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 (s...

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.
<p class="jove_cont...

Watch this Scientific Journal Video about The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex M

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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20.0K vistas.  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