The authors have no acknowledgements.

This protocol was approved by the UCL Ethics Review Board (#249/001) for non-invasive brain stimulation of neurologically normal human volunteers.
1. Create the TMS protocol
Almost all TMS experiments in cognitive neuroscience use bi-phasic stimulation in conjunction with a figure-of-eight shaped coil. This provides the ability to deliver rapid trains of pulses (&#62; 1 Hz) and target a cortical site as precisely as possible. It is possible to use mono-phasic stimulation35 or a different coil shape36, but here the standard configuration was applied.
<ol>
	<li>Choose a frequency and duration of stimulation. 
	NOTE: A common choice in cognitive neuroscience is to use 10 Hz stimulation for 500 msec from onset of the stimulus37-40.</li>
	<li>Choose a level of intensity based on extensive pilot testing. Hold it constant across participants. 
	NOTE: For the equipment used here, commonly used intensities range between 50 - 70% of the maximum stimulator output11,41-44.</li>
	<li>Choose an inter-trial interval. For both practical and safety reasons, separate the stimulation trials by a minimum of 3 - 5 sec27,45.</li>
</ol>
2. Perform Head Registration
<ol>
	<li>Acquire a high-resolution, T1-weighted anatomical magnetic resonance imaging (MRI) scan for each participant on a separate session prior to the TMS. Include the fiducial points in the image that will be used in step 2.3.&#160;</li>
	<li>Load the scan into the frameless stereotaxy system before the TMS session to enable accurate targeting of stimulation sites in each participant. Mark the stimulation sites on the head at the beginning of the experiment or monitor continually throughout the experiment.</li>
	<li>Mark four fiducial points on the participant&#8217;s image. Typically these include the tip of the nose, the bridge of the nose, and the notch above the tragus of each ear.</li>
	<li>Provide the participant information about TMS in order for them to give informed consent to participate in the experiment.</li>
	<li>Ask the participant to complete a TMS Safety Screen form that has been approved by the Institutional Review Board.&#160; 
	NOTE: Permanent contradictions to TMS include a personal or family history of epilepsy, a clinical history of neurological or psychiatric problems, or implanted medical devices such as a cardiac pacemaker or cochlear implants. Not following TMS safety requirements can potentially induce syncope and seizure.</li>
	<li>Place the subject tracker on the participant&#8217;s head; it will act as a reference when measuring fiducial points. Touch each fiducial point on the subject&#8217;s head with a pointer that comes with the stereotaxy system and save the corresponding co-ordinates on the computer. Calibrate subject&#8217;s head with the MRI image. Check the quality of registration and repeat the process if necessary.</li>
	<li>Ask the participant to wear earplugs during stimulation to attenuate the sound of the coil discharge and avoid damage to participants&#8217; hearing46.</li>
	<li>Set up the TMS machine according to the choices made in Section 1.&#160;</li>
	<li>Introduce the participant to stimulation before testing to ensure that the participant is familiarized with its sensation and tolerates well. First demonstrate stimulation on the researcher&#8217;s arm and then on the participant&#8217;s arm to acclimate the person with the sensation. 
	NOTE: This is especially important for participants who are experiencing TMS for the first time.</li>
	<li>Demonstrate the stimulation protocol on each of the testing sites as the sensation may be different at different locations. Place the coil on the first site as identified by the frameless stereotaxy system such that the coil is tangential to the scalp and the line of maximum magnetic flux intersects the stimulated site.&#160; 
	NOTE: Stimulation sometimes affects facial nerves or muscles and may lead to discomfort so it is important to test whether the participant tolerates it well.</li>
</ol>
3. Perform Functional Localization
<ol>
	<li>Optimize the stimulation site by customizing it to each participant. Mark several potential stimulation sites within the brain region of interest on the participant&#8217;s structural image. Locate targets at least 10mm from each other given the spatial resolution of TMS47 using a grid or anatomical marking (Figure 1).</li>
	<li>Choose a localizer task that taps into the cognitive function of interest and has a measurable behavior (e.g., reaction times, accuracy, eye-movements). Repeat the task multiple times when testing the possible sites and create different versions of the task to avoid constant repetition of the stimuli.</li>
	<li>Allow the participant to practice the task without stimulation until they are comfortable with it. Then introduce a second practice session with TMS randomly (or pseudo-randomly) presented on 50% of the trials so that the participant gets used to performing the task without being distracted by stimulation.</li>
	<li>Choose a testing site and run one version of the localizer task. Immediately afterwards check the results to see whether stimulation affected performance.&#160; 
	NOTE: In many cases, stimulating an &#8220;incorrect&#8221; site will actually facilitate responses relative to no stimulation due to inter-sensory facilitation2, in this case due to hearing the clicks and feeling the sensation of stimulation in the scalp. In addition, large effects of stimulation (i.e., over 100 msec) are often artifactual and require re-testing. If they replicate and are specific to a particularly testing site, then they may be genuine effects. Be sure to choose a robust measure of a TMS effect to be confident in localization.&#160;</li>
	<li>If no effect is observed, choose a new testing site and repeat, otherwise test the same site again to determine whether it replicates. Test multiple sites back-to-back in the same session to ensure that they do not all produce an effect as this would indicate a non-specific TMS effect. Counterbalance the order that sites are stimulated across participants.</li>
</ol>
4. Main Task
<ol>
	<li>After localization and in the same session, run the main experiment using the target site that was functionally localized. 
	NOTE: This will involve a different task to the one used in localization but one that shares the key process of interest. For example, a rhyme judgment task can be used for localizing an area sensitive to processing the sounds of words while a homophone judgment task might be used for the main experiment. In this example, both tasks require phonological processing of written words although the specific task demands and stimuli differ.</li>
	<li>Include sufficient control conditions to rule out non-specific effects of TMS.</li>
	<li>Test the same site on a control task that does not include the process of interest to demonstrate functional specificity in processing.</li>
	<li>Test a different site on the main task to demonstrate anatomical specificity of the effect.</li>
	<li>Include additional control conditions such as sham TMS, control stimuli, or multiple time windows.</li>
	<li>Conduct a traditional &#8220;virtual lesion&#8221; experiment using the same TMS parameters used during localization (e.g., intensity, frequency, and duration of stimulation). For chronometric TMS experiment, use the same intensity but replace the train of pulses used during localization by either a single48 or double pulse49 delivered at different onset latencies.</li>
</ol>

The authors declare that they have no competing financial interests.

This article presents a protocol for assessing the causal and temporal involvement of brain regions in cognitive processes using online TMS. This discussion highlights first the critical steps for creating a successful TMS protocol and then the limitations that need to be considered when designing a TMS experiment.
Because TMS protocols have a large number of free parameters, ensuring the optimal stimulation parameters is a critical step in preparing a TMS experiment. Normally, this is achieved through extensive pilot testing in order to determine the stimulation frequency, duration, intensity, inter-trial interval, and coil orientation necessary to produce robust effects. To create an effective &#8220;virtual lesion&#8221; the frequency must induce a robust effect that covers a sufficiently large time window to encompass the cognitive process of interest. As a result, both frequency and duration vary across studies. Similarly, the &#8220;right&#8221; stimulation intensity is one that ensures the magnetic field affects neural processing in the target brain region and here the main factor is the distance from the coil to the stimulation site51. Many studies identify the intensity of stimulation necessary to produce a motor response when stimulating the hand area of primary motor cortex and use this to normalize intensity across participants52,53-55. This measure, however, is not a reliable index of the optimal intensity for non-motor areas42,51,56. Another option is to use the same intensity for all participants. The chosen intensity should be effective across all pilot subjects after experimenting with a range of stimulation intensities. Additionally, the coil orientation is an important parameter that requires consideration. The specific coil orientation affects the distribution of the induced electric field within the stimulated neuronal population and therefore may influence behavior. In general, published protocols can provide a starting point that is iteratively modified during pilot testing to suit the specific experiment. Often, however, information about this pilot testing is omitted from the final manuscript, which has the unfortunate effect of hiding some key aspects of the protocol design process.
Choosing a localization procedure is also essential to ensure that stimulation is administered to the optimal site. Although many studies have successfully localized stimulation sites using anatomy-based methods that target a single location across individual participants 57,58, customizing the stimulation site for each subject individually reduces between-subject variance in behavioral results yielding a more efficient method31. Here we presented a TMS-based functional localization procedure that offers advantages over fMRI-based localization. Specifically, it avoids the problem of different spatial biases between fMRI (i.e., draining veins59) and TMS (i.e., the orientation of axons within the magnetic field6,60) that may result in the same neural response being localized to different locations. In addition, it is well known that the specific location of activation &#8220;peaks&#8221; in fMRI can vary considerably, making them sub-optimal as TMS targets55,61. Even so, a variety of different localization procedures are demonstrably effective, so the specific choice is less important that ensuring that whichever method is used provides reliable, reproducible effects.
Although the experiment data presented here used reaction times as the dependent measure, there are many other options available. For instance, some studies use accuracy instead9,12,62. In these cases, normal performance without TMS is already below ceiling levels so the disruption induced by stimulation is reflected in the accuracy scores. Other studies have measured the effects of stimulation on eye movements63,64. Most cognitive neuroscience experiments with TMS, however, use reaction times as their dependent measure13,48,65,66. Typically, the effects are on the order of tens of msec, or roughly a 10% change in reaction times67. Whatever dependent measure is used should be robust and consistent so that relatively small changes can be easily observed.
Like any experimental technique, TMS has important limitations that need to be considered when choosing this methodology. The most common ones are: i) the spatial resolution of TMS, ii) the non-specific effects associated with stimulation, and iii) safety aspects of the methodology. First, TMS has a limited depth of stimulation because the magnetic field reduces in intensity the further away it is from the coil. Consequently, it is most effective at stimulating brain regions near scalp (~2 - 3 cm)68,69 and is ineffective in stimulating deep brain structures. As a result, the only regions directly accessible to TMS are limited to the cortical mantle, although different shaped coils are being developed to reach deeper regions such as the basal ganglia69. TMS also has a spatial resolution of approximately 0.5 - 1 cm47,70-72. Thus, the method cannot be used to investigate the functional contributions from fine-grained spatial structures such as cortical columns.
A second limitation of TMS is that stimulation introduces concurrent sensory side effects as a result of the rapidly-changing magnetic field. Most notably, each magnetic pulse is accompanied by an auditory click and a tapping sensation. Therefore TMS may be inappropriate for certain auditory or somatosensory experiments where these side effects may interfere with task performance. Note, however, that online TMS has been used successfully in some auditory experiments73,74 and is therefore feasible in at least some tasks. Another consideration is that the intensity of the sensory effects differs across head locations. For example, stimulation that is administered to a location close to the ear will sound louder than locations further away. Similarly more ventral locations on the head produce greater muscle contraction than dorsal areas75,76. Because these site differences can induce experimental confounds, it is important to use either a control site with similar side-effects to the main site such as contralateral homologues77 or include control conditions/tasks that do not tap into the process of interest24,62,73,78,79.
Finally, safety considerations must always be taken into account when designing TMS experiments as it can potentially induce syncope and seizures27. To minimize this risk, internationally accepted guidelines for stimulation intensity, frequency, and duration exists, as well as for the total number of pulses and the inter-trial intervals27,28. Protocols that stay within these guidelines are believed to be safe for neurologically normal participants. It is worth noting, however, that these are as yet incomplete and that often novel TMS protocols are introduced that also prove safe. In general, the evidence suggests that when published guidelines are followed, TMS is a safe procedure with no dangerous side effects. One consequence of these limits, however, is that behavioral protocols will often need to be adjusted before they can be used with TMS. This has implications for several aspects of the design, including the length of the experiment, number of trials, number of conditions and stimulation sites that can be tested. Some of these limitations may be overcome by splitting the experiment into separate sessions such as testing different stimulation sites on different days. In those cases, it is important to ensure that localization and testing of a site are done within the same session. This minimizes experimental variance by maximizing the accuracy of the targeting. When deciding whether to use one or more testing session, the fundamental limitation is the safety of the participant &#8211; specifically, the amount of stimulation that is safe in a single session. The total stimulation involves familiarization, practice, localization (if using TMS), and testing, potentially over multiple sites, and critically depends on the number of trials per condition. Where this figure exceeds the guidelines for a single session, it is necessary to break the experiment into multiple sessions, conducted a minimum of 24 hr apart. There are no hard-and-fast rules regarding the minimum number of trials necessary for TMS experiments, but like any experiment, these can be computed using standard power calculations based on the effect size, variance, &#945;-level (typically 0.05) and desired sensitivity. Often reasonable estimates of the effect size and variance are available as a result of the extensive pilot testing done to optimize the experimental protocol.
In summary, TMS has become an important tool with broad applications to cognitive neuroscience. This article provides a basic protocol for online TMS in conjunction with a behavioral task for investigating causal brain-behavioral relationships both in &#8220;virtual lesion&#8221; mode and also a chronometric tool for exploring the temporal dynamics of regionally-specific neural information processing.

Transcranial magnetic stimulation (TMS) is a safe and non-invasive tool used for brain stimulation. It uses a rapidly changing electric current within a conducting coil to generate a strong, but relatively focal, magnetic field. When applied to the scalp, the magnetic field induces electrical activity in the underlying brain tissue, temporarily disrupting local cortical information processing. This transient interference effectively creates a short-lasting &#8220;virtual lesion&#8221;1,2. This technique offers a non-invasive method for drawing causal brain-behavior inferences and investigating the temporal dynamics of online neural information processing in both healthy adults and neurological patients.
By selectively interfering with regionally-specific cortical processing, TMS can be used to draw causal links between brain regions and specific behaviors3,4. That is, if stimulating a cortical area significantly affects task performance relative to appropriate control conditions, this indicates that the stimulated area is necessary to perform the task normally. Causal inferences of this sort are one of the major advantages of TMS over neuroimaging methods such as functional magnetic resonance imaging (fMRI) or positron emission tomography (PET). Unlike neuroimaging techniques that measure neural activity and correlate it with behavior, TMS offers the opportunity to perturb neural information processing and measure its effects on behavior. In this sense, it is more like traditional lesion-deficit analyses in patients with brain damage except that TMS is non-invasive and the effects are temporary and reversible. TMS also has several advantages over lesion studies. For instance, the effects of stimulation are generally more spatially precise than naturally occurring lesions, which are often large and vary greatly across patients. In addition, participants can be used as their own controls, thereby avoiding the issue of potential differences in pre-morbid abilities between patients and controls. Finally, there is insufficient time for functional reorganization to take place during TMS, meaning that recovery processes are unlikely to confound the results5. In other words, TMS offers a powerful tool for investigating causal brain-behavior relations that complements correlative techniques such as functional neuroimaging.
TMS can also be used to investigate the time course of neural information processing by using very short bursts of stimulation and varying the onset of stimulation6. Typically this involves either a single or double pulse TMS delivered to a region at different points of time within a trial. Because the effect of an individual TMS pulse occurs immediately and lasts somewhere between 5 and 40 msec7-10, this enables the researcher to map the temporal dynamics of regional neuronal activity including its onset, duration, and offset11,12. The duration of this disruption limits the temporal resolution of the technique to 10s of msec, roughly an order of magnitude coarser than electroencephalography (EEG) and magnetoencephalography (MEG). On the other hand, the timings observed in chronometric TMS studies tend to match those from invasive neurophysiological recordings better than EEG and MEG9,13. Presumably this is because EEG and MEG measure large scale neuronal synchrony that lags behind the earliest onset of activity14. In addition, like fMRI and PET, EEG and MEG are correlative measures of whole brain activity whereas chronometric TMS can not only provide important information about regional temporal dynamics but also about the necessity of the region for a given behavior.
Although TMS was originally developed for investigating the physiology of the motor system15, it was quickly adopted as a valuable tool for cognitive neuroscience. One of its earliest uses as a &#8220;virtual lesion&#8221; technique was to induce speech arrest by stimulating the left inferior frontal cortex16-18. The results confirmed the importance of Broca&#8217;s area for speech production and suggested a potential alternative to Wada testing to determine language dominance subsequent to neurosurgical interventions16,19. Now TMS is used in virtually all areas of cognitive neuroscience including attention20, memory21, visual processing22, action planning23, decision making24, and language processing25. Typically TMS induces either increased error rates or slower reaction times (RTs), both of which are taken as indicators of causal relations between brain and behavior3,4. Some studies use TMS in both its virtual lesion mode and as a chronometric tool. For example, Pitcher and colleagues11 first showed that repetitive TMS (rTMS) delivered to the occipital face area disrupted accurate facial discrimination and then used chronometric TMS to determine that this effect was only present when TMS was delivered at 60 and 100 msec, demonstrating that this particular brain region processes face-part information at an early stage of face recognition. In all of the examples mentioned here, TMS is administered &#8220;on-line&#8221;, that is during task performance, so that the effects of TMS are immediate and short lived (i.e., the effects last as long as the duration of stimulation). This contrasts with &#8220;off-line&#8221; TMS which involves either long runs of low frequency stimulation21 or short bursts of patterned stimulation26 before starting a task. In off-line TMS the effects last well beyond the duration of the TMS application itself. This article focuses exclusively on the &#8220;on-line&#8221; approach.
The initial steps in preparing any TMS experiment include identifying a stimulation protocol and choosing a localization method. Stimulation parameters include intensity, frequency and duration of TMS and are constrained by internationally defined safety requirements27,28. Each TMS experiment also requires a suitable localization procedure for positioning and orienting the coil accurately over the stimulation site. Localization can be based on standard space coordinates29 or 10 - 20 localization system30, but typically is customized to each individual participant31. For the latter, there are many options that include targeting stimulation based on each individual&#8217;s anatomy32, functionally localizing using fMRI33 or functionally localizing using TMS34. The protocol presented here advocates functional localization with TMS as part of a general protocol for on-line TMS experiments. Then an illustrative example is presented of how TMS can be used to investigate the functional contributions of the left supramarginal gyrus (SMG) to phonological processing in reading.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:640px;">1) Magstim Rapid2 stimulator (Magstim, Carmarthenshire, UK)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">2) 70-mm diameter figure-of-eight coil</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">3) Brainsight frameless stereotaxy system (RogueResearch, Montreal, Canada)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">4) Polaris Vicra infrared camera (Northern Digital, Waterloo, ON, Canada)</td>
		</tr>
	</tbody>
</table>

transcranial magnetic stimulation for investigating causal brain-behavioral relationships and their time course

Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily disrupt information processing in a brain region, generating a short-lived &#8220;virtual lesion.&#8221; Stimulation that interferes with task performance indicates that the affected brain region is necessary to perform the task normally. In other words, unlike neuroimaging methods such as functional magnetic resonance imaging (fMRI) that indicate correlations between brain and behavior, TMS can be used to demonstrate causal brain-behavior relations. Furthermore, by varying the duration and onset of the virtual lesion, TMS can also reveal the time course of normal processing. As a result, TMS has become an important tool in cognitive neuroscience. Advantages of the technique over lesion-deficit studies include better spatial-temporal precision of the disruption effect, the ability to use participants as their own control subjects, and the accessibility of participants. Limitations include concurrent auditory and somatosensory stimulation that may influence task performance, limited access to structures more than a few centimeters from the surface of the scalp, and the relatively large space of free parameters that need to be optimized in order for the experiment to work. Experimental designs that give careful consideration to appropriate control conditions help to address these concerns. This article illustrates these issues with TMS results that investigate the spatial and temporal contributions of the left supramarginal gyrus (SMG) to reading.

Figure 2 illustrates the results of two TMS experiments mentioned as examples. Namely, the first investigated whether the left SMG is causally involved in processing the sounds of words while the second investigated the temporal dynamics of this involvement. Figure 2A shows representative results of the first experiment where rTMS (10 Hz, 5 pulses, 55% of maximum intensity) was delivered to SMG during three tasks. The phonological task focused attention on the sounds of words (&#8220;Do these two words sound the same? knows-nose&#8221;) while the semantic task focused on their meaning (&#8220;Do these two words mean the same thing? idea-notion&#8221;). A third control task presented pairs of consonant letter strings and asked whether they were identical (&#8220;wsrft-wsrft&#8221;). Each task consisted of 100 trials. The results demonstrated that TMS significantly increased RTs relative to no stimulation in the phonological task by an average of 37 msec. In contrast, SMG stimulation had no significant effect on the RTs in the semantic or orthographic control tasks. In other words, a &#8220;virtual lesion&#8221; of the left SMG selectively interfered with processing the sounds of words, indicating the necessity of SMG in processing phonological aspects of written words44.
Figure 2B shows representative results of the chronometric experiment exploring the time course of phonological processing within SMG. Here, double pulse TMS was delivered at five different time windows after stimulus onset during the same phonological task with 100 trials divided into five equal blocks each testing different time window. When compared to the baseline condition (40/80 msec), a significant increase in RTs was observed when TMS was delivered 80/120, 120/160, and 160/200 msec after the onset of the stimulus. These results demonstrated SMG was engaged in phonological processing between 80 and 200 msec post-stimulus onset, indicating both early and sustained involvement in phonological processing44.
<img alt="Figure 1" src="/files/ftp_upload/51735/51735fig1highres.jpg" /> 
Figure 1.&#160;Two common methods of marking potential stimulation sites.&#160;(A)&#160;A first method involves placing a grid of markers over a hand motor area and testing each until TMS produces the expected effect. This approach is common for identifying a motor &#8220;hot spot&#8221;- that is, the place where stimulation produces the strongest, most reliable muscle contraction. (B)&#160;A second method applies additional anatomical constrains by placing a set of markers within a well-defined brain region. In this example, the location of the three markers is restricted to the anterior region of&#160;SMG. The first one is located superior to the termination of the posterior ascending ramus of the Sylvian fissure; the second one is at the ventral end of the anterior SMG; and the third one is approximately halfway between the other two sites. Stimulation markers are shown on a parasagittal plane of an individual MRI scan using frameless stereotaxy system. The black scale bar in the left bottom corner indicates a distance of 1 cm.
<img alt="Figure 2" src="/files/ftp_upload/51735/51735fig2highres.jpg" /> 
Figure 2.&#160;Reaction times (RTs) from the onset of the stimulus. (A)&#160;noTMS (light bars) and TMS (dark bars) conditions in three different language tasks. (B)&#160;Five stimulation timing conditions in the phonological task. In the example presented here, double pulses were delivered at either 40/80 msec, 80/120 msec, 120/160 msec, 160/200 msec, and 200/240 msec post stimulus onset. The first time window, 40/80 msec, was used as a baseline control condition because visual information was not expected to arrive at the SMG that rapidly. Error bars represent the standard error of the mean adjusted to correctly reflect within-subject variance50. The first experiment contains data from 12 participants and the second from 32 participants. *p &#60; 0.05.

Watch this Scientific Journal Video about Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course at JoVE.com

Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course

Transcranial magnetic stimulation (TMS) is a technique for non-invasively disrupting neural information processing and measuring its effect on behavior. When TMS interferes with a task, it indicates that the stimulated brain region is necessary for normal task performance, allowing one to systematically relate brain regions to cognitive functions.

Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily ...

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42.8K Views.  University College London.  Stimolazione Magnetica Transcranica (TMS) è una tecnica per non invasivo interrompere l'elaborazione delle informazioni neurali e misurare il suo effetto sul comportamento. Quando TMS interferisce con un compito, indica che la regione del cervello stimolata è necessaria per prestazioni normali attività, permettendo di riferiscono sistematicamente regioni cerebrali alle funzioni cognitive. 

Video: Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Simultaneous EEG Monitoring During Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that delivers weak electric currents through the scalp. This constant electric current induces shifts in neuronal membrane excitability, resulting in secondary changes in cortical activity. Although tDCS has most of its neuromodulatory effects on the underlying cortex, tDCS effects can also be observed in distant neural networks. Therefore, concomitant EEG monitoring of the effects of tDCS can provide valuable information on the mechanisms of tDCS. In addition, EEG findings can be an important surrogate marker for the effects of tDCS and thus can be used to optimize its parameters. This combined EEG-tDCS system can also be used for preventive treatment of neurological conditions characterized by abnormal peaks of cortical excitability, such as seizures. Such a system would be the basis of a non-invasive closed-loop device. In this article, we present a novel device that is capable of utilizing tDCS and EEG simultaneously. For that, we describe in a step-by-step fashion the main procedures of the application of this device using schematic figures, tables and video demonstrations. Additionally, we provide a literature review on clinical uses of tDCS and its cortical effects measured by EEG techniques.

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has shown initial therapeutic effects in several neurological conditions. The main mechanism underlying these therapeutic effects is the modulation of cortical excitability. Therefore, online monitoring of cortical excitability would help guide stimulation parameters and optimize its therapeutic effects. In the present article we review the use of a novel device that combines simultaneous tDCS and EEG monitoring in real time.

Transcranial  direct current stimulation (tDCS) is a technique that delivers weak electric  currents through the scalp. This constant electric current ...

Stimulating the Lip Motor Cortex with Transcranial Magnetic Stimulation

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception. Researchers have used single-pulse and repetitive TMS to stimulate the lip representation in the motor cortex. The excitability of the lip motor representation can be investigated by applying single TMS pulses over this cortical area and recording TMS-induced motor evoked potentials (MEPs) via electrodes attached to the lip muscles (electromyography; EMG). Larger MEPs reflect increased cortical excitability. Studies have shown that excitability increases during listening to speech as well as during&#160;viewing speech-related movements. TMS can be used also to disrupt the lip motor representation. A 15-min train of low-frequency sub-threshold repetitive stimulation has been shown to suppress motor excitability for a further 15-20 min. This TMS-induced disruption of the motor lip representation impairs subsequent performance in demanding speech perception tasks and modulates auditory-cortex responses to speech sounds. These findings are consistent with the suggestion that the motor cortex contributes to speech perception. This article describes how to localize the lip representation in the motor cortex and how to define the appropriate stimulation intensity for carrying out both single-pulse and repetitive TMS experiments.

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception.&#160; This article describes how to record motor evoked potentials (MEPs) from the lip muscles and how to disrupt the motor lip representation using repetitive TMS.

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception. ...

Transcranial Direct Current Stimulation and Simultaneous  Functional Magnetic Resonance Imaging

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses weak electrical currents administered to the scalp to manipulate cortical excitability and, consequently, behavior and brain function. In the last decade, numerous studies have addressed short-term and long-term effects of tDCS on different measures of behavioral performance during motor and cognitive tasks, both in healthy individuals and in a number of different patient populations. So far, however, little is known about the neural underpinnings of tDCS-action in humans with regard to large-scale brain networks. This issue can be addressed by combining tDCS with functional brain imaging techniques like functional magnetic resonance imaging (fMRI) or electroencephalography (EEG).
In particular, fMRI is the most widely used brain imaging technique to investigate the neural mechanisms underlying cognition and motor functions. Application of tDCS during fMRI allows analysis of the neural mechanisms underlying behavioral tDCS effects with high spatial resolution across the entire brain. Recent studies using this technique identified stimulation induced changes in task-related functional brain activity at the stimulation site&#160;and also in more distant brain regions, which were associated with behavioral improvement. In addition, tDCS administered during resting-state fMRI allowed identification of widespread changes in whole brain functional connectivity.
Future studies using this combined protocol should yield new insights into the mechanisms of tDCS action in health and disease and new options for more targeted application of tDCS in research and clinical settings. The present manuscript describes this novel technique in a step-by-step fashion, with a focus on technical aspects of tDCS administered during fMRI.

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique. It has successfully been used in basic research and clinical settings to modulate brain function in humans. This article describes the implementation of tDCS and simultaneous functional magnetic resonance imaging (fMRI), to investigate the neural basis of tDCS effects.

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses weak electrical currents administered to the scalp ...

Modulating Cognition Using Transcranial Direct Current Stimulation of the Cerebellum

Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain regions involved in working memory and attention using transcranial electrical brain stimulation. Some researchers now believe the cerebellum supports cognition, possibly via a remote neuromodulatory effect on the prefrontal cortex. This paper describes a procedure for investigating a role for the cerebellum in cognition using transcranial direct current stimulation (tDCS), and a selection of information-processing tasks of varying task difficulty, which have previously been shown to involve working memory, attention and cerebellar functioning. One task is called the Paced Auditory Serial Addition Task (PASAT) and the other a novel variant of this task called the Paced Auditory Serial Subtraction Task (PASST). A verb generation task and its two controls (noun and verb reading) were also investigated. All five tasks were performed by three separate groups of participants, before and after the modulation of cortico-cerebellar connectivity using anodal, cathodal or sham tDCS over the right cerebellar cortex. The procedure demonstrates how performance (accuracy, verbal response latency and variability) could be selectively improved after cathodal stimulation, but only during tasks that the participants rated as difficult, and not easy. Performance was unchanged by anodal or sham stimulation. These findings demonstrate a role for the cerebellum in cognition, whereby activity in the left prefrontal cortex is likely dis-inhibited by cathodal tDCS over the right cerebellar cortex. Transcranial brain stimulation is growing in popularity in various labs and clinics. However, the after-effects of tDCS are inconsistent between individuals and not always polarity-specific, and may even be task- or load-specific, all of which requires further study. Future efforts might also be guided towards neuro-enhancement in cerebellar patients presenting with cognitive impairment once a better understanding of brain stimulation mechanisms has emerged.

Transcranial direct current stimulation (tDCS) over the cerebellum exerts a remote effect on the prefrontal cortex, which can modulate cognition and performance. This was demonstrated using two information-processing tasks of varying complexity, whereby only cathodal tDCS improved performance when the task was difficult, but not easy.

Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain ...

Multifunctional Setup for Studying Human Motor Control Using Transcranial Magnetic Stimulation, Electromyography, Motion Capture, and Virtual Reality

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular function include transcranial magnetic stimulation, electromyography, and three-dimensional motion capture. The advent of readily available and cost-effective virtual reality solutions has expanded the capabilities of researchers in recreating &#8220;real-world&#8221; environments and movements in a laboratory setting. Naturalistic movement analysis will not only garner a greater understanding of motor control in healthy individuals, but also permit the design of experiments and rehabilitation strategies that target specific motor impairments (e.g. stroke). The combined use of these tools will lead to increasingly deeper understanding of neural mechanisms of motor control. A key requirement when combining these data acquisition systems is fine temporal correspondence between the various data streams. This protocol describes a multifunctional system&#8217;s overall connectivity, intersystem signaling, and the temporal synchronization of recorded data. Synchronization of the component systems is primarily accomplished through the use of a customizable circuit, readily made with off the shelf components and minimal electronics assembly skills.

Transcranial magnetic stimulation, electromyography, and 3D motion capture are commonly used non-invasive techniques for investigating neuromuscular function in humans. In this paper, we describe a protocol that synchronously samples data generated by all three of these tools along with the unique addition of virtual reality stimulus presentation and feedback.

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular ...

Neuronavigation-guided Repetitive Transcranial Magnetic Stimulation for Aphasia

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential therapeutic effects. Brain excitability is non-invasively modulated by rTMS, and rTMS to the language areas has proved its potential effects on treatment of aphasia. In our protocol, we aim to artificially induce virtual aphasia in healthy subjects by inhibiting Brodmann area 44 and 45 using neuronavigational TMS (nTMS), and F3 of the International 10-20 EEG system for conventional TMS (cTMS). To measure the degree of aphasia, changes in reaction time to a picture naming task pre- and post-stimulation are measured and compare the delay in reaction time between nTMS and cTMS. Accuracy of the two TMS stimulation methods is compared by averaging the Talairach coordinates of the target and the actual stimulation. Consistency of stimulation is demonstrated by the error range from the target. The purpose of this study is to demonstrate use of nTMS and to describe the benefits and limitations of the nTMS compared to those of cTMS.

This study is designed to test the hypothesis that neuronavigational system-guided transcranial magnetic stimulation has higher accuracy for targeting the intended target as demonstrated by eliciting a greater degree of virtual aphasia in healthy subjects, measured by delay in reaction time to picture naming.

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential ...

Concurrent Electroencephalography Recording During Transcranial Alternating Current Stimulation (tACS)

Oscillatory brain activities are considered to reflect the basis of rhythmic changes in transmission efficacy across brain networks and are assumed to integrate cognitive neural processes. Transcranial alternating current stimulation (tACS) holds the promise to elucidate the causal link between specific frequencies of oscillatory brain activity and cognitive processes. Simultaneous electroencephalography (EEG) recording during tACS would offer an opportunity to directly explore immediate neurophysiological effects of tACS. However, it is not trivial to measure EEG signals during tACS, as tACS creates a huge artifact in EEG data. Here we explain how to set up concurrent tACS-EEG experiments. Two necessary considerations for successful EEG recording while applying tACS are highlighted. First, bridging of the tACS and EEG electrodes via leaking EEG gel immediately saturates the EEG amplifier. To avoid bridging via gel, the viscosity of the EEG gel is the most important parameter. The EEG gel must be viscous to avoid bridging, but at the same time sufficiently fluid to create contact between the tACS electrode and the scalp. Second, due to the large amplitude of the tACS artifact, it is important to consider using an EEG system with a high resolution analog-to-digital (A/D) converter. In particular, the magnitude of the tACS artifact can exceed 100 mV at the vicinity of a stimulation electrode when 1 mA tACS is applied. The resolution of the A/D converter is of importance to measure good quality EEG data from the vicinity of the stimulation site. By following these guidelines for the procedures and technical considerations, successful concurrent EEG recording during tACS will be realized.

Concurrent Electroencephalography Recording During Transcranial Alternating Current Stimulation tACS

In this article we explain how to set up a concurrent transcranial alternating current stimulation and EEG experiment.

Oscillatory brain activities are considered to reflect the basis of rhythmic changes in transmission efficacy across brain networks and are assumed to ...

Treating Clinical Depression with Repetitive Deep Transcranial Magnetic Stimulation Using the Brainsway H1-coil

Repetitive transcranial magnetic stimulation (rTMS) is an emerging non-pharmacological approach to treating many brain-based disorders. rTMS uses electromagnetic coils to stimulate areas of the brain non-invasively. Deep transcranial magnetic stimulation (dTMS) with the Brainsway H1-coil system specifically is a type of rTMS indicated for treating patients with major depressive disorder (MDD) who are resistant to medication. The unique H1-coil design of this device is able to stimulate neuronal pathways that lie deeper in the targeted brain areas than those reached by conventional rTMS coils. dTMS is considered to be low-risk and well tolerated, making it a viable treatment option for people who have not responded to medication or psychotherapy trials for their depression.
Randomized, sham-control studies have demonstrated that dTMS produces significantly greater improvement in depressive symptoms than sham dTMS treatment in patients with major depression that has not responded to antidepressant medication. In this paper, we will review the methodology for treating major depression with dTMS using an H1-coil.

Here we present a protocol outlining the methodology for treatment of Major Depressive Disorder using the deep Transcranial Magnetic Stimulation (dTMS) system.

Repetitive transcranial magnetic stimulation (rTMS) is an emerging non-pharmacological approach to treating many brain-based disorders. rTMS uses ...

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...