This work was funded in part by a grant from the Osteopathic Heritage Foundations to BC Clark. We would like to state a special thank you to Marissa McGinley for her assistance in creating many of the figure graphics.

1. Single and Paired-Pulse TMS of the FCR and ES Muscles
<ol>
	<li>Basic Safety Precautions: Prior to performing TMS on a human subject it is necessary to first screen them for basic safety precautions as it pertains to exposure to a magnetic field. In our laboratory we follow the screening guidelines set forth by the Institute for Magnetic Resonance Safety, Education and Research 10. In our laboratory we also routinely exclude individuals with a family history of epilepsy seizures. We also require subjects undergoing TMS of the ES muscles to wear earplugs and a mouth guard due to the less focal and stronger stimulation intensities.</li>
	<li>Electrical Recordings: To examine TMS responses in the motor system it is necessary to record electromyographic (EMG) signals from skeletal muscles. For the FCR muscle we place surface electrodes on the forearm using a bipolar electrode arrangement located longitudinally over the muscle on shaved and abraded skin as we have previously described 7,11. For the erector spinae muscles we use a similar electrode arrangement located longitudinally over the muscles at the L3-L5 vertebral level on shaved and abraded skin 8.</li>
	<li>TMS Coil Orientation: To predominantly activate corticospinal neurons transsynaptically it is necessary to position the TMS coil appropriately 12. For the FCR muscles we place a 70-mm figure-of-eight TMS coil tangential to the scalp and 45-degrees to the midline so that the induced current flows in a lateral-posterior to medial-anterior direction. For the ES muscles we use a double-cone coil that has greater penetration depth and is needed due to the representation of these muscles being deeper in the homunculus. Here, the coil is positioned such that the current flows in an anterior to posterior direction. We have custom-modified our coil with a laser attachment system to assist us in subsequent re-positioning of the double cone coil.</li>
	<li>Identifying 'Hotspot': It is necessary to determine the stimulation location that elicits the largest motor evoked potential. For the FCR muscle we do this by subtly moving the TMS coil around in very small increments and determining where we observe the largest motor evoked potential amplitude. Once located we note this area with indelible ink on either the scalp or a lycra cap. TMS of the ES muscles is considerably more uncomfortable to human subjects than TMS of upper limb muscles. Accordingly, we have streamlined our TMS protocol for the ES muscles to increase it's tolerability and feasibility. Here, instead of locating the "hotspot" we use anthropometric measurements to identify the vertex of the skull. Specifically, we identify the vertex as the intersection of the skull in the sagittal (between the nasinon and inion) and coronal (between the tragus) planes.</li>
	<li>Biomechanical Positioning: In our laboratory when we perform TMS of the FCR muscles subjects are seated with the arm resting in an extended position in a BioDex System 4 Dynamometer (Figure 2). However, we should note that this is only an example of one possible set-up for measuring exerted forces. For the ES muscles subjects are asked to sit with an upright posture while their hands rest in their lap (Figure 3). They are seated in a swivel base chair with the thigh at 90&deg; relative to the trunk, the lower leg at ~45&deg; relative to the thigh, and the lumbar spine in a neutral posture8.</li>
	<li>Quantifying Motor Threshold: For the FCR, we determine motor threshold (MT) by delivering single pulses at gradually increasing stimulation intensities until motor evoked potentials have peak-to-peak amplitudes greater than 50 microvolts in more than 50% of trials (Figure 4). To streamline the TMS protocol and increase tolerability and feasibility we do not determine motor threshold in the ES muscles with the same precision as when we test the upper limb musculature. Rather, we begin the TMS protocol by delivering an initial single pulse at 50% of the maximum stimulator output to determine if this stimulus intensity is above or below motor threshold. If an MEP is observed at this stimulus intensity-defined as discernable MEP relative to the level of the background EMG-the intensity is reduced to 40% of stimulator output to determine if this stimulus intensity is sub- or supra-threshold 8.</li>
	<li>Quantifying MEP Amplitude using Single-Pulse TMS: To examine the motor evoked potential amplitude of the FCR we deliver a single TMS pulse to the 'hotspot' at an intensity equal to 130% of motor threshold, and calculate the peak-to-peak amplitude. Generally, we normalize this outcome to the maximal compound muscle fiber action potential observed following supramaximal electrical stimulation of the median nerve. We should note that the MEP size is very dependent on the degree of cortical excitability. Accordingly, when the TMS pulse is delivered during a background contraction, when cortical excitability is increased, the MEP size will dramatically increase. For the ES muscles, we deliver a single TMS pulse to the vertex at an intensity 40 or 50% above the sub-motor threshold intensity 8. Unfortunately, because peripheral nerves innervating the ES muscles are not accessible to electrical stimulation we are not able to normalize these motor evoked potentials to the compound muscle fiber action potential.</li>
	<li>Quantifying Silent Period Duration using Single-Pulse TMS: When a TMS pulse to the cortex is delivered during a muscle contraction it will produce a motor evoked potential followed by electrical quiescence before activity resumes that is indicative of corticospinal inhibition and commonly referred to as the silent period 13 (Figure 5). To quantify the silent period we deliver a single TMS pulse to the 'hotspot' at an intensity equal to 130% of motor threshold while the study participant is performing a wrist flexion muscle contraction at 15% of their maximal strength. We have not previously quantified the silent period duration of the ES muscles; however, we should note that we have anecdotally observed its existence in this muscle group when the TMS pulse id delivered during a background contraction.</li>
	<li>Quantifying Intracortical Facilitation using Paired-Pulse TMS: We use paired-pulse TMS to quantify intracortical facilitation 6,7 (Figure 6 and 7 represents this measurement for the FCR and ES muscles, respectively). For the FCR muscle we first determine the stimulus intensity needed to elicit a motor evoked potential that is between 0.5-1.0 mV. Next, we deliver a subthreshold conditioning pulse-which in our laboratory is commonly set equal to 70% of motor threshold- 15-msec before the suprathreshold test pulse. This conditioning pulse delivered at this time period prior to the test pulse will increase, or facilitate, the amplitude of the motor evoked potential more than a single unconditioned pulse of the same intensity. For the ES muscle group the conditioning pulse intensity is set to the observed sub-motor threshold intensity (either 40% or 50% of stimulator output) and the test pulse intensity is set to 40% above the sub-motor threshold level (80% or 90% of stimulator output)8. We should note that the intensity of the conditioning pulses could be varied depending on the objective of the study. Similarly, the pulse intervals can vary depending on the muscle and its location relative to the cortex.</li>
	<li>Quantifying Short-Interval Intracortical Inhibition using Paired-Pulse TMS: We also use paired-pulse TMS to quantify short-interval intracortical inhibition 6,7 (Figure 6 and 7 represents this measurement for the FCR and ES muscles, respectively). Here, for both the FCR and ES muscles, the procedures are the same as described for measuring intracortical facilitation with the exception that the interstimulus interval between the two pulses is reduced to 3 msec. This conditioning pulse delivered at this time period prior to the test pulse will decrease, or inhibit, the amplitude of the motor evoked potential more than a single unconditioned pulse of the same intensity.</li>
	<li>Quantifying Long-Interval Intracortical Inhibition using Paired-Pulse TMS: Delivering two identical suprathreshold test pulses that are separated by 100 milliseconds can also be used to assess long-interval intracortical inhibition 6,7. In this case-for the FCR muscle- the motor evoked potential associated with the second pulse will be smaller, or inhibited more, than that associated with the first (Figure 8). We have not previously quantified long-interval intracortical inhibition in the ES muscles due to concerns over subject tolerability.</li>
</ol>
2. Representative Results:
Following the delivery of a suprathreshold TMS pulse, the muscles being stimulated should demonstrate an easily observable EMG response (the MEP) (illustrated in Figures 4-8). The latency between the stimulus onset and the MEP will vary between the muscle groups being examined, but for the FCR it is generally 16-19 msec (Figure 6) and for the ES it is 17-22 msec (Figure 7; although it should be noted that in some subjects definitive MEP onset in the ES muscles is more difficult to visually identify). It should be noted that when testing the ES muscle group several other muscle groups are also visibly and dramatically stimulated concomitantly (including the muscles of the lower extremity, which are represented within the same general region of the homunculus). During the measurement of intracortical facilitation the MEP amplitude is generally larger than that observed with a single unconditioned pulse (Figure 6 and 7). However, it is our experience that the degree of facilitation varies between muscles groups with some muscle groups-such as the FCR- showing only modest facilitation in many subjects. For the measurement of short-interval and long-interval intracortical inhibition a decrease in the MEP amplitude is generally observed in comparison to a single unconditioned pulse of the same intensity (Figures 6-8).
<img alt="Figure 1" src="/files/ftp_upload/3387/3387fig1.jpg" /> 
Figure 1. The basic mechanisms of TMS. The TMS coil induces a magnetic field, which penetrates the scalp and induces an Eddy current within the motor cortex. This eddy current is then able to stimulate neurons within the brain. Figure reprinted from McGinley and Clark, In Press14.
<img alt="Figure 2" src="/files/ftp_upload/3387/3387fig2.jpg" /> 
Figure 2. Setup for performing TMS on the FCR muscle. Note the recording of electromyogram (EMG) signals from the forearm, and the TMS paddle over the motor cortex. We generally also record muscle forces, and use electrical peripheral nerve stimulation to obtain the maximal compound muscle fiber action potential, as this is useful in interpreting amplitude values (e.g., one can express and MEP relative to the maximal muscle response as opposed to a absolute mV value which can be heavily influenced by non-physiologic factors such as subcutaneous adipose tissue). Figure reprinted from the following: Clark et al. 20089, Clark et al., 20106, and McGinley et al. 20107.
<img alt="Figure 3" src="/files/ftp_upload/3387/3387fig3.jpg" /> 
Figure 3. Setup for performing TMS on the erector spinale muscles. Figure reprinted from Goss et al. 20118.
<img alt="Figure 4" src="/files/ftp_upload/3387/3387fig4.jpg" /> 
Figure 4. Example of the motor threshold determination. The EMG traces represent the motor evoked potential (MEP) response to gradually increasing stimulus intensities (represented as a percentage of stimulator output (SO)). Note that at the lower intensities (28-30% of SO) very small MEPs were elicited (sub-threshold), but that at 32% SO a MEP was elicited that reached motor threshold (typically defined as an MEP with a p-p amplitude &gt; 50 &mu;V).
Figure reprinted from McGinley and Clark, In Press14.
<img alt="Figure 5" src="/files/ftp_upload/3387/3387fig5.jpg" /> 
Figure 5. TMS during a contraction: motor evoked potential &amp; silent period. The silent period is observed when a subject performs a slight contraction and a single stimulus is applied to the motor cortex. The first part of the silent period is due to spinal cord inhibition and the latter part is attributed to cortical inhibition, specifically GABAB receptors. There is no consensus way to quantify the duration of the silent period, but our findings indicate that either defining it from stimulus onset or MEP onset to the return of the voluntary interference electromyogram signal is the most reliable15. 
Figure reprinted from Clark and Quick, 201116, and McGinley and Clark, In Press14.
<img alt="Figure 6" src="/files/ftp_upload/3387/3387fig6.jpg" /> 
Figure 6. Change in motor evoked potential sized ith paired pulse TMS of the FCR muscle. Measurement of short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF). To quantify SICI and ICF the conditioning pulse (CP) is set below motor threshold, and the test pulse (TP) is set to evoke MEP's between 0.5-1 mV. At short interstimulus intervals (e.g., 3-msec) the CP inhibits the MEP in comparison to the TP only (SICI), whereas at longer interstimulus intervals (e.g., 15-msec) it facilitates the MEP (ICF).
CP: conditioning pulse, TP: test pulse Figure reprinted from Clark et al., 20106, McGinley et al. 201014, Clark and Quick, 201116, and McGinley and Clark, In Press14.
<img alt="Figure 7" src="/files/ftp_upload/3387/3387fig7.jpg" /> 
Figure 7. Change in motor evoked potential sized with paired pulse TMS of the ES muscle. Example of EMG traces from the erector spinae muscles and the measurement of short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF). 
Figure reprinted from Goss et al. 20118.
<img alt="Figure 8" src="/files/ftp_upload/3387/3387fig8.jpg" /> 
Figure 8. Change in motor evoked potential sized with paired pulse TMS. Measurement of long-interval intracortical inhibition (LICI). To quantify LICI two test pulses are delivered at an interstimulus interval of 100-msec. This results in the second MEP being inhibited in comparison to the first MEP. 
Figure reprinted from Clark et al., 20106, McGinley et al. 20107 and McGinley and Clark, In Press14.

No conflicts of interest declared.

The overall goal of this article is to provide scientists and clinicians a visual account of our laboratories use of transcranial magnetic stimulation. However, in addition to providing a visualization of these experiments, below we discuss basic issues to consider when performing TMS in this manner, provide a brief overview of the physiology of TMS responses, and also discuss our use of TMS with regards to the usage of others.
General Issues To Be Aware of When Performing TMS As Described in th...

<table border="1">
<tbody>
<tr>
<th>Name of the Equipment</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments (optional)</th>
</tr>
<tr>
<td>Transcranial Magnetic Stimulator 2002 
Transcranial Magnetic Stimulator Bi-Stim2 
Figure-Eight 70-mm coil 
Double Cone Coil</td>
<td>The Magstim Company</td>
<td>NA</td>
<td>TMS equipment (including coils)</td>
</tr>
<tr>
<td>Biodex System 4 </td>
<td>Biodex</td>
<td>NA</td>
<td>Dynamometer</td>
</tr>
<tr>
<td>Biopac MP150 Data Acquisition System</td>
<td>Biopac</td>
<td>MP150WSW</td>
<td>A-D converter for EMG and force</td>
</tr>
<tr>
<td>AcqKnowledge 4.0 Data acquisition software</td>
<td>Biopac</td>
<td>ACK100W</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nikomed Trace 1 ECG electrodes</td>
<td>Nikomed</td>
<td>2015</td>
<td>EMG electrodes</td>
</tr>
<tr>
<td>Constant Current Stimulator</td>
<td>Digitimer</td>
<td>DS7A</td>
<td>Peripheral nerve stimulator</td>
</tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System at JoVE.com

Utilizing Transcranial Magnetic Stimulation 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 ...

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26.5K 조회수.  Ohio University. Transcranial 자기 자극 (TMS)은 인간의 신경 시스템의 생리와 기능에 대한 통찰력을 얻을 수있는 비침습적인 도구입니다. 여기, 우리는 상부 사지와 허리 근육의 외피 흥분을 공부하기 위해 TMS 기술을 제시한다.

비디오: Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
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A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

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Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
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Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes.
Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets.

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation HD-tDCS

High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation.

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Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

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Using Retinal Imaging to Study Dementia

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Surgical Labyrinthectomy of the Rat to Study the Vestibular System

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To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

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Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Employing the Forced Oscillation Technique for the Assessment of Respiratory Mechanics in Adults

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and diseased individuals. FOT, a complementary method to traditional pulmonary function testing, utilizes a range of oscillatory frequencies superimposed on tidal breathing to measure the functional relationship between airway pressure and flow. This passive assessment provides an estimate of respiratory system resistance (Rrs) and reactance (Xrs) that reflect airway caliber and energy storage and dissipation, respectively. Despite the recent increase in popularity and updated Technical Standards, clinical adoption has been slow which relates, in part, to the lack of standardization regarding the acquisition and reporting of FOT data. The goal of this article is to address the lack of standardization across laboratories by providing a comprehensive written protocol for FOT and an accompanying video. To illustrate that this protocol can be utilized irrespective of a particular device, three separate FOT devices have been employed in the case examples and video demonstration. This effort is intended to standardize the use and interpretation of FOT, provide practical suggestions, as well as highlight future questions that need to be addressed.

As the use of forced oscillation technique (FOT) is increasingly utilized to characterize respiratory mechanics, there is a need to standardize methods with respect to nascent technical guidelines and various manufacturer's recommendations. A detailed protocol is provided including FOT assessment and interpretation for two cases to facilitate the standardization of methods.

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and ...