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).

MMS, SWG, CJC and BSC have nothing to disclose. APL serves on the scientific advisory boards for Nexstim, Neuronix, Starlab Neuroscience, Neuroelectrics, and Neosync, and is listed as an inventor on several issued and pending patents on the real-time integration of transcranial magnetic stimulation (TMS) with electroencephalography (EEG) and magnetic resonance imaging (MRI).

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 spatially and temporally targeted manipulations of brain activity in specific cortical regions; when combined with EEG, TMS can be used to assess the brain&#39;s response to stimulation across different brain regions. Consequently, TMS-EEG can be applied to regions with altered fMRI functional connectivity to assess whether the observed alterations in connectivity have a physiologic correlate that might relate to the underlying disease pathophysiology.
This article presents a protocol using connectivity-guided TMS-EEG to assess cortical excitability in patients with epilepsy due to a malformation of brain development, periventricular nodular heterotopia, which is associated with the development of abnormal functional connectivity networks37. This protocol is used to demonstrate that patients with active epilepsy have cortical hyperexcitability that is specific to the regions with altered resting-state fMRI functional connectivity, and that hyperexcitability can be assessed on an individual subject level. In a patient with seizures previously captured on EEG, the abnormal late TMS-evoked activity is seen in the same region (distant from the stimulation site) from which the patient&#39;s seizures originate, suggesting that the region of abnormal functional connectivity is indeed part of the seizure-generating network.
There are a number of critical steps to successful completion of this protocol. Technical expertise with resting-state fMRI data collection, high-quality resting-state data, and experience with rs-fcMRI data processing and analysis techniques are essential for accurate determination of connectivity-based targets. Another important constraint in designing and executing TMS-EEG studies is the need for TMS-compatible EEG equipment; furthermore, for studies where precise targeting is critical, neuronavigation equipment is also necessary. Another limitation is that TMS often generates substantial EEG artifacts, particularly when stimulating over the frontopolar and lateral temporal regions, and therefore it may be difficult to obtain high-quality data when the stimulation target is located in these regions. The data collection and EEG recording process also needs to be optimized to minimize artifacts in the EEG signal, and experiments should ideally be run by persons familiar with EEG data so that artifacts that do arise (e.g., poor impedance as conducting paste dries) can be rapidly identified and minimized. One important step involves demonstrating the effects of eye blinks, muscle contraction and movement on the EEG to the research subject, as this can be critical in helping the subject to understand and minimize these types of artifacts.
Another important consideration is the minimization of biological artifacts that may limit interpretation of the results. One particularly important such biological artifact is the auditory evoked-potential produced by the TMS coil &#34;click&#34;, which is known to contribute to the magnitude of the TMS-evoked potential, particularly at 100 and 180 milliseconds55,67,68 when the TMS-evoked potential is also typically maximal. One method that has been shown to minimize the TMS auditory evoked potential is noise masking via the use of white or colored noise, with the addition of a thin layer of foam between the coil and scalp10,55. In the absence of such noise masking, differences in the TMS auditory-evoked potential could potentially contribute to differences in the evoked activity between subject groups (although not the observed differences in the evoked potentials between different sites within subjects.)
Finally, even if care is taken to optimize the recording, significant preprocessing is often necessary to recover clean data for analysis. Fortunately, validated methods for removing artifacts from TMS-EEG recordings have been published60; however, even with these techniques, recovery of very early signals (&#60; 15 msec) can be very difficult or unreliable. An additional challenge is that EEG data is high-dimensional and complex, and therefore a clear prior hypothesis is often necessary to extract meaningful information. Furthermore, because TMS effects and EEG signals can vary significantly between subjects because of a wide range of non-cerebral factors that are difficult or impossible to control (e.g., skull thickness, skull-cortex distance, concomitant medications, quality of sleep the night prior), outcome measures that are less reliant on the raw magnitude of evoked responses are likely to be more informative or meaningful.
Although technically challenging, the integration of rs-fcMRI, TMS and EEG together in one experiment enables tests of a wide array of hypotheses regarding the significance of specific connectivity findings on cortical physiology. In disease states, these technologies can be integrated together to assess the relationship between fMRI network connectivity changes, pathophysiologic alterations in cortical excitability and evoked brain activity, and disease expression. Notably, this protocol can be used to investigate cortical physiology via common outcome measures even when the focus of abnormal connectivity (and thus the stimulated region) differs from one subject to another, providing an output that may be meaningful at the individual subject level, and opening up the possibility of a personalized approach to investigation and ultimately treatment.
The protocol described in this study could also be expanded to assess specific features of cortical physiology in different subject groups. For example, a number of recent studies have suggested that the N45 component of the TMS-evoked EEG response represents activity of GABA-A receptors69, whereas the N100 component of the TMS-evoked EEG response is a measure of GABA-B mediated inhibition21,69. Paired-pulse TMS-EEG with a long-interval intracortical inhibition protocol provides another measure of GABAergic activity, and has been shown to be altered in frontal regions in patients with schizophrenia relative to controls70. Thus, the above protocol could be modified to specifically address questions regarding GABAergic activity in regions with altered functional connectivity. Additionally, source localization of the peaks in the TMS-evoked potential may identify distant regions that are engaged by stimulation, and thus help inform which of the functional connections identified by conventional resting-state fMRI are capable of causally transmitting evoked activity. For situations in which the key network hubs are deep, rs-fcMRI can also be used to identify cortical targets that are accessible to stimulation, and thereby enable modulation of specific brain networks involved in normal behavior and in disease states35,36,71. In such cases, the techniques described in this study can be used to assess local and distributed single-pulse TMS-evoked activity before and after a repetitive plasticity protocol, to determine whether the plasticity protocol has indeed changed cortical excitability locally, and/or network excitability distally.
In summary, the integration of rs-fcMRI, TMS and EEG enables the exploration of how brain connectivity influences cortical physiology and behavior in human subjects. Moreover, these techniques can also be combined to assess how alterations in connectivity are related to pathophysiology in disease states, as illustrated in the protocol described above.

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><tbody><tr><td>3T MRI scanner</td><td></td><td></td><td></td></tr><tr><td>MRI functional connectivity software</td><td></td><td></td><td></td></tr><tr><td>MRI image viewing software</td><td></td><td></td><td>MRICron</td></tr><tr><td>Transcranial Magnetic Stimulator</td><td>Nexstim</td><td>eXimia Stimulator</td><td>Can use stimulators from other suppliers, &lt;i&gt;e.g.&lt;/i&gt;, Magventure, Magstim</td></tr><tr><td>MRI neuronavigation system</td><td>Nexstim</td><td>NBS v3.2.1</td><td>Alternative MRI neuronavigation system, &lt;i&gt;e.g.&lt;/i&gt;, Brainsight, Localite</td></tr><tr><td>TMS-compatible EEG system</td><td>Nexstim</td><td>Eximia EEG</td><td>Alternatives: Brain Products, Synamps, ANT</td></tr><tr><td>Matlab</td><td>Mathworks</td><td>R2012b</td><td>Alternatives: Octave</td></tr><tr><td>EEGLab</td><td></td><td></td><td></td></tr><tr><td>Minimum Norm Estimate (MNE) software</td><td></td><td></td><td></td></tr><tr><td>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 the evoked EEG results compared to the EEG potentials produced by stimulation of a control non-connected target in the same patients. Furthermore, the same regions can be targeted in healthy control subjects (Figure 2) to determine whether the abnormal functional connectivity seen in the PNH patients has pathophysiologic significance for the patients&#39; clinical epilepsy syndrome. Specifically, the presence of cortical hyperexcitability can be assessed on the individual patient level by determining the normalized area-under-the-curve of the Global Mean Field Potential, and then evaluating whether this value is larger for the epilepsy patient than his or her matched control (Figure 2). Source localization of the abnormal late peaks in the TMS-evoked potentials in patients with epilepsy can identify the brain regions from which the abnormal activity arises, and may spatially co-localize with the patient&#39;s seizure focus (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/53727/53727fig1.jpg" /> 
Figure 1. Resting-state Functional Connectivity and TMS Targets. (A, B) Regions with significant correlations in functional activation (blue/green) to the resting-state BOLD signal in the heterotopic nodules in two patients with periventricular nodular heterotopia and epilepsy. (C, D) The connected target site (red) and the non-connected target site (blue) in these two patients. (Modified with permission from Shafi et al., 201537). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53727/53727fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53727/53727fig2.jpg" /> 
Figure 2. TMS-evoked Potentials and Global Mean Field Potentials. (A) The TMS-evoked potential produced by stimulation of the connected target in a patient with PNH and epilepsy. (B) The TMS-evoked potential produced by stimulation of the same region in the above patient&#39;s matched healthy control subject. (C) The global mean field potential (GMFP) produced by stimulation of the connected and non-connected targets for this patient and his matched control. (D) The normalized area-under-the-curve of the GMFP produced by stimulation of the connected and non-connected targets for this subject pair. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53727/53727fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53727/53727fig3.jpg" /> 
Figure 3. Source Localization of TMS-evoked Activity and Seizure Onsets. (A) Electrical source imaging results of a late TMS-evoked peak in a patient with epilepsy; scale is the estimated currents multiplied by 10-11. (B) Electrical source imaging results of a previously captured seizure onset in that same patient. (Modified with permission from Shafi et al, 201537) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53727/53727fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-multimodal-imaging-stimulation-based-method-evaluating-connectivity

Nanyang Technological University

Research

JoVE Journal

Medicine

11.0K Views.  Harvard Medical School. 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. 

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

Performing Behavioral Tasks in Subjects with Intracranial Electrodes

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in different areas of their brain for the localization of their seizure focus and eloquent areas. After implantation, the patient must remain in the hospital until the pathological area of brain is found and possibly resected. During this time, these patients offer a unique opportunity to the research community because any number of behavioral paradigms can be performed to uncover the neural correlates that guide behavior. Here we present a method for recording brain activity from intracranial implants as subjects perform a behavioral task designed to assess decision-making and reward encoding. All electrophysiological data from the intracranial electrodes are recorded during the behavioral task, allowing for the examination of the many brain areas involved in a single function at time scales relevant to behavior. Moreover, and unlike animal studies, human patients can learn a wide variety of behavioral tasks quickly, allowing for the ability to perform more than one task in the same subject or for performing controls. Despite the many advantages of this technique for understanding human brain function, there are also methodological limitations that we discuss, including environmental factors, analgesic effects, time constraints and recordings from diseased tissue. This method may be easily implemented by any institution that performs intracranial assessments; providing the opportunity to directly examine human brain function during behavior.

Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population.

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in ...

Behavior

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Neuroscience

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

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

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

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

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer's Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

Integrating MEG and HD-EEG in Pediatric Epilepsy Localization

Electromagnetic Source Imaging in Presurgical Evaluation of Children with Drug-Resistant Epilepsy

For children with drug-resistant epilepsy (DRE), seizure freedom relies on the delineation and resection (or ablation/disconnection) of the epileptogenic zone (EZ) while preserving the eloquent brain areas. The development of a reliable and noninvasive localization method that provides clinically useful information for the localization of the EZ is, therefore, crucial to achieving successful surgical outcomes. Electric and magnetic source imaging (ESI and MSI) have been increasingly utilized in the presurgical evaluation of these patients showing promising findings in the delineation of epileptogenic as well as eloquent brain areas. Moreover, the combination of ESI and MSI into a single solution, namely electromagnetic source imaging (EMSI), performed on simultaneous high-density electroencephalography (HD-EEG) and magnetoencephalography (MEG) recordings has shown higher source localization accuracy than either modality alone. Despite these encouraging findings, such techniques are performed in only a few tertiary epilepsy centers, are rarely recorded simultaneously, and are underutilized in pediatric cohorts. This study illustrates the experimental setup for recording simultaneous MEG and HD-EEG data as well as the methodological framework for analyzing these data aiming to localize the irritative zone, the seizure onset zone, and eloquent brain areas in children with DRE. More specifically, the experimental setups are presented for (i) recording and localizing interictal and ictal epileptiform activity during sleep and (ii) recording visual-, motor-, auditory-, and somatosensory-evoked responses and mapping relevant eloquent brain areas (i.e., visual, motor, auditory, and somatosensory) during visuomotor task, as well as auditory and somatosensory stimulations. Detailed steps of the data analysis pipeline are further presented for performing EMSI as well as individual ESI and MSI using equivalent current dipole (ECD) and dynamic statistical parametric mapping (dSPM).

Author Spotlight: Advancing Pediatric Epilepsy Surgery in Children Through Novel Biomarkers and Enhanced Localization

Magnetoencephalography (MEG) and high-density electroencephalography (HD-EEG) are rarely recorded simultaneously, although they yield confirmatory and complementary information. Here, we illustrate the experimental setup for recording simultaneous MEG and HD-EEG and the methodology for analyzing these data aiming to localize epileptogenic and eloquent brain areas in children with drug-resistant epilepsy.

For children with drug-resistant epilepsy (DRE), seizure freedom relies on the delineation and resection (or ablation/disconnection) of the epileptogenic ...

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Flexible Multi-Region Neural Recording Device for Epilepsy Research

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the propagation of epileptic signals through associated neural circuits. Therefore, in vivo recording of local field potential (LFP) from the critical brain regions is&#160;essential for deciphering the circuits involved in seizure propagation. However, current methods for electrode fabrication and implantation lack flexibility. Here, we present a handy device designed for electrophysiological recordings (LFPs and electroencephalography [EEG]) across multiple regions. Additionally, we seamlessly integrated optogenetic manipulation and calcium signaling recording with LFP recording. Robust after-discharges were observed in several separate regions during epileptic seizures, accompanied by increasing&#160;calcium signaling. The approach used in this study offers a convenient and flexible strategy for synchronous neural recordings across diverse regions of the brain. It holds the potential for advancing research on neurological disorders by providing insights into the neural profiles of multiple regions involved in these disorders.

Author Spotlight: Unraveling Neural Communication and Circuit Interactions in Health and Disease

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the ...

Monitoring the Brain Cortex with a Multi-Electrode Array in Response to Seizure-Inducing Agents

Source: Lu, H. C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53709">Direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation.</a> J. Vis. Exp. (2016).
In this video, the effects of transcranial direct-current stimulation (tDCS) on brain slices containing the anterior cingulate cortex (ACC) and thalamus regions are investigated, using a multi-electrode array (MEA) to measure electric signals. By delivering electric pulses and infusing seizure-inducing drugs, the study investigates the ACC response to seizures.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparing Experimental ...

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

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