The authors would like to thank Emily L. Thorn, B.A., for her assistance with the Source estimation of evoked electrical activity Section. MMS was supported by a KL2/Catalyst Medical Research Investigator Training award from Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health Award KL2 TR001100). CJC was supported by a grant from the National Institutes of Health (5K12NS066225). APL was supported in part by grants from the Sidney R. Baer Jr. Foundation, the National Institutes of Health (R01 HD069776, R01 NS073601, R21 MH099196, R21 NS082870, R21 NS085491, R21 HD07616), and Harvard Catalyst/The Harvard Clinical and Translational Science Center (NCRR and the NCATS, NIH UL1 RR025758). BSC was supported by the National Institute of Neurological Disorders and Stroke (R01 NS073601).

The protocol described here was approved by the institutional review boards of the Beth Israel Deaconess Medical Center and the Massachusetts Institute of Technology.
1. Subject Selection
<ol>
	<li>Patient selection for research protocol.
	<ol>
		<li>Identify patients with active epilepsy (seizures within the past year) or a history of remote epilepsy (prior seizures, but with no seizures in the past five years either on or off medication) and periventricular nodular heterotopia on structura...

Resting-state functional connectivity MRI has been used to identify network connectivity in the human brain, and to identify alterations of connectivity that occur in different disease states26,31,32. However, as fMRI functional connectivity is based on identifying correlations in the BOLD signal, and as blood oxygenation changes have a non-trivial relationship with underlying neural activity, the causal significance and physiological relevance of these fMRI connectivity findings is unclear. TMS enables spatia...

Transcranial magnetic stimulation (TMS) is a means of noninvasively stimulating regions of cortex via electromagnetic induction. In TMS, a large but spatially restricted magnetic flux is used to induce an electrical field in a target cortical area, and thereby modulate the activity of the underlying neural tissue. TMS to motor cortex results in motor evoked potentials that can be measured peripherally via electromyography (EMG). When applied in pairs or triplets of pulses, TMS can be used to assess the activity of specific intracortical GABAergic and glutaminergic circuits1-3, and thus assess the balance of excitation and inhibition in vivo in human patients. In epilepsy specifically, TMS studies have shown that cortical hyperexcitability is present in patients with epilepsy4,5, and may normalize with successful anti-epileptic drug therapy and thus predict response to medication6. Furthermore, TMS measures of cortical excitability show intermediate values in patients with a single seizure7 and in siblings of patients with both idiopathic generalized and acquired focal epilepsies8. These findings suggest that TMS measures of cortical excitability may allow us to identify endophenotypes for epilepsy. However, the sensitivity and specificity of these measures are limited, likely because TMS-EMG can only be assessed with stimulation of motor cortical circuits, and many patients with epilepsy have seizure foci outside the motor cortex.
Electroencephalography (EEG) provides an opportunity to directly measure the cerebral response to TMS, and can be used to assess cerebral reactivity across wide areas of neocortex. Studies integrating TMS with EEG (TMS-EEG) have shown that TMS produces waves of activity that reverberate throughout the cortex9,10 and that are reproducible and reliable11-13. By evaluating the propagation of evoked activity in different behavioral states and in different tasks, TMS-EEG has been used to causally probe the dynamic effective connectivity of human brain networks10,14-16. TMS-EEG measures have shown significant abnormalities in diseases ranging from schizophrenia17 to ADHD18, and in disorders of consciousness such as persistent vegetative state19. Furthermore, several groups have identified EEG correlates of the paired-pulse TMS-EMG metrics that are abnormal in patients with epilepsy20,21. Of particular relevance, previous studies have also suggested that abnormal stimulation-evoked EEG activity is seen in patients with epilepsy22-25.
Another means of evaluating brain circuits is via resting-state functional connectivity MRI (rs-fcMRI), a technique that evaluates the correlations over time in the blood oxygenation level dependent (BOLD) signal from different brain regions26. Studies using rs-fcMRI have demonstrated that the human brain is organized into distinct networks of interacting regions26-29, that neuropsychiatric diseases may occur within specific large-scale distributed neural networks identified by rs-fcMRI30, and that the brain networks identified via rs-fcMRI are often abnormal in neuropsychiatric disease states31,32. In terms of potential clinical applications, rs-fcMRI has several advantages over conventional task-based fMRI application33, including less reliance on subject cooperation and concern over variable performance. Consequently, there has recently been an explosion of studies exploring rs-fcMRI changes in different disease states. However, one of the limitations of rs-fcMRI is the difficulty in determining whether and how correlations (or anticorrelations) in the BOLD signal relate to the electrophysiological interactions that form the basis of neuronal communication. A related problem is that it is often unclear whether the rs-fcMRI changes seen in various disease states have physiologic significance. In particular with regards to epilepsy, it is unclear whether abnormalities in rs-fcMRI are due solely to interictal epileptiform transients, or exist independently of such electrophysiological abnormalities; simultaneous EEG-fMRI is needed to help evaluate between these possibilities34.
As TMS can be used to produce transient or sustained changes in the activations of different cortical regions, TMS studies provide a means of causally assessing the significance of different resting-state fMRI connectivity patterns. One approach is to use rs-fcMRI to guide therapeutic stimulation efforts in different disease states; it could be expected that TMS targeted to regions that are functionally connected to areas known to be involved in different disease states is more likely to be therapeutically effective than TMS targeted to regions without such functional connectivity, and indeed several studies have found preliminary evidence for this35,36. Another approach would involve using TMS-EEG to causally assess the physiologic significance of different resting-state fcMRI patterns. Specifically, one can test the hypothesis that regions that show abnormal functional connectivity in a specific disease state should show a different response to stimulation in patients than in healthy subjects, and that these physiologic abnormalities are present specifically (or primarily) with stimulation of the abnormally connected region.
To illustrate the above, we provide an example of a recent study in which rs-fcMRI, TMS and EEG were combined to explore cortical hyperexcitability in patients with epilepsy due to the developmental brain abnormality periventricular nodular heterotopia (PNH)37. Patients with PNH present clinically with adolescent- or adult-onset epilepsy, reading disability, and normal intelligence, and have abnormal nodules of gray matter adjacent to the lateral ventricles on neuroimaging38,39. Previous studies have shown that these periventricular nodules of heterotopic gray matter are structurally and functionally connected to discrete foci in the neocortex40,41, and that epileptic seizures may originate from neocortical regions, heterotopic gray matter, or both simultaneously42, suggesting that epileptogenesis in these patients is a circuit phenomenon. By using resting-state fc-MRI to guide TMS-EEG, we demonstrated that patients with active epilepsy due to PNH have evidence of cortical hyperexcitability, and that this hyperexcitability appears to be limited to regions with abnormal functional connectivity to the deep nodules.
The protocol is conducted in two separate sessions. During the first session, structural and resting-state blood-oxygenation level-dependent (BOLD) contrast MRI sequences are acquired (for patients), or just structural MRI sequences (for the healthy controls). Between the first and second sessions, resting-state functional connectivity analysis is used to define the cortical targets for the patients, and the MNI coordinates for these targets are obtained. The equivalent cortical targets (based on MNI coordinates) are then identified for each healthy control subject. In the second session, the TMS-EEG data is obtained.
In the example given in this paper, functional-connectivity MRI analyses were performed using an in-house software toolbox and the MRI software43,44. Neuro-navigated TMS was performed with a transcranial magnetic stimulator with real-time MRI neuronavigation. EEG was recorded with a 60-channel TMS-compatible system, which utilizes a sample-and-hold circuit to avoid amplifier saturation by TMS. EEG data were analyzed using custom scripts and the EEGLAB toolbox45 (version 12.0.2.4b) running in MATLAB R2012b.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3T MRI scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MRI functional connectivity software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MRI image viewing software</td>
			<td></td>
			<td></td>
			<td>MRICron</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transcranial Magnetic Stimulator</td>
			<td>Nexstim</td>
			<td>eXimia Stimulator&#160;</td>
			<td>Can use stimulators from other suppliers e.g. Magventure, Magstim</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MRI neuronavigation system</td>
			<td>Nexstim</td>
			<td>NBS v3.2.1</td>
			<td>Alternative MRI neuronavigation system e.g. Brainsight, Localite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMS-compatible EEG system</td>
			<td>Nexstim</td>
			<td>Eximia EEG</td>
			<td>Alternatives: Brain Products, Synamps, ANT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>Mathworks</td>
			<td>R2012b</td>
			<td>Alternatives: Octave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EEGLab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Minimum Norm Estimate (MNE) software</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FreeSurfer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a multimodal imaging- and stimulation-based method of evaluating connectivity-related brain excitability in patients with epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional connectivity fMRI can be used to identify regions of cortex that demonstrate high functional connectivity with the heterotopic periventricular gray matter nodules (Figure 1), and control regions without such connectivity. To determine whether such abnormal functional connectivity has physiologic significance, the cortical region with correlated resting-state activity can be chosen as the &#34;connected&#34; target sites for neuronavigated TMS, and...

Watch this Scientific Journal Video about A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy at JoVE.com

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

The overall goal of this experiment is to evaluate regional cortical hyperexcitability in patients with epilepsy using resting-state functional connectivity, MRI-guided, transcranial magnetic stimulation in combination with simultaneous EEG recording. This method can help answer key questions in the field of epilepsy and neurophysiology, such as whether patients with epilepsy have evidence of hyperexcitability in regions that are believed to be part of the epileptogenic network. The main advantage of this technique is that it can be used to assess differences in cerebral excitability as a function of connectivity and can be used to assess cortical reactivity across a variety of different brain regions.This technique has implications for the diagnosis and treatment of epilepsy, as cortical hyperexcitability can be identified even when the routine EEG is normal, and epileptogenic circuits can be targeted therapeutically. Demonstrating the procedure will be Tamara Gedankian, a research assistant in my laboratory. Prior to testing, determine the two TMS target regions by superimposing each subject's functional connectivity map onto each subject's structural image.To begin the experimental session, bring the subject into the testing room and have them sit in the chair. Measure the subject's head, and select an Electroencephalography, or EEG, cap, of appropriate size to enable low electrode impedances. Next, thoroughly clean the skin under each electrode using a cotton tip applicator and alcohol.Add conductive gel to each electrode, and press down on the electrode to ensure good contact between the scalp, gel, and electrode. To minimize charging artifacts, ensure that the gel does not spread outside of the electrode holder. Place the reference and ground electrodes on the forehead, and as far from the stimulation coil as possible, to minimize the possibility of a TMS-induced electrode artifact contaminating the entire recording.Place these electrodes within a few centimeters of each other to minimize common-mode noise. Then press the measure impedances button on the EEG system. Check electrode impedances by plugging the EEG output cables into the impedance jack of the EEG recording system.Ensure that the electrode impedance is not greater than five kilomes. Next, prepare the electromyography electrodes on the contralateral hand. Give the subject earplugs to minimize risk of hearing loss and tinnitus.Then place the infrared detectors on the subject's head, ensuring that the detectors are placed in a way to minimized risk of movement during the experimental session. Coregister the subject's head with the MRI images by identifying the location of the preselected, external anatomic fiducial markers on the subject using the pointer that is included with the neuronavigation equipment. Familiarize the subject with stimulation by applying a pulse elsewhere or by applying a low-intensity stimulation pulse to the scalp.Determine the resting motor threshold by locating the subject's motor cortex on the hemisphere ipsilateral to the functional connectivity-base targets. Angle the coil perpendicular to the gyrus with the handle pointing occipitally, and begin stimulation at an intensity that is expected to be sub-threshold. Then increase stimulation intensity in steps of 5%max stimulator output until TMS consistently evokes motor-evoked potentials with amplitudes greater than 50 microvolts in each trial.Decrease stimulation intensity in steps of 1%max stimulator output until less than five positive responses out of 10 are recorded. Finally, set the TMS intensity to the desired value. Apply single pulses of TMS to each of the target regions using the neuronavigation software, with variable intervals between pulses to minimize cortical plasticity and subject expectancy effects.Begin by performing an initial round of an Independent Component Analysis, or ICA, and remove the one to two components representing the large TMS-induced, initial muscle activation. To do this, run ICA using the fast ICA method with the symmetric approach, and the 10 contrast function, using the command shown here. Identify components consistent with the TMS artifact by selecting Tools, Reject data using ICA, and remove components by map, which will plot topographic maps of all the ICA components.Then click on the number for each component to plot component details. Next, delete the artifactual components by selecting Tools, Remove components, and entering the relevant component numbers in the field for Component(s)to remove from data. In the confirmation box that pops up, press Plot ERPs to review the event-related potentials, or ERPs, that result from deletion of the selected components.To review single trial effects, press Plot single trials. After reviewing the ERP, as in single trials, press the Accept button to delete the selected components. Perform a second round of ICA and remove components corresponding to artifacts of decay, blinks, muscle, and electrode noise.To do this, run ICA using the fast ICA method with the symmetric approach and the tan contrast function just as done for the first round of ICA. Likewise, evaluate component properties just as done with the topographic map in the first round of ICA. Then mark components consistent with residual TMS decay artifacts, blink artifacts, and muscle artifacts.Additionally, mark components consistent with channel noise based on spatial and temporal distribution. Finally, remove marked components, just as done in the first round of ICA, by selecting Tools, Remove components, and entering the relevant component numbers in the field for Component(s)to remove from data. Resting-state functional connectivity MRI is used to identify regions on the cortical surface with connectivity to the regions of heterotopia.TMS to these regions produces abnormally increased delayed activity relative to regions that do not have abnormal connectivity and relative to the same sights in healthy controls. Here, source localization of the abnormal late peaks in the TMS-evoked potentials in patients with epilepsy can identify brain regions from which the abnormal activity arises. It may spatially colocalize with the patient's seizure-focus.After watching this video, you should have a good understanding of how to use resting-state functional connectivity, MRI-guided, TMS EEG to evaluate brain excitability in different regions in patients with epilepsy and other neuropsychiatric disorders. Following this procedure, other methods, like repetitive TMS, can be performed to determine if decreasing the cortical excitability in brain regions that are part of the pathogenic network can modify disease activity.

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10.9K visualizações.  Harvard Medical School. The overall goal of this experiment is to evaluate regional cortical hyperexcitability in patients with epilepsy using resting-state functional connectivity, MRI-guided, transcranial magnetic stimulation in combination with simultaneous EEG recording. This method can help answer key questions in the field of epilepsy and neurophysiology, such as whether patients with epilepsy have evidence of hyperexcitability in regions that are believed to be part of the epileptogenic network. The main adva...