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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Subject Selection
- Patient selection for research protocol.
- 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 structural brain imaging.
- Exclude patients without any history of seizures. Also, exclude patients with alternative possible etiologies for seizures (e.g., a history of traumatic brain injury, stroke, meningoencephalitis) or with EEG findings consistent with an alternative diagnosis (e.g., idiopathic generalized epilepsy, mesial temporal lobe epilepsy).
- Exclude patients with additional neurologic or psychiatric disease, or with any other unstable medical condition. Also, exclude patients with a history of prior brain surgery, inability to tolerate magnetic resonance imaging (MRI), recent illicit substance or heavy alcohol use, or a specific MRI or TMS contraindication.
- Healthy control subject selection.
- For each periventricular nodular heterotopia (PNH) patient (in our prior published study, 8 patients, ages 20 - 43 years mean 30.25; 3 male, 5 female), identify an age- and gender-matched healthy control.
- Exclude subjects with any ongoing neurologic or psychiatric disease or on psychoactive medications, any other unstable medical condition, a history of prior brain surgery, inability to tolerate MRI, illicit substance or heavy alcohol use, or any other specific MRI or TMS contraindication.
2. Generating the Stimulation Targets
- Using a 3T MRI system, acquire high-resolution structural whole-brain slices using a T1-weighted sequence. Use the following acquisition parameters: 128 slices per slab, a 256 x 256 matrix, field of view (FOV) 256 mm, slice thickness 1.33 mm with 0.63 mm interslice gap, voxel size 1 x 1 x 1.33 mm3, repetition time (TR) 2,530 msec, inversion time 1,100 msec, echo time (TE) 3.39 msec, flip angle 7°.
- Using a 3T MRI system, acquire resting-state functional images using an echo-planar sequence sensitive to blood-oxygenation level-dependent (BOLD) contrast. While performing this scan, instruct the patients to rest quietly with their eyes open without performing any specific task. Use the following acquisition parameters: FOV 256 mm, voxel size 2.0 x 2.0 x 2.0 mm, TR 6,000 msec, TE 30 msec, flip angle 90°, acquisition time 6.4 min.
- Using MRICroN software, identify each discrete region of nodular heterotopia (either each individual nodule or an inseparable contiguous cluster of nodules). Use the Pen tool to manually outline heterotopia regions of interest (ROIs), slice by slice in the axial plane on T1-weighted structural images.
- Use the CONN functional connectivity software toolbox to perform four sequential steps in resting-state functional data processing: Setup, Preprocessing, Analysis, and Results.
- For Setup, use the menu choices to start a new project and enter basic experiment information. Load the functional images, realigned and co-registered to the anatomic images for each subject.
- Load the structural images. Load heterotopia ROI files created in step 2.3. Enter details of the experimental condition; since this is a resting state, enter a single condition with onset 0 seconds and duration equal to the complete duration of each session. The toolbox will extract the heterotopia ROI BOLD time-series. Inspect for possible inconsistencies.
- For Preprocessing, confounding sources of BOLD variation include respiratory-induced modulations of the main magnetic field and cardiac pulsations, as well as subject motion. Remove confounders via the integrated principal component-based method that analyzes time-series data from regions unlikely to be associated with neural activity, such as ventricles and large vessels, to identify physiological noise processes. Preview the total variance explained by each of the possible confounding sources. Apply a band-pass frequency filter (0.01 Hz < f < 0.1 Hz) and Gaussian smoothing (6 mm full width at half-maximum).
NOTE: The toolbox will by default identify sources of possible confounders, including BOLD signal from the white matter and cerebrospinal fluid and realignment parameters (subject motion). - For Analysis and Results, identify the sources of interest as the heterotopia ROIs. Preview the connectivity measure of correlation (rather than regression), and display using threshold values for correlation coefficients.
- For each subject, create seed-to-voxel connectivity maps utilizing each discrete region of heterotopic gray matter as a seed ROI, demonstrating the correlation between the average BOLD signal time series of the ROI and every other brain voxel.
- Perform second-level analyses for between-subject or between-source contrasts (optional). Display the results using height (voxel-level) and extent (cluster-level) thresholds; uncorrected and false discovery rate-corrected p-values are shown.
- Use MRICroN software to manually outline two targets of interest, a connected target and a non-connected target, for TMS, using the Pen tool. Using the "Overlay" function, superimpose the functional connectivity maps created above onto the structural images for each subject.
- Ensure that the target region is a region of cortex that has significant functional connectivity to the gray matter heterotopia as described above. Ensure that the non-connected target is a similar-sized region that does not demonstrate significant functional connectivity to any heterotopia ROI and is located at least 2.5 cm away from the connected target on the cortical surface to minimize the risk of neighborhood stimulation effects during TMS.
- Choose targets such that the likelihood of large TMS-induced artifacts is small. Specifically, avoid selecting targets in the lateral temporal or frontopolar regions, as these are likely to produce large muscle contraction and/or eye movement artifacts that can obscure the early TMS-EEG signal. Save the outlined targets as new target ROIs.
- Determine the MNI (Montreal Neurological Institute) coordinates for each target ROI in each subject. Then use these coordinates to identify the equivalent two target sites in each subject's matched healthy control subject.
3. TMS-EEG Experimental Setup
- Upload structural scans (typically high-resolution T1-weighted 3D volumetric images) into the neuronavigation system.
- Using the neuronavigation software, mark the desired targets on the images. Also mark external anatomic markers (the nasion, bilateral tragus) that will be used for coregistration and neuronavigation during the stimulation session. If using an EEG cap with rotatable electrodes and electrode wires, orient the wires perpendicular to the long axis of the TMS coil52.
- Contact the subject prior to the experimental session to remind him or her not to use conditioners or other hair products (shampoo is acceptable) on the day of the TMS-EEG session, to avoid alcoholic drinks the evening prior to the TMS-EEG session, and to drink his or her usual daily caffeine consumption prior to the TMS session.
4. Experimental Session
- Confirm that the subject passes TMS safety criteria, ideally via a structured questionnaire. Confirm that the subject did not consume alcoholic beverages the prior night, did not drink significantly more or less than his or her usual daily caffeine consumption, did not consume over-the-counter sleep aids that alter cortical excitability (such as diphenhydramine) the prior night, and received a typical night's sleep (as sleep deprivation can increase cortical excitability).
- Ask the subject to sit in a comfortable chair.
- Mount the EEG cap on the subject and prepare the electrodes.
- Measure the subject's head and select an EEG cap of appropriate size to help enable low electrode impedances.
- Thoroughly clean the skin underneath each electrode using a cotton-tip applicator and alcohol.
- Add conductive gel to each electrode. Do not add too much gel so that it leaks between electrodes, as that may create a bridge and lead to a common signal between different electrodes.
- If necessary, to ensure good contact between the scalp, the gel, and the electrode, try pressing down on each electrode after adding the gel. To minimize charging artifacts, ensure that the gel does not spread outside the electrode holder. Homogeneously reduced the conductance levels to minimize recording artifacts.
- Place the reference and ground electrodes as far from the stimulation coil as possible to minimize the possibility of TMS-induced electrode artifact contaminating the entire recording. It is preferable to place these electrodes above bony structures, in presumably "inactive" zones with minimal cortical activity.
NOTE: Even in studies for which the target locations are variable, frontopolar regions are unlikely to be selected as targets because TMS to these regions can result in large eye movements, contraction of the frontalis and facial muscles, and, frequently, scalp pain and headache; consequently, the TMS-EEG signal during stimulation of these regions is often obscured by large artifacts. - Since these regions are thus unlikely to be chosen as targets for stimulation, use the forehead for placement of the reference and ground electrodes. Place them within a few centimeters of each other to minimize common-mode noise.
NOTE: In situations where all the stimulation targets are in one hemisphere, the contralateral mastoid would be another option. - Check electrode impedances as follows: plug the EEG output cables into the "impedance" jack of the EEG recording system, then press the "measure impedances" button on the EEG system. Ensure that the electrode impedance is not greater than 5 kΩ.
- Prepare the EMG electrodes on the contralateral hand (use the first dorsal interosseous or abductor pollicis brevis muscles; utilize the same muscle across subjects in a single study).
- Give the subject earplugs to minimize the risk of hearing loss and tinnitus.
NOTE: Another option would be to utilize earphones playing white noise or colored noise (with spectral features matching those of the TMS click) throughout the recording process, at a volume sufficient to mask the auditory click produced by TMS; this would have the added benefit of minimizing the potential confound of TMS-induced auditory evoked potentials. Of note, a thin layer of foam between the coil and scalp is also necessary to minimize the auditory evoked potential. - Place the infrared detectors on the subject's head, ensuring that the detectors are placed in a way to minimize the risk of movement during the experimental session.
- Coregister the subject's head with the MRI images by identifying the location of the pre-selected external anatomic fiducial markers (section 3.2) on the subject using the pointer that is included with the neuronavigation equipment.
- Familiarize the subject with stimulation by applying a pulse elsewhere (e.g., the subject's arm), or by applying a low-intensity stimulation pulse (e.g., 5% max stimulator output) to the scalp.
- Determine the resting motor threshold (the minimum intensity that produces a motor-evoked potential at least 50 µV in size on 5/10 trials). One such method, the relative frequency method, is as follows.
- Determine the location of the subject's motor cortex on the hemisphere ipsilateral to the fMRI connectivity-based targets. When using neuronavigation, this is generally in the region of the "Omega" in the precentral gyrus. Angle the coil perpendicular to the gyrus, with the handle pointing occipitally.
- Begin stimulation at an intensity that is expected to be subthreshold (e.g., 35% maximum stimulator output).
- Increase stimulation intensity in steps of 5% max stimulator output until TMS consistently evokes motor evoked potentials (MEPs) with amplitudes > 50 µV in each trial.
- Then decrease stimulation intensity in steps of 1% maximum stimulator output until less than 5 positive responses out of 10 are recorded.
NOTE: This stimulation intensity plus 1 is defined as the motor threshold. Alternatively, use adaptive threshold techniques to identify the motor threshold with fewer stimuli.
- For stimulation of the target areas, set the TMS intensity to the desired value (e.g., 120% resting motor threshold).
NOTE: However, in cases where there are significant regional variations in scalp-cortex distance (e.g., in patients with frontal lobe atrophy), such a technique may result in subthreshold stimulation. Alternatively, with appropriate neuronavigation systems capable of performing online estimations of the induced electric field, the intensity of the stimulation can also be set at a specific amplitude of the calculated induced electric field (in V/m) on the cortical surface. - Apply single pulses of TMS to each of the target regions using the neuronavigation software, with a variable interval between pulses to minimize cortical plasticity and subject expectancy effects (e.g., every 4 - 6 sec, with an interval of at least 3 sec to avoid cumulative effects). To maximize consistency, angle the coil perpendicularly to the long axis of the underlying gyrus, with the handle pointed posterolaterally.