This works was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. ST was supported by a Vanier Canada Graduate scholarship from the Canadian Institutes of Health Research. MM acknowledges the support from Biotechnology Research Center (BTRC) grant P41 RR008079 and P41 EB015894 (NIBIB), and NCC P30 NS076408.
We would like to acknowledge Romain Valabr&#232;gue (Centre de NeuroImagerie de Recherche &#8211; CENIR, Paris, France) and Brice Tiret (Centre Recherche de l&#8217;Institut Universiatire de G&#233;riatrie (CRIUGM), Montr&#233;al, Canada; Commissariat &#224; l&#39;&#233;nergie atomique et aux &#233;nergies alternatives (CEA), Paris, France) for developing processing tools, and Edward J. Auerbach (Center for Magnetic Resonance Research and Department of Radiology, University of Minnesota, USA). The MEGA-PRESS and FASTESTMAP sequences were developed by Edward J. Auerbach and Ma&#322;gorzata Marja&#324;ska and were provided by the University of Minnesota under a C2P agreement.

The study was approved by the Research and Community Ethics Boards of Unit&#233; de Neuroimagerie Fonctionnelle and University of Montr&#233;al and was done in compliance with the code of ethics as stated in the Declaration of Helsinki. All subjects gave written informed consent following careful screening for MRI compatibility and were financially compensated for their participation.
1. tDCS Material
<ol>
	<li>Make sure all necessary materials are available before starting the experiment (see Figure 1 for list). 
	Note: Different electrode sizes are available for tDCS. For this study, two 5 x 7 cm rubber electrodes will be used. Other sizes can be chosen depending on the area of stimulation and the desired focality of the stimulation 48.</li>
	<li>Make sure to check that the batteries of the DC-stimulator are charged and to periodically charge them since the device cannot be charged or plugged-in during stimulation for safety reasons.</li>
</ol>
2. Planning of the Conditions for Stimulation
<ol>
	<li>Turn on the tDCS device according to the instructions included with the device. Pre-set the tDCS device for two different stimulation modes (active and sham).</li>
	<li>As some devices do not have a pre-set mode, select the appropriate sham parameters before starting stimulation.
	<ol>
		<li>Pre-define a set of parameters by loading a setting. Press the button 2 or 4 to select from the main menu the &#34;system&#34; option (see Figure 2).</li>
		<li>Move the cursor to line 2 on the display by pressing the button 3.</li>
		<li>Press the button 2 or 4 until the &#34;load setting&#34; shows on the display. Press the button 3.</li>
		<li>Select the letter of the setting (A, B, C or D) by pressing the button 2 or 4.</li>
		<li>Move the cursor upwards with the button 1. The display will automatically show the &#34;parameters&#34; option.</li>
		<li>Set the tDCS device to a current of 1 mA. To do so, press the button 1 to select the line 3 of the &#34;parameters&#34; menu of the display. Select the &#34;current&#34; option by pressing the button 2 or 4. Press the button 3 to reach the line 4 and modify the intensity to 1,000 &#956;A by pressing the button 2 or 4.</li>
		<li>Press the button 1 to go back to the line 3. Select the &#34;fade in&#34; option from the screen menu of the device by pressing the button 2 or 4. Press the button 3 to go to the line 4 and press the buttons 2 and 4 to adjust the duration to 15 s. 
		Note: Fade in durations can be modified.</li>
		<li>Press the button 1 to go back to the line 3. Select the &#34;fade out&#34; option from the screen menu of the device by pressing the buttons 2 or 4. Press the button 3 to go to the line 4 and press the buttons 2 and 4 to adjust the duration to 15 s. 
		Note: Fade in durations can be modified.</li>
		<li>Press the button 1 to go back to the line 3. Press the button 2 or 4 until the &#34;duration&#34; option shows on the display menu. Press the button 3 to go to the line 4 and press the button 2 and 4 to adjust the duration to the minimum duration available on the device (15 s for the present device; see Figure 3b). 
		Note: This will induce a tingling sensation similar to the active stimulation.</li>
	</ol>
	</li>
	<li>Press the buttons 1 and 3 simultaneously to save the changes of the setting.</li>
	<li>Pre-program the active stimulation parameters. To do so, follow the same instructions as for the setting of the sham stimulation, but program the duration to 1,200 sec (20 min; see Figure 3a).</li>
	<li>Pre-program the test stimulation parameters. To do so, follow the same instructions as for the setting of the sham stimulation but program the duration to 45 sec. 
	Note: The test stimulation will be used for the measurement of the impedance prior to the experimentation.</li>
	<li>Pseudo-randomly assign the conditions of stimulation to participants.</li>
	<li>Assign a number to each of the three conditions for a blind experimentation: 1) bilateral: anodal right, cathodal left; 2) bilateral: anodal left, cathodal right; 3) sham: anodal right, cathodal left.</li>
</ol>
3. Consenting the Participants
<ol>
	<li>Inform the participant of the procedure and sign consent form.
	<ol>
		<li>Verify that participants do not have any contraindication to tDCS: a psychiatric or neurological history, the presence of a pacemaker, metal implanted in skull, a history of fainting, a history of seizures, a history of substance abuse, a family history of seizure, a history of febrile fits, a lack of sleep in the preceding night, a history of skin sensitivity, and any alcohol consumption the previous day.</li>
		<li>Inform the participant of the most reported side-effects of tDCS: mild tingling; moderate fatigue; light sensation of itching under the electrodes; slight burning sensation.</li>
	</ol>
	</li>
	<li>Inform the participant of the usual MR contraindications and side effects.</li>
</ol>
4. Measurements for Electrodes Placement
<ol>
	<li>Use the 10/20 international system to find the following landmarks on the participant head: nasion and inion (Figure 4a), preauricular points, and the two targeted areas: C3 and C4 (Figure 4b).
	<ol>
		<li>Locate the nasion as the distinct depressed area located on the bridge of the nose at the level between both eyes. Locate the inion as the most prominent projection of the occipital bone located at the lower part of the skull. Locate the preauricular point near each ear; it is the indentation above the zygomatic notch. Locate the C3 and C4 based on measurements as described below.</li>
	</ol>
	</li>
	<li>Use a measuring tape to measure the distance between the nasion and inion along the midline of the head and make a mark at 50% of the distance with a non-permanent hydro marker.</li>
	<li>Use a measuring tape to measure the distance between the two preauricular points and make a mark with a non-permanent hydro marker at 50% of the distance in line with the previous mark. This point corresponds to Cz (vertex).</li>
	<li>From the Cz, along the line created between the preauricular points, mark two points, one on each side, with a non-permanent hydro marker that correspond to 20% of the total distance. These marks correspond to the target areas (C3 and C4, Figure 4b). 
	Note: Other methods such as TMS or neuronavigation can also be used to localize M1.</li>
</ol>
5. Placement of Electrodes
<ol>
	<li>Move as much hair as possible away from the targeted areas that will be stimulated. Apply an EEG-type exfoliating gel with a cotton-swab to clean the targeted areas.</li>
	<li>Clean the targeted areas with a 70% isopropyl alcohol and pumice prepping pad to enhance electrode contact.</li>
	<li>Generously cover the entire electrode with an EEG-type conductive paste. Ensure that the paste is approximately 5 mm thick across the entire surface. Make sure the entire rubber area is covered with paste. Lightly wet the target areas and the conductive paste on the electrodes with a saline solution.</li>
	<li>Position the electrodes as shown in Figure 4b and press the electrodes firmly onto the targeted areas. Place a rubber band around the head of the participant to ensure optimal stability of the electrodes. Adjust it in such a way that the participant will experience no pain or discomfort during the scanning session.</li>
	<li>Make sure that the leads do not come in contact with the skin to avoid potential burns.</li>
</ol>
6. tDCS Test Outside the Scanner Room
<ol>
	<li>Use a multimeter to verify the proper functioning of the electrode cable and resistance.</li>
	<li>Turn on the tDCS device and load the test stimulation settings.
	<ol>
		<li>Press the button 2 or 4 to select from the main menu the &#34;system&#34; option. Move the cursor to line 2 on the display by pressing the button 3. Press the button 2 or 4 until the &#34;load setting&#34; shows on the display. Press the button 3. Select the letter of the pre-programmed test setting (A, B, C or D) by pressing the button 2 or 4.</li>
		<li>Move the cursor upwards with the button 1. The display will automatically show &#34;parameters&#34; option. On the first line, press the button 2. The display will show &#34;stimulation?&#34; with the different pre-programmed parameters.</li>
	</ol>
	</li>
	<li>Press the button 1 to start the stimulation. The display will show the impedance level and automatically stop if it reaches more than 20 k&#937;. If the impedance level is over 20 k&#937;, unplug the electrode wires from the inner box and exit the scanning room to verify the positioning of the electrodes.</li>
	<li>Redo the test stimulation. When a good level of impedance is reached and when the test stimulation is over, unplug the electrodes from the inner box.</li>
</ol>
7. tDCS Setup
<ol>
	<li>As shown in Figure 5, place the tDCS device and the outer box in the scanner control room. 
	Note: The tDCS device and the outer box are not MR compatible and should not be taken into the magnet environment.</li>
	<li>Plug the outer box wires into the tDCS device and then plug the long box cable into the outer box.</li>
	<li>Run the tDCS box cable from the scanner control room into the MRI room. Make sure to run this cable as straight as possible, avoiding any kinks or loops, along the wall of the MRI room towards the back of the MRI scanner. Put multiple MR compatible sandbags on the cable to ensure its stability, as shown in Figure 5.</li>
	<li>Bring the inner box into the MRI room and plug the long box cable into it (Figure 5).</li>
</ol>
8. MRI Scan Preparation
<ol>
	<li>Ask the participant to enter the MRI room, if not already in there from the tDCS test, and to put in earplugs.</li>
	<li>Put a thin cushion under the coil area of the MRI table. Ask the participant to lie down on the table. Put a cushion under the legs of the participant for comfort and a blanket if needed. Give the participant the alarm button for security purposes.</li>
	<li>Put separate headphones over both ears to allow transmission of information from the scanner control room to the participant in the MRI room.</li>
	<li>Position the participant&#8217;s head as high as possible under the area where the head coil will be positioned (top of the head as close as possible to the top of the table where the coil will be placed). Put the electrode wires along the right side of the head of the participant, as recommended by the tDCS device company.</li>
	<li>Place the 32-channel receive-only coil around the head of the participant. Run the electrode cables through the right side of the coil. Position the head of the participant as straight as possible using a red positioning laser (built-in feature of the scanner).</li>
	<li>Ask the participant to move arms and legs into a comfortable position, while making sure that hands do not touch. Make sure to remind the participant to stay as still as possible during the entire session. When the participant is ready, move the table past the middle line to reach the electrode wires at the back of the scanner.</li>
	<li>Use medical tape to stabilize the electrode cable on the right side of the back of the coil. Plug the electrode wires located inside the scanner into the tDCS inner box. Put the inner box on the right side of the scanner with a sandbag on it for maximal stability.</li>
	<li>Move the table back into its final position. Keep the tDCS turned on and the electrodes plugged into the outer box for the entire MRI session.</li>
</ol>
9. Pre-tDCS 1H-MRS Session
<ol>
	<li>Run a localizer sequence to acquire images needed to verify the proper positioning of the head and to compare to a second localizer which will be acquired at the end of the session to check for overall movement.</li>
	<li>Acquire anatomical T1-weighted MPRAGE images for the positioning of the M1 voxel and detection of possible structural abnormalities (TR = 2,300 msec; TE = 2.91 msec; FA: 9&#176;; FOV = 256 x 256 mm; 256 x 256 matrix; TI : 900 msec; 176 slices; orientation: sagittal; acquisition time: 4 min 12 sec).</li>
	<li>Perform a multi-planner reconstruction of the images in planes that are more appropriate for visualization of the spectroscopy volume-of-interest (VOI).
	<ol>
		<li>In the 3D card, browse the MPRAGE raw images (sagittal orientation). From the &#34;creating parallel ranges&#34; window select &#34;axial 2X2&#34;. Adjust the position of the parallel lines and click on save to create the axial orthogonal view.</li>
		<li>From the &#34;creating parallel ranges&#34; window select &#34;coronal 2X2&#34;. Adjust the position of the parallel lines and click on &#34;save&#34; to create the coronal orthogonal view.</li>
	</ol>
	</li>
	<li>Locate the left M1 based on Yousry and collaborators&#8217; 49 anatomical landmarks on the three orientation slices. Then, position the VOI (30 x 30 x 30 mm3) on the area without any angulation relative to the scanner axis (figure 6).</li>
	<li>Acquire a line-width scan (21 s).
	<ol>
		<li>Select the spectroscopy card to measure water line-width on the real part of the signal from this line-width scan. Load the line-width raw data from the browser. Load the line-width measurement protocol (protocols menu: select the protocol).</li>
		<li>Adjust the phase using the scanner software interactive post-processing tools. Select the phase correction section and adjust the phase for the baseline with the cursor.</li>
		<li>In order to reduce the line-width, run the FAST(EST)MAP 50 sequence three times. Repeat the line-width scan and the line-width measurement (step 9.5). Note the final water line-width.</li>
	</ol>
	</li>
	<li>Start 4 blocks of 64 metabolite scans (32 &#34;EDIT OFF&#34; and 32 &#34;EDIT ON&#34;, interleaved) with a MEGA-PRESS sequence 44,45, where VAPOR 51, OVS 51 and individual storage of FIDs are enabled (TR = 3 s, TE = 68 msec, total acquisition time: 12 min)</li>
	<li>Acquire a water reference using MEGA-PRESS sequence without MEGA water suppression, with VAPOR suppression (&#34;only RF off&#34;) and with a delta measurement at 0 ppm. Acquire a single block of 4 metabolite scans instead of 64 (acquisition time: 42 sec).</li>
</ol>
10. tDCS Procedure
<ol>
	<li>Inform the participant that the tDCS stimulation will start and that the scanner will be silent for the entire stimulation.</li>
	<li>Select one of the two previously programmed parameters according to the condition and start the stimulation. Keep track of the impedance and voltage during the 20 min of stimulation. When the stimulation is over, notify the participant that the post-tDCS MRS session will begin. Do not turn off the tDCS device.</li>
</ol>
11. Post-tDCS 1H-MRS Session
<ol>
	<li>Run the same metabolite scans with MEGA-PRESS sequence as the pre-tDCS scan but double the blocks of acquisition (8 blocks of 64 scans (32 &#34;EDIT OFF&#34; and 32 &#34;EDIT ON&#34;, interleaved)) to acquire the metabolites at two different time points post-tDCS.</li>
	<li>As with the pre-tDCS session, acquire a water reference scan using the same parameters. Finish the session with a localizer sequence.</li>
	<li>Visually compare the localizer images acquired at the beginning and end of the scanning session as an index of head motion.</li>
	<li>Access the viewing card and go to the browser menu. Select the first and second localizer raw images. Load the images in the viewing card and compare both images. Export data in the dicom format through the server.</li>
</ol>
12. Analysis of the 1H-MRS Data
<ol>
	<li>Import data using a programming and processing software, and adjust frequency and phase of individually stored FIDs using tCr and tCho signal between 2.85 and 3.40 ppm. To do so, use the software&#8217;s lsqnonlin function to fit frequency and phase of each individual Fourier-transformed FIDs (spectra) to the average spectra of the session. 
	Note: this is a site-specific approach and other methods for importing and analyzing data will not necessarily affect data quality.</li>
	<li>To obtain the final spectra, subtract the signals from alternate scans with the selective double-banded pulses that were applied at 4.7 ppm and 7.5 ppm (&#34;EDIT OFF&#34;) and at 1.9 ppm and 4.7 ppm (&#34;EDIT ON&#34;) (Figure 7).</li>
	<li>Use the LCModel 52 for the analysis of both difference and &#34;EDIT OFF&#34; spectra. Deactivate default simulations and baseline modeling.</li>
	<li>Perform a visual inspection of the spectra to exclude sessions with contamination from subscapular lipid signal (see Figure 9).</li>
	<li>As part of quality control, exclude spectra with linewidth of tCr-CH3 above 10 Hz. Only include in the analysis metabolites (GABA, Glx, tCr, tNAA) which were quantified with Cramer-Rao lower bounds (CRLB) lower than 35%. 
	Note: CRLB provide estimated error of the metabolite quantification. CRLB &#62; 50% is not reliable and is a recommended cut-off by LCModel manual. Many in the field have used a CRLB lower than 35% as a standard. 53-55 Additionally, the CRLB should be kept in mind when interpreting the results.</li>
	<li>Obtain GABA and Glx quantifications from the &#34;DIFF&#34; spectra, tCr from the &#34;EDIT OFF&#34; spectra, and tNAA from both &#34;EDIT OFF&#34; and &#34;DIFF&#34;. Express concentrations of the different metabolites of interest as ratios over tCr. For GABA and Glx, multiply the ratio by the following group-averaged correction factor to account for the different basis set used for the numerator and denominator (tNAA from &#34;EDIT OFF&#34; spectra / tNAA from &#34;DIFF&#34; spectra). 
	Note: Note: GABA and Glx concentrations can also be quantified using water or NAA signal.</li>
</ol>

The authors have nothing to disclose.

The present paper aimed to describe a standard protocol for combining tDCS and 1H-MRS using a 3 T scanner. In the next section, methodological factors will be discussed.
Critical Steps 
Contraindications Screening 
Previous to the experiment, it is crucial to screen participants for any contraindication regarding the use of tDCS and 1H-MRS. The use of the following exclusion criteria is recommended for tDCS: a psychiatric or neurologic...

The idea of applying electricity to the human brain to modulate its activity has been studied since ancient times. In fact, writings from as early as the 11th century have been found that describe the use of the torpedo electric fish in the treatment of epileptic seizures1. Yet, it is not until recently that non-invasive brain stimulation has received widespread interest in the scientific community as it was shown to produce modulatory effects on cognitive function and motor response2. While transcranial magnetic stimulation (TMS) has been extensively studied since the early 1980&#39;s3, recent interest in transcranial direct current stimulation (tDCS) has increased as it is now considered a viable treatment option for a wide range of neuropathologies, such as stroke4, alcohol addiction5, and chronic pain6. tDCS has many advantages over neurostimulation techniques like TMS, for example, since it is relatively inexpensive, painless, well tolerated by patients, and portable, thus making it possible to administer at bedside7. In fact, only a small percentage of patients experience a mild tingling sensation during stimulation8. However, this sensation usually disappears after a few seconds9. Consequently, tDCS allows robust double-blind, sham-controlled studies since a majority of participants cannot differentiate sham stimulation from real stimulation9,10.
tDCS involves the induction of a constant low-amperage electric current (1-2 mA) applied to the cortex via surface electrodes positioned on the scalp of the subject. The electrodes are usually placed into saline-soaked sponges or directly on the scalp with an EEG-type paste. To conduct a tDCS study, four main parameters need to be controlled by the experimenter: 1) the duration of stimulation; 2) the intensity of stimulation; 3) the electrode size; and 4) the electrode montage. In standard protocols, the &#8220;active&#8221; electrode is positioned over the region of interest while the reference electrode is usually placed over the supraorbital region. The current flows from the positively charged anode towards the negatively charged cathode. The effect of tDCS on primary motor cortex (M1) is determined by the polarity of the stimulation where anodal stimulation enhances the excitability of a population of neurons and cathodal stimulation reduces it 11. Unlike TMS, the induced current is insufficient to produce action potentials in cortical neurons. The changes in cortical excitability are believed to be due to the modulation of the membrane neuronal threshold leading to either the hyperpolarization of membrane potentials or a facilitation of depolarization of neurons depending on the direction of the current flow 8,11. The duration of the excitability changes can persist for up to 90 min after the offset of stimulation, depending on stimulation duration 11,12.
tDCS and Motor Rehabilitation
The M1 has been extensively used as a target of stimulation since excitability changes elicited by tDCS can be quantified through motor evoked potentials (MEPs) induced by single pulse TMS 3. Early studies showing the possibility of measuring polarity-specific excitability changes induced by tDCS have used M1 as a target of stimulation 11,12. Since then, M1 has remained one of the primary targets of tDCS in studies involving both clinical populations and healthy subjects because of its importance in motor function, memory formation, and consolidation of motor skills 12.
The brain relies on a complex interaction between motor regions of both hemispheres to perform a movement 14. When one area is damaged, after suffering a stroke for example, inter-hemispheric interactions are altered. Studies on brain plasticity have shown that the motor areas of the brain adapt to this modification in different ways 15. First, the intact, surrounding regions of the damaged area can become overactived, leading to inhibition of the damaged area - a process called intra-hemispheric inhibition. Second, the homologous region of the damaged area can become overactivated and exert inhibition on the injured hemisphere - a process called inter-hemispheric inhibition. The affected M1 can therefore be twice penalized: first by the lesion and second by the inhibition coming from both the unaffected M1 and the surrounding region of the affected M1 16. A recent study has shown that increased excitability in the unaffected hemisphere is linked to slower rehabilitation 17, which has been described as maladaptive inter-hemispheric competition 18.
Understanding the plasticity occurring after a stroke may lead to the development of neuromodulation protocols that can restore interhemispheric interactions 19. Three main tDCS treatments have been proposed in patients with motor deficits following stroke 20,21. The first treatment aims to reactivate the injured motor cortex by unilateral anodal stimulation (a-tDCS). In this case, stimulation aims at directly increasing activity in perilesional areas, which are believed to be essential for recovery. In fact, studies have shown improvement of the paretic upper or lower limb following this treatment 22-26. The second treatment was developed with the aim of reducing the over-activation of the contralesional hemisphere by applying unilateral cathodal tDCS (c-tDCS) over the intact M1. Here, stimulation aims at indirectly increasing activity in perilesional areas through interhemispehric interactions. Results from these studies have shown improvement of motor function after c-tDCS 4,27-29. Finally, the third treatment aims at combining the excitatory effects of a-tDCS over the injured M1 with the inhibitory effects of c-tDCS over the unaffected M1 using bilateral tDCS. Results have shown improvements in motor function after bilateral tDCS 27,30,31. Moreover, one study demonstrated greater improvements following bilateral tDCS compared to both unilateral methods 32.
Physiological Mechanisms of tDCS
Despite the increasing use of tDCS in the treatment of stroke, the physiological mechanism underlying its effects remains unknown 33. A better understanding of the physiological effects could help develop better treatment options and could lead to standardized protocols. As mentioned earlier, the effects of tDCS can last for up to 90 min after the offset of stimulation 11,12. Therefore, hyperpolarization/depolarization processes cannot completely explain long lasting effects 33,34. Different hypotheses have been suggested regarding the physiological mechanism underlying tDCS after-effects on M1 including changes in neurotransmitter release, protein synthesis, ion channel function, or receptor activity 34,35. Insights into this matter were first acquired through pharmacological studies showing a suppression of the after effects of anodal and cathodal stimulation on M1 excitability by the glutamatergic N-methyl-D-aspartate (NMDA) receptor antagonist dextromethorphan36,37 whereas the opposite effect was shown using a NMDA receptor agonist38. NMDA receptors are thought to be involved in learning and memory function through long term potentiation (LTP) and long term depression (LTD), both mediated by glutamatergic and GABAergic neurons39,40. Animal studies are in line with this hypothesis as they have shown that a-tDCS induces LTP13.
Despite the important progress made in our understanding of the mechanisms of action underlying tDCS effects, pharmacological protocols present important limitations. Indeed, drug action cannot be as spatially specific as tDCS, especially in the context of human experimentation, and the mechanism of action of their effects is mostly due to post-synaptic receptors 34. Therefore, there is a need to investigate more directly the effects of tDCS on the human brain. Proton magnetic resonance spectroscopy (1H-MRS) is a good candidate as it allows non-invasive in vivo detection of neurotransmitter concentrations in a specific region of interest. This method is based on the principle that every proton-containing neurochemical in the brain has a specific molecular structure and consequently, produces chemically specific resonances that can be detected by 1H-MRS 41. The acquired signal from the brain&#8217;s volume of interest is generated from all protons that resonate between 1 and 5 ppm. The acquired neurochemicals are represented on a spectrum and plotted as a function of their chemical shift with some clearly distinguishable peaks, but where many resonances from the different neurochemicals overlap. The signal intensity of each peak is proportional to the concentration of the neurometabolite 41. The amount of neurochemicals that can be quantified depends on the strength of the magnetic field 42,43. However, low-concentration metabolites, which are obscured by very strong resonances, are hard to quantify at lower field strength such as 3 T. One way to obtain information about such overlapping signals is to remove the strong resonances via spectral editing. One of such techniques is a MEGA-PRESS sequence, which allows detection of &#947;-aminobutyric acid (GABA) signals 44,45.
Only a few studies have investigated the effect of tDCS on the brain metabolism using 1H-MRS in motor 34,46 and non-motor regions47. Stagg and collaborators 34 assessed the effects of a-tDCS, c-tDCS, and sham stimulation on M1 metabolism. They found a significant reduction in GABA concentration following a-tDCS, and a significant reduction of glutamate+glutamine (Glx) and GABA following c-tDCS. In another study, it was reported that the amount of changes in GABA concentration induced by a-tDCS over M1 was related to motor learning 46.
These studies highlight the potential of combining 1H-MRS with tDCS to increase our understanding of the physiological mechanism underlying the effect of tDCS on motor function. In addition, the use of clinical protocols such as a-tDCS and c-tDCS over M1 is useful because their behavioral effects are well studied and can be directly related to physiological results. Therefore, a standard protocol for combining bilateral tDCS and 1H-MRS is demonstrated in healthy participants using a 3 T MRI system. Bihemispheric tDCS is presented to contrast data with a previous MRS study where unilateral cathodal or unilateral anodal tDCS were applied over motor cortex 34. The protocol is described specifically for stimulation with a NeuroConn stimulator in a Siemens 3 T scanner performing MEGA-PRESS 1H-MRS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DC-stimulator plus</td>
			<td>NeuroConn</td>
			<td>30DCS01E</td>
			<td>MR compatible device</td>
		</tr>
		<tr>
			<td>NuPrep preparation gel</td>
			<td>Weaver and Co.</td>
			<td>#10-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Ten20 conductive paste</td>
			<td>Weaver and Co.</td>
			<td>#10-20-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode prepping pad</td>
			<td>Grass technologies</td>
			<td>MD0017</td>
			<td>70% isopropyl alcohol and pumice</td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Local drugstore sample</td>
			<td></td>
			<td>0.9% sodium chloride</td>
		</tr>
		<tr>
			<td>Non permanent hydro-marker</td>
			<td>Sharpie</td>
			<td>SHPE20WH</td>
			<td></td>
		</tr>
		<tr>
			<td>SYNGO MR VB17</td>
			<td>Siemens AG</td>
			<td></td>
			<td>MRI software</td>
		</tr>
		<tr>
			<td>MAGNETOM Trio A Tim System</td>
			<td>Siemens AG</td>
			<td></td>
			<td>MRI scanner version</td>
		</tr>
		<tr>
			<td>Matlab 2013a (Version 8.1)</td>
			<td>MathWorks Inc</td>
			<td></td>
			<td>processing and analysis software</td>
		</tr>
		<tr>
			<td>LCModel 6.3</td>
			<td>LC MODEL inc</td>
			<td></td>
			<td>see: s-provencher.com</td>
		</tr>
		<tr>
			<td>FASTESTMAP</td>
			<td colspan="2">Developers: Edward J. Auerbach and Ma&#322;gorzata Marja&#324;ska</td>
			<td>shimming sequence</td>
		</tr>
		<tr>
			<td>MEGA-PRESS</td>
			<td colspan="2">Developers: Edward J. Auerbach and Ma&#322;gorzata Marja&#324;ska</td>
			<td>MRS sequence</td>
		</tr>
	</tbody>
</table>

the use of magnetic resonance spectroscopy as a tool for the measurement of bi-hemispheric transcranial electric stimulation effects on primary motor cortex metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

Figure 6 shows the position of the VOI located on the representation of hand in M1 where all MRS measures were taken. In figure 6D, a 3D visualization shows a clear representation of the tDCS electrodes positioned on the scalp over the putative primary motor cortex. Figure 7 shows representative &#8220;EDIT OFF&#8221; and difference (&#8220;DIFF&#8221;) spectra acquired in M1. Peaks corresponding to Glx, GABA+MM as well as NAA can be clearly seen.
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The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

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20.1K Views.  University of Montréal. This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Video: The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

A Novel Approach for Documenting Phosphenes Induced by Transcranial Magnetic Stimulation

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical stimulation of the occipital cortex, but also by non-invasive Transcranial Magnetic Stimulation (TMS)1 of the same cortical regions. The intensity at which a phosphene is induced (phosphene threshold) is a well established measure of visual cortical excitability and is used to study cortico-cortical interactions, functional organization 2, susceptibility to pathology 3,4 and visual processing 5-7. Phosphenes are typically defined by three characteristics: they are observed in the visual hemifield contralateral to stimulation; they are induced when the subject s eyes are open or closed, and their spatial location changes with the direction of gaze 2. Various methods have been used to document phosphenes, but a standardized methodology is lacking. We demonstrate a reliable procedure to obtain phosphene threshold values and introduce a novel system for the documentation and analysis of phosphenes. We developed the Laser Tracking and Painting system (LTaP), a low cost, easily built and operated system that records the location and size of perceived phosphenes in real-time. The LTaP system provides a stable and customizable environment for quantification and analysis of phosphenes.

Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical ...

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

The Combined Use of Transcranial Direct Current Stimulation and Robotic Therapy for the Upper Limb

Neurologic disorders such as stroke and cerebral palsy are leading causes of long-term disability and can lead to severe incapacity and restriction of daily activities due to lower and upper limb impairments. Intensive physical and occupational therapy are still considered main treatments, but new adjunct therapies to standard rehabilitation that may optimize functional outcomes are being studied.
Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that polarizes underlying brain regions through the application of weak direct currents through electrodes on the scalp, modulating cortical excitability. Increased interest in this technique can be attributed to its low cost, ease of use, and effects on human neural plasticity. Recent research has been performed to determine the clinical potential of tDCS in diverse conditions such as depression, Parkinson&#39;s disease, and motor rehabilitation after stroke. tDCS helps enhance brain plasticity and seems to be a promising technique in rehabilitation programs.
A number of robotic devices have been developed to assist in the rehabilitation of upper limb function after stroke. The rehabilitation of motor deficits is often a long process requiring multidisciplinary approaches for a patient to achieve maximum independence. These devices do not intend to replace manual rehabilitation therapy; instead, they were designed as an additional tool to rehabilitation programs, allowing immediate perception of results and tracking of improvements, thus helping patients to stay motivated.
Both tDSC and robot-assisted therapy are promising add-ons to stroke rehabilitation and target the modulation of brain plasticity, with several reports describing their use to be associated with conventional therapy and the improvement of therapeutic outcomes. However, more recently, some small clinical trials have been developed that describe the associated use of tDCS and robot-assisted therapy in stroke rehabilitation. In this article, we describe the combined methods used in our institute for improving motor performance after stroke.

The combined use of transcranial direct current stimulation and robotic therapy as an add-on for conventional rehabilitation therapy may result in improved therapeutic outcomes due to modulation of brain plasticity. In this article, we describe the combined methods used in our institute for improving motor performance after stroke.

Neurologic disorders such as stroke and cerebral palsy are leading causes of long-term disability and can lead to severe incapacity and restriction of ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

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