The authors have no acknowledgements.

The experimental procedures outlined received Institutional ethical approval and are in accordance with the Declaration of Helsinki. Data were collected from a representative participant who was aware of the procedures and gave his written informed consent.
1. Instrument Preparation
<ol>
	<li>Clean the skin at the electrode location by shaving, and remove the dirt with alcohol to obtain low impedance (&#60;5 k&#937;).</li>
	<li>Place two AgCl surface electrodes (recording diameter of 10 mm) at 2/3 of the line between the medial condylis of the femur to the medial malleolus for the soleus muscle; on the most prominent bulge of the muscle for the medial gastrocnemius; at 1/3 of the distance along a line between the head of the fibula and the heel for the lateral gastrocnemius; and at 1/3 of the distance along a line between the tip of the fibula and the tip of the medial malleolus for the tibialis anterior muscle, with an interelectrode distance (center-to-center) of 2 cm, according to the SENIAM recommendations30. 
	Note: The soleus muscle electrodes have to be positioned under the distal insertion of gastrocnemii muscles to ensure that they are not recording activity from the heads of the gastrocnemii muscles (cross-talk).</li>
	<li>Place a reference electrode in a central position on the same leg (between stimulation and recording sites).</li>
	<li>Adjust the height and the depth of the chair to obtain an ankle angle of 90&#176; (0&#176; = full plantar flexion), so that the soleus and gastrocnemii muscles are not stretched and the H-reflex is not altered11,12.
	<ol>
		<li>Set the knee angle at 90&#176; (0&#176; = full knee extension) due to the biarticular nature of the gastrocnemii muscles. However, the optimal ankle angle to perform a maximal voluntary torque of the plantar flexors is 70-80&#176; (0&#176; = full plantar flexion)26. Thus, ankle angle will depend on the parameter of interest (electrophysiological versus mechanical recordings). 
		Note: Regardless of the chosen initial angle, it must remain constant throughout the experiment to standardize neuromuscular excitability11,12,27,28.</li>
		<li>Pay particular attention when monitoring the subjects&#8217; posture during the test to maintain constant cortico-vestibular influences on the excitability of the motor pool29.</li>
	</ol>
	</li>
	<li>Firmly strap the ankle to an ergometer, with the anatomical axis of the joint (external malleolus) aligned with the axis of rotation of the ergometer25.
	<ol>
		<li>Have the subject exert pressure on a footplate attached to the ergometer to record plantar flexor torque. Keep the foot immobile throughout the experiment so that small changes in torque can be detected.</li>
	</ol>
	</li>
	<li>Note: Under certain circumstances, the heel may lift slightly off the force plate if the foot and ankle are not secured, which may lead to an incomplete transmission of the torque against the plate. Figure 3 presents a description of the experimental setup.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52974/52974fig3.jpg" /> 
Figure 3: Experimental setup. Classical experimental setup to record electromyographic (EMG) and torque signals.
<ol start="6">
	<li>Connect the electrodes to the amplifier with cables.</li>
	<li>Set the sampling rate for torque and EMG measurements to 2-5 kHz. Record the EMG signal using an Analog-to-Digital (AD) conversion system. The signal is displayed on a monitor with a data acquisition system, which instantaneously gives values of several parameters (e.g. maximal value, peak-to-peak amplitude, duration). The spectrum of the EMG signal can range between 5 Hz and 2 kHz frequencies, but is mainly contained between 10 Hz and 1 kHz31. Thus, sampling frequency must be high enough to preserve signal shape during EMG recording. Amplify and filter EMG signals (gain = 500-100) using a bandwidth frequency between 10 Hz and 1 kHz8,21,32.</li>
	<li>Place the anode for the electrical stimulation over the patellar tendon.</li>
	<li>Determine the best stimulation site of the posterior tibial nerve to obtain an optimal soleus H reflex for a given intensity, using a hand-held cathode ball electrode in the popliteal fossa. Test several stimulation sites with the cathode ball electrode until a maximal value of the H reflex is reached.
	<ol>
		<li>Record tibialis anterior EMG activity to ensure that the common peroneal nerve is not activated to avoid influence from antagonist Ia afferents12. Set the pulse width at 1 msec to provide an optimal activation of the nervous fibers, especially afferent fibers10.</li>
	</ol>
	</li>
	<li>Place a self-adhesive AgCl cathode at the location of the stimulation site to ensure constant stimulus condition (e.g. pressure, orientation). 
	Note: All of these parameters (subject position, electrode location and stimulation site) do not change for the assessment of the different electrophysiological measurements. Only the intensity of the stimulation and the condition (rest versus contraction) vary.</li>
</ol>
2. Testing Procedures at Rest
<ol start="1">
	<li>Instruct the subject to remain relaxed and to keep his/her muscles at rest.</li>
	<li>Adjust the stimulation intensity to obtain maximal soleus H-reflex amplitude (Hmax; usual range: 20-50 mA). An M-wave of the soleus muscle can be observed at Hmax intensity. 
	Note: For repeated measurements (e.g. before and after a fatiguing protocol), the optimal intensity to obtain a Hmax response may vary during the session. As keeping a constant intensity can lead to an underestimation of Hmax amplitude, it is recommended that the experimenter regularly reevaluates Hmax intensity33.</li>
	<li>Record a minimum of 3 soleus H-reflex responses at this intensity with a minimum interval of 3 sec to avoid post-activation depression34. 
	Note: Although recording several responses is more suitable due to the particular sensitivity of the H-reflex, a single stimulation may be enough under some circumstances, for example when trying to avoid the effects of rapid recovery (e.g. during a fatiguing protocol).</li>
	<li>Increase the stimulation intensity to obtain maximal soleus M-wave amplitude (Mmax; usual range: 40-100 mA). Usually, set the increment in stimulation intensity at 2-4 mA, with an interval of 8-10 sec between two stimuli12,35. The desired intensity is reached when Mmax is obtained, and no H-reflex response can be observed.</li>
	<li>Set the final intensity to 120-150% of Mmax stimulus intensity to ensure that the M-wave attains a plateau of its maximal value. This intensity is called supramaximal intensity in the instructions below.</li>
	<li>Keep constant stimulation intensity for soleus M-wave recordings throughout the session.</li>
	<li>Record 3 soleus M-waves and 3 associated twitch torques at this intensity.</li>
</ol>
3. Testing Procedures During Voluntary Contraction
<ol start="1">
	<li>As a warm-up, ask the subject to perform 10 brief and non-fatiguing submaximal contractions of the plantar flexor muscles, with a few seconds rest between each of the contractions. At the end of the warm-up, take a minimum 1 min rest to avoid any fatiguing effects11.</li>
	<li>Continuously record triceps surae EMG activity. Recording soleus and gastrocnemii muscles allows the analysis of the behavior of different muscle typologies for a single stimulation site24.</li>
	<li>Instruct the subject to perform an isometric maximal voluntary contraction (MVC) of the plantar flexors. The subject has to push as hard as possible against the ergometer by contracting his plantar flexor muscles. Give visual feedback to the subject during the effort, and standardized verbal encouragement19. The MVC is reached when a plateau is observed.</li>
	<li>Deliver a paired stimulation (100 Hz frequency) at supramaximal intensity during the plateau of the MVC (superimposed doublet), and another paired stimulation when the muscle is fully relaxed immediately after the contraction (potentiated doublet) to evaluate the voluntary activation level. Deliver this paired stimulation through a specific device (e.g. Digitimer D185 MultiPulse Stimulator) or through a stimulation program associated with a single pulse stimulator.</li>
	<li>Instruct the subject to perform a second MVC of the plantar flexor with at least 1 min rest between each trial11. If the peak torque from the second trial is not within 5% of the first, additional trials must be performed36. The greatest torque achieved by the subject is taken as the MVC torque.</li>
</ol>
4. Data Analysis
<ol start="1">
	<li>Data Analysis at Rest
	<ol>
		<li>Select a time window including the EMG response associated with the twitch at rest (H-wave or M-wave).</li>
		<li>Measure the peak-to-peak amplitude, peak-to-peak duration, and/or the area of the waves (Figure 4A). If the amplitude is not directly provided by the software, subtract the minimum to the maximum values.
		<ol>
			<li>For the duration, measure the time frame starting from the maximal peak and ending to the minimal peak. For the area, calculate the integral of EMG signal starting from the beginning of the wave and ending to the end of the wave. 
			Note: Peak-to-peak amplitude can reflect: 1) neuromuscular transmission, 2) motor unit action potential amplitude and/or 3) temporal dispersion of motor unit action potential37. M-wave duration reflects neuromuscular propagation37.</li>
		</ol>
		</li>
		<li>For multiple trials, calculate the average of the waves. If the average cannot be directly provided by the software, use spreadsheet software (e.g. the formula function in a spreadsheet program) to calculate this value from several trials (at least 3).</li>
		<li>Select the resting twitch.</li>
		<li>Measure the peak torque associated with the resting twitch (Figure 4B).</li>
		<li>For multiple trials, calculate the average peak torque of the resting twitches. If the average cannot be directly provided by the software, use spreadsheet software (e.g. the formula function in a spreadsheet program) to calculate this value from the several trials (at least 3).</li>
		<li>Repeat these procedures described in point 4.1.2 for the other desired parameters (contraction time or half-relaxation time). The analysis of twitch parameters provides indications as to the excitation-contraction coupling efficiency17. In particular, contraction time provides an index of contraction kinetics8, which can depend upon the chosen muscle group38.</li>
		<li>Calculate the ratio between the peak torque and the sum of M-waves using spreadsheet software (e.g. Excel), to quantify the electromechanical efficiency (Pt/M). As the mechanical responses evoked by posterior tibial nerve stimulation correspond to the activation of the triceps surae as a whole, amplitudes of soleus and gastrocnemii M-waves must be summed39.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/52974/52974fig4.jpg" /> 
Figure 4: Explanation of electrophysiological and mechanical responses. (A) Measurement of peak-to-peak amplitude (mV), latency (ms) and area (mV.ms) of a typical M-wave. (B) Measurement of peak twitch torque (Nm), contraction time (ms) and half-relaxation time (msec) of a twitch. <a href="https://www.jove.com/files/ftp_upload/52974/52974fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Data analysis in contraction
	<ol>
		<li>Select a 500 msec time window of soleus EMG activity during the plateau of MVC torque including the peak torque but excluding the time between the stimulation artefact and the end of the silent period of EMG. The silent period corresponds to the suppression of the ongoing voluntary EMG activity following stimulation.</li>
		<li>If the root mean square (RMS) is not directly provided by the software, calculate the RMS to quantify EMG activity using the following formula40: RMSEMG 
		<img alt="Equation 1" src="/files/ftp_upload/52974/52974eq1.jpg" /></li>
		<li>Measure or calculate the RMS of Mmax at rest over the duration of the wave.</li>
		<li>Calculate the RMSEMG/RMSMmax ratio using spreadsheet software. RMSEMG value and RMSMmax value have to be selected from the same muscle.</li>
		<li>Measure the maximal peak torque of the MVC from the baseline of torque at rest to the maximal value of MVC excluding the superimposed torque induced by the doublet stimulation (Figure 5).</li>
		<li>Measure the superimposed torque induced by the doublet stimulation during the MVC, from the voluntary torque value at the onset of the stimulation to the peak of the evoked response (Figure 5).</li>
		<li>Select the potentiated doublet.</li>
		<li>Measure the peak torque associated with the potentiated doublet.</li>
		<li>Calculate the voluntary activation level (VAL) using the following formula40: 
		<img alt="Equation 1" src="/files/ftp_upload/52974/52974eq2.jpg" /></li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/52974/52974fig5.jpg" /> 
Figure 5: Measurement of superimposed and potentiated doublet on mechanical signal. To record the superimposed peak torque (Pts), stimulation doublet is evoked during the plateau of isometric maximal voluntary contraction (MVC). To record potentiated peak torque (PtP), stimulation doublet is evoked at rest after the offset of MVC.

The authors have nothing to disclose.

Percutaneous nerve stimulation enables the quantification of numerous characteristics of the neuromuscular system not only to understand the fundamental control of neuromotor function in healthy humans, but also to be able to analyze acute or chronic adaptations through fatigue or training17. This is very beneficial especially for fatiguing protocols, where measurements must be performed as soon as possible after exercise end to avoid the effects of rapid recovery42.
Alth...

Percutaneous electrical nerve stimulation is widely used to assess neuromuscular function1. The basic principle consists of inducing an electrical stimulus to a peripheral motor nerve to evoke a muscular contraction. Mechanical (torque measurement) and electrophysiological (electromyographic activity) responses are simultaneously recorded. Torque, recorded at the considered joint, is assessed using an ergometer. The electromyographic (EMG) signal recorded using surface electrodes has been demonstrated to represent the activity of the muscle2. This non-invasive method is not painful and more easily implemented than intramuscular recordings. Both monopolar and bipolar electrodes can be used. The monopolar electrode configuration has been shown to be more sensitive to changes in muscle activity3, which can be useful for small muscles. However, bipolar electrodes have been shown to be more effective in improving the signal-to-noise ratio4 and are most commonly used as a method of recording and quantifying motor unit activity. The methodology described below will focus on bipolar recordings. EMG activity is an indicator of the efficacy and integrity of the neuromuscular system. The use of percutaneous nerve stimulation offers further insights into neuromuscular function, i.e. changes at muscular, spinal, or supra-spinal level (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/52974/52974fig1.jpg" /> 
Figure 1: Overview of the neuromuscular measurements. STIM: nerve stimulation. EMG: Electromyography. VAL: Voluntary Activation Level. RMS: Root Mean Square. Mmax: Maximal M-wave amplitude.
At rest, the compound muscle action potential, also called M-wave, is the short-latency response observed after stimulus artefact, and represents excitable muscle mass by the direct activation of motor axons leading to the muscle (Figure 2, number 3). M-wave amplitude increases with intensity until reaching a plateau of its maximal value. This response, called Mmax, represents the synchronous summation of all motor units and/or muscle fiber action potentials recorded under the surface EMG electrodes5. The evolution of the peak-to-peak amplitude or wave area is used to identify alterations of neuromuscular transmission6. Changes in the mechanical responses associated with the M-wave, i.e. peak twitch torque/force, may be due to alterations in muscle excitability and/or within the muscle fibres7. The association of Mmax amplitude and peak twitch torque amplitude (Pt/M ratio) provides an index of electromechanical efficiency of the muscle8, i.e. mechanical response for a given electrical motor command.
<img alt="Figure 2" src="/files/ftp_upload/52974/52974fig2.jpg" /> 
Figure 2: Motor and reflexive pathways activated by nerve stimulation. Electrical stimulation of a mixed (motor/sensory) nerve (STIM) induces a depolarization of both motor axon and Ia afferent firing. Depolarization of Ia afferents towards the spinal cord activates an alpha motoneuron, which in turn evokes an H-reflex response (pathway 1+2+3). Depending upon the stimulus intensity, motor axon depolarization evokes a direct muscular response: M-wave (pathway 3). At maximal M-wave intensity, an antidromic current is also generated (3&#39;) and collides with reflex volley (2). This collision partially or totally cancels the H-reflex response.
The H-reflex is an electrophysiological response used to assess changes in the Ia-&#945; motoneuron synapse9. This parameter can be assessed at rest or during voluntary contractions. H-reflex represents a variant of the stretch reflex (Figure 2, number 1-3). The H-reflex activates motor units monosynaptically recruited by Ia afferent pathways10,11, and can be subjected to peripheral and central influences12. The method of evoking a H-reflex is known to have a high intra-subject reliability to assess spinal excitability at rest13,14 and during isometric contractions15.
During a voluntary contraction, the magnitude of the voluntary neural drive can be assessed using the amplitude of the EMG signal, generally quantified using the Root Mean Square (RMS). RMSEMG is commonly used a means of quantifying the level of excitation of the motor system during voluntary contraction (Figure 1). Because of the intra- and inter-subject variability16, RMSEMG has to be normalized using the EMG recorded during a muscle-specific maximal voluntary contraction (RMSEMGmax). In addition, because changes in EMG signal may be due to alterations at peripheral level, normalization using a peripheral parameter such as M-wave is required to assess only the central component of EMG signal. This can be done by dividing the RMSEMG by the maximal amplitude or the RMSMmax of the M-wave. Normalization using RMSMmax (i.e. RMSEMG/RMSMmax) is the preferred method as it takes into consideration the possible change of the M-wave duration17.
Motor commands can also be evaluated by calculating the voluntary activation level (VAL). This method uses the twitch interpolation technique18 by superimposing an electrical stimulation at Mmax intensity during a maximal voluntary contraction. The extra-torque induced by stimulating the nerve is compared to a control twitch produced by identical nerve stimulation in a relaxed potentiated muscle19. To evaluate maximal VAL, the original twitch interpolation technique described by Merton18 involves a single stimulus interpolated over a voluntary contraction. Recently, the use of paired stimulation has become more popular because the evoked torque increments are larger, more readily detected, and less variable compared to single stimulation responses20. The VAL provides an index of the capacity of the central nervous system to maximally activate the working muscles21. Currently, VAL evaluated using the twitch interpolation technique is the most valuable method of assessing the level of muscle activation22. Furthermore, peak torque assessed using an ergometer is the most properly studied strength testing parameter applicable of use in research and clinical settings23.
Electrical nerve stimulation can be used in a variety of muscle groups (e.g. elbow flexors, wrist flexors, knee extensors, plantar flexors). However, nerve accessibility makes the technique difficult in some muscles groups. The plantar flexor muscles, especially triceps surae (soleus and gastrocnemii) muscles, are frequently investigated in the literature24. Indeed, these muscles are involved in locomotion, justifying their particular interest. The distance between stimulation site and recording electrodes allows the identification of the different evoked waves of the triceps surae muscles. The superficial part of the posterior tibial nerve in the popliteal fossa and the large number of spindles make it easier to record reflex responses compared to other muscles24. For these reasons, the currently presented reflex methodology focuses on the triceps surae group of muscles (soleus and gastrocnemius). The aim of this protocol is therefore to describe percutaneous nerve stimulation technique to investigate neuromuscular function in the triceps surae.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Biodex dynamometer</td>
			<td>Biodex Medical System Inc., New York, USA</td>
			<td></td>
			<td><a href="http://www.biodex.com/">www.biodex.com</a></td>
		</tr>
		<tr>
			<td>MP150 Data Acquisition System</td>
			<td>Biopac Systems Inc., Goleta, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acknowledge 4.1.0 software</td>
			<td>Biopac Systems Inc., Goleta, USA</td>
			<td></td>
			<td><a href="http://www.biopac.com/">www.biopac.com</a></td>
		</tr>
		<tr>
			<td>DS7A constant current high voltage stimulator</td>
			<td>Digitimer, Hertfordshire, UK</td>
			<td></td>
			<td><a href="http://www.digitimer.com/">www.digitimer.com</a></td>
		</tr>
		<tr>
			<td>Silver chloride surface electrodes</td>
			<td>Control Graphique Medical, Brie-Comte-Robert, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 Cable for connecting the Biodex to the MP150</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 Cable for connecting the Digitimer to the MP150</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 Cable for connecting the MP150 to the computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of neuromuscular function using percutaneous electrical nerve stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

Increasing stimulus intensity leads to a different evolution of response amplitudes between H- and M-waves. At rest, the H-reflex reaches a maximum value before being totally absent from EMG signal, while M wave progressively increases until reaching a plateau at maximal intensity (see Figure 4 for a graphical depiction of the M-wave and Figure 6 for the evolution of M-waves and H-reflex with intensity). For the soleus muscle, the latency between the stimulus onset and M-wave is about 10...

Watch this Scientific Journal Video about Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation at JoVE.com

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

assessment-neuromuscular-function-using-percutaneous-electrical-nerve

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JoVE Journal

Neuroscience

21.7K vistas.  Univ. Bourgogne Franche–Comté. We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). 

Video: Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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

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

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

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

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...