will record MEPs evoked by TMS over the primary motor cortex while subjects observe the movements of others compared with control stimuli.