The authors have no acknowledgements or financial disclosures.

Ensure that each patient is reviewed for suitability for the research study, and appropriate patients must be consented for participation in the study according to local IRB procedures.
1. Patient Selection for SEEG and Research
<ol>
	<li>Patient Selection for SEEG 
	Note: Epilepsy patients must be clinically assessed by a multidisciplinary team consisting of epileptologists, neuropsychologists and neurosurgeons.
	<ol>
		<li>Ensure that the patient has medically refractory focal epilepsy, defined as failure to respond to at least 2 adequate trials of anti-epileptic medications.</li>
		<li>Ensure that non-invasive techniques have failed to localize the epileptogenic foci.</li>
		<li>Confirm with multidisciplinary team that patient is not suitable for investigation only with subdural grid electrodes.</li>
		<li>Confirm with multidisciplinary team that there is clinical suspicion of a deep seizure onset zone.</li>
	</ol>
	</li>
	<li>Patient selection for research task
	<ol>
		<li>Ensure that subject is between the ages of 13 and 65 years.</li>
		<li>Obtain consent or assent (together with parental consent if below the age of 18 years) from the patient.</li>
		<li>Ensure that subjects are able to attend to the task and cooperate with the testing.</li>
	</ol>
	</li>
</ol>
2. Preparation and Implantation Technique
<ol>
	<li>Perform a volumetric T2 and contrast enhanced volumetric T1 MRI preoperatively and transfer the images to the stereotactic navigation software, according to manufacturer&#8217;s protocol.
	<ol>
		<li>Plan the depth electrode targets based on the MRI and clinical suspicion of seizure foci. 
		Note: The examples provided are based on the BrainLab navigation software and are thus specific to this system. However, any stereotactic navigation software can be used to plan depth electrode trajectories and placement.</li>
		<li>Define the anatomical region of interest as the target point within the &#8220;Stereotactic Planning&#8221; function in the stereotactic navigation software.</li>
		<li>For example, use dACC as the target of interest. To define its trajectory, press &#8220;New Trajectory,&#8221; then press &#8220;target&#8221; and click on the dACC. Center the target in the middle of dACC by examining dACC in all 3 planes (axial, coronal and sagittal) and clicking on the middle of dACC in each plane.
		<ol>
			<li>Define the entry point on the scalp within the &#8220;Stereotactic Planning&#8221; function in the stereotactic navigation software.</li>
		</ol>
		</li>
		<li>For example, choose a point on the scalp that appears to be the shortest path to the dACC. Press &#8220;entry&#8221; and choose the point on the scalp to make the entry point.</li>
		<li>Click and drag the &#8220;target&#8221; and &#8220;entry&#8221; points to modify the defined trajectory to avoid cortical and subcortical vascular structures as well as any potential eloquent cerebral regions.</li>
		<li>Repeat for all planned depth electrode targets (Figure 1).</li>
		<li>Admit the patient on the morning of the surgery, bring to the operating room, and induce under general anesthesia26,27.</li>
		<li>Attach a Cosman-Roberts-Wells (CRW) stereotactic headframe to the patient&#8217;s head with skull screws.</li>
		<li>Obtain a volumetric CT with the headframe in place.</li>
		<li>Load the volumetric CT and MRI images into the stereotactic navigation software via the &#8220;Load and Import&#8221; function.</li>
		<li>Click on the &#8220;Localization&#8221; function within the stereotactic navigation software.</li>
		<li>Localize the CRW headframe by clicking on all of the images defined by the stereotactic navigation software as containing the headframe and then pushing the &#8220;Assign localizer&#8221; button.</li>
		<li>Click on the &#8220;AC/PC Localization&#8221; function within the stereotactic navigation software.</li>
		<li>Identify the anterior and posterior commissures based on their anatomical location.</li>
		<li>Designate the anterior and posterior commissures via the &#8220;Set AC/PC system&#8221; function within the stereotactic navigation software.</li>
		<li>Click on the &#8220;Image Fusion&#8221; function within the stereotactic navigation software.</li>
		<li>Merge the CT images with the MRI images in the stereotactic navigation software28,29. Click on the paired volumetric CT and MRI images underneath the &#8220;Fusion&#8221; tab and then click &#8220;Auto Fusion&#8221;. 
		Note: This places the MRI within the stereotactic frame coordinates.</li>
		<li>Click on the &#8220;Stereotactic Planning&#8221; function within the stereotactic navigation software and confirm the planned trajectories from steps 2.1.2 - 2.1.6.</li>
		<li>Choose the volumetric CT as the stereotactic reference under the &#8220;Stereotactic Functions&#8221; tab.</li>
		<li>Click the &#8220;print&#8221; icon in the vertical column of icons to print the final stereotactic coordinates for each depth electrode trajectory 30,31.</li>
	</ol>
	</li>
	<li>Implantation technique
	<ol>
		<li>Return the patient to the operating room following the CT scan.</li>
		<li>Prepare and drape the surgical field using routine sterile methods32,33.</li>
		<li>Ensure that a fluoroscope is in the operating room and draped along with the rest of the surgical field.</li>
		<li>Using the printed stereotactic coordinates from step 2.1.20, set the coordinates for the first depth electrode on the headframe. 
		Note: The stereotactic coordinates are given in 3 planes: lateral (x), vertical (y) and anterior-posterior (z). For example, the printed coordinates for a target in the right dACC are +48.2 mm A-P, 6.6 mm Lateral and +2.2 mm Vertical. The headframe is then set to those coordinates accordingly.</li>
		<li>Extend the guideblock down to the skin and mark the location of the burr hole on the scalp with a marking pen. Fix the guideblock in place based on the stereotactic coordinates and as such, no landmarks are necessary to mark the incision.</li>
		<li>Inject 2 - 3 ml&#160;of 0.5% bupivicaine in a 1:100,000 dilution of epinephrine into the marked incision.</li>
		<li>Make a nick in the scalp with a scalpel down to the skull into the marked incision.</li>
		<li>Cauterize the dermis and deep tissue using monopolar cautery directed with a coated obturator in order to minimize any bleeding from vessels in the skin or subcutaneous tissue.</li>
		<li>Drill a burr hole using a 2.1 mm twist drill bit in the middle of the incision.</li>
		<li>Open the dura with a rigid obturator probe. Screw an anchor bolt into the skull. Place a pre-measured stylet probe through the anchor bolt to make a track for the electrode.</li>
		<li>Carefully advance the electrode to the pre-calculated depth. Tighten the anchor bolt cap down to secure the electrode.</li>
		<li>Repeat this process for all of the depth electrodes.</li>
		<li>Place the fluoroscope underneath and surrounding the patient&#8217;s head in both AP and lateral planes to obtain fluoroscopic images to ensure adequate placement trajectories of all electrodes.</li>
		<li>Connect the electrodes to the clinical EEG system to verify appropriate impedances.</li>
		<li>Wake the patient from anesthesia and transport to the recovery room, and subsequently to the EMU.</li>
		<li>In the EMU, monitor the patient via closed circuit monitoring for clinical seizures and via ECoG for electrographic evidence of seizures.</li>
	</ol>
	</li>
</ol>
3. Behavioral Task and Data Acquisition
<ol>
	<li>Behavioral task
	<ol>
		<li>Open behavioral software on computer devoted solely to running the behavioral software. 
		Note: The instructions provided are based on MonkeyLogic, a MATLAB toolbox designed for the presentation and execution of temporally precise psychophysical tasks34,35 and are thus specific to that behavioral software platform. This program is run on Matlab version 2010a and requires the &#8220;Data Acquisition Toolbox.&#8221; However, any behavioral software platform capable of presenting visual stimuli and recording electrophysiological data could be used.</li>
		<li>Set the conditions file designed to run the MSIT task to include all four trial types of equal frequency. 
		Note: The MSIT task consists of presenting the subject with a cue of three numbers between 0 and 3, where two of the numbers, the &#8216;distractors&#8217;, are the same and one number, the &#8216;target&#8217;, is different.
		<ol>
			<li>Instruct the subject to identify the &#8216;target&#8217; by pressing the corresponding button on a button box. If &#8216;1&#8217; is the target, the left button is the correct choice. If &#8216;2&#8217;, the middle button, and if &#8216;3&#8217;, the right button. &#8216;0&#8217; does not correspond to a possible button (Figure 2).</li>
		</ol>
		</li>
		<li>Press the &#8220;Set conditions&#8221; button and choose the desired conditions file set in the prior step. 
		Note: There are two types of cognitive interference that induce conflict during the decision making process. Flanker interference trials occur when the distractors are possible (1, 2, or 3, rather than 0) button choices (e.g., 121), while spatial interference trials occur when the target number&#8217;s spatial location differs from the response location (e.g., 200, in which the middle button is the correct response, despite the fact that the target number is in the left position). There are four trial types based on the presence or absence of these two interference types.</li>
		<li>Test the behavioral display monitor by clicking &#8220;Test&#8221; in the display box. The display monitor should show the test visual stimulus for 2 - 3 sec.</li>
		<li>Connect the subject interface device (button box) to the analog inputs on the data acquisition board on the computer devoted to recording the electrophysiological data via three standard BNC cables.</li>
		<li>Connect the button box to a power source.</li>
		<li>Connect the data acquisition board to the 512-channel neural signal processor via a ribbon cable split into 9 ribbons. 8 of the ribbons are connected to ports 0 -&#160;7 on the digital I/O portion of the data acquisition board while the 9th ribbon is connected to port 0 on the digital PFI portion of the data acquisition board. 
		Note: The ribbons send 8-bit digital markers (ports 0 - 7, digital I/O) and a strobe pulse (port 0, digital PFI), to the neural signal processor.</li>
		<li>Set the desired sampling rate in the neural signal processor software.
		<ol>
			<li>In this example, set the desired sampling rate to 50,000 samples per second, alias and down-sample online to 1,000 samples per second. Adjust the sample rate to fit the specific goals of the task. Sub-millisecond precision timing requires an extremely high sampling rate.</li>
		</ol>
		</li>
		<li>Connect the amplifier to the neural signal processor via fiber optic cable.</li>
		<li>Connect the neural signal processor to the data streamer and the optical PCI card in the neural data acquisition computer via fiber optic cable.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Use the research rig for EMU electrophysiology containing a 512-channel neural signal processor for processing and filtering digitized, pre-amplified electrical signals from the depth electrodes. 
		Note: While there are 512 channels for processing, in practice, there are never more than 15 - 20 electrodes placed for clinical purposes. Therefore, we recommend recording from as many electrodes as feasible as data size and spatial resolution is never an issue.</li>
		<li>Transport the rig to the patient&#8217;s room, place the behavioral monitor in front of the patient on a portable table and connect to the behavioral control computer running the behavioral software using a standard DVI cable.</li>
		<li>Place the recording rig behind or to the side of the patient&#8217;s bed in order to remain as unobtrusive as possible.</li>
		<li>Connect the research system to the splitter box that separates the research recording from the clinical system.</li>
		<li>Control recording parameters&#160;using the neural signal processor software34,35. 
		Note: This system enables sub-millisecond control over behavioral events34,35. Synchronization between neural and behavioral data can be accomplished with either analog pulses coding for task events or digital markers. Both signals can be sent from either the digital or analog outputs on the data acquisition board to the analog or digital inputs on the neural signal processor.</li>
		<li>Hand the patient the subject interface device (button box) and give task instructions.</li>
		<li>Click &#8220;Run&#8221; to run the task.</li>
		<li>Allow the patient to complete 2 blocks of 150 trials each.</li>
	</ol>
	</li>
</ol>
4. Data Analysis
<ol>
	<li>Open software package that allows for visualization of electrophysiological data. 
	Note: The instructions provided below are specific to Matlab version 2010a but any software that allows for visualization and manipulation of electrophysiological data can be used.</li>
	<li>Open .edf file containing raw electrophysiological data from the trial session.</li>
	<li>Visualize SEEG signal from the session to ensure there is no visible artifact such as epileptiform discharges or movement artifact (Figure 3A).</li>
	<li>Overlay the timing pulses from the behavioral task onto the raw LFP trace (Figure 3B) to illustrate how analog pulses can delineate trial structure.</li>
	<li>Using the timing pulses, align the SEEG trace to cue presentation for each trial (Figure 3C).</li>
	<li>Remove outliers (&#62;4 standard deviations) and artifact traces (Figure 3D).</li>
	<li>Save all aligned trials in a matrix for further analyses (20 trials shown stacked in Figure 3E).</li>
	<li>Average LFP activity across trials to reduce effect of noise, artifact, or EEG activity not related to the presented stimuli, and to increase the signal of interest (Figure 3F).</li>
	<li>Create the raw, trial-averaged spectrogram using multi-tapered spectral analysis36-38. 
	Note: Time-frequency analysis can be used in order to investigate the specific spectro-temporal dynamics across single or multiple trials. This method enables the investigation of neuronal oscillations at different frequencies over time.</li>
	<li>Pad the signal from each trial with zeroes to the next largest power of 2 to avoid edge effects.</li>
	<li>Apply a 800 ms sliding window with 5 leading tapers and a time-bandwidth product of 9 every 10 ms through the duration of the signal to create the spectrogram (Figure 4A).</li>
	<li>Multiply the log of the spectrogram by 10 and normalize to display higher frequency information. 
	Note: Spectrograms can be normalized by a theoretical frequency distribution (i.e., each frequency value raised to the negative 2nd power)(Figure 4B), the mean spectrum of some baseline activity (Figure 4C), or by dividing by the mean and subtracting the standard deviation of the values in each frequency band (Figure 4D). This procedure allows for the examination of specific frequency bands in both raw and normalized forms over time for changes specific to the task. For example, high gamma band activation (70 - 150 Hz), which is shown in Figure 3E, is thought to reflect local excitatory activity of the local neuronal population surrounding the electrode39,40.</li>
</ol>

The authors have no conflict of interest to disclose.

In this paper SEEG was used to investigate the activity of local neuronal populations within the dACC during a decision-making task in humans. Previous work has investigated the activity of individual neurons in the dACC using intraoperative microelectode recordings 14 and demonstrated that dACC activity is modulated by previous activity. Microelectrode studies enable the investigation of the spiking activity of individual neurons. SEEG measures LFPs, which are related to the summated synaptic potentials acros...

Epilepsy, a common neurological disorder characterized by multiple recurrent seizures over time, accounts for 1% of the worldwide burden of diseases 1. Anti-epileptic medications fail to control seizures in 20 - 30% of patients 2,3. In these medically intractable patients, epilepsy surgery is often indicated 4,5. The decision to proceed with surgery requires locating the seizure focus, a prerequisite to formulating a surgical plan. Initially, non-invasive techniques are used to lateralize and localize the seizure focus. Electroencephalography (EEG), for example, measures cortical electrical activity recorded from electrodes placed on the scalp and can often provide sufficient information about the location of the seizure focus. In addition, magnetic resonance imaging (MRI) can demonstrate discrete lesions, such as hippocampal sclerosis, the classic pathology seen in the most common form of medically intractable epilepsy, mesial temporal lobe epilepsy (MTLE).
Frequently, however, the noninvasive workup is unable to identify a seizure focus. In these cases, invasive electrocorticography (ECoG) with intracerebral electrodes is required to localize the focus and guide further surgical treatment 6. ECoG is a neurophysiological technique used to measure electrical activity using electrodes placed in direct contact with the brain. Grids or strips of surface (subdural) electrodes are placed over the surface of the brain, a process that requires a craniotomy (removal of a bone flap) and large opening of the dura. These surface electrodes can be placed over the putative area(s) of seizure onset. The distal ends of the electrodes are tunneled through small openings in the skin and connected to the recording equipment in the epilepsy monitoring unit (EMU). In the EMU, the patient is monitored for clinical seizure activity through continuous video and ECoG recordings. This technique is useful for collecting long-term (days to weeks) recordings of ictal and interictal electrical discharges over relatively large areas of the cortical surface. While these intracranial recordings are invaluable clinically for investigating seizure foci and propagation, they also provide us with the opportunity to investigate cognitive function and neurophysiology in humans undergoing specifically designed behavioral tasks.
ECoG using subdural grid electrodes has been used to investigate various aspects of cortical function, including sensory and language processing. As one of many examples, Bouchard et al demonstrated the temporal coordination of the oral musculature in the formation of syllables for spoken language in the ventral sensorimotor cortex, a region identified as the human speech sensorimotor cortex 7. Furthermore, ECoG with subdural grid placement has also been utilized to study the mechanisms by which humans are able to attend to a particular voice within a crowd: the so-called &#8216;cocktail party effect&#8217; 8,9. ECoG recordings demonstrated that there are two distinct neuronal bands that dynamically track speech streams, both low frequency phase and high-gamma amplitude fluctuations, and that there are distinct processing sites - one &#8216;modulation&#8217; site that tracks both speakers, and one &#8216;selection&#8217; site that tracks the attended talker 5.
Another emerging application of ECoG with subdural electrode placement is the potential for use with Brain Computer Interfaces (BCIs), which &#8220;decode&#8221; neuronal activity in order to drive an external output. This technology has the potential of allowing patients with severe brain or spinal cord injuries to communicate with the world and manipulate prostheses 10,11.
While subdural grid placement has contributed greatly to our understanding of superficial cortical areas and is useful in identifying cortical epileptogenic foci, this technique does require a craniotomy and its attendant risks, and is generally limited to studying the outer surface of the brain. Stereotactic electroencephalography (SEEG) is a technique that enables the assessment of deep epileptogenic foci12. With a long history of use in France and Italy, it is also increasingly being used in the US 13. SEEG involves the placement of multiple electrodes (typically 10 - 16) deep within the substance of the brain through small (few mm) twist drill burr holes. Advantages of SEEG over subdural grid placement include its less invasive nature, the ease of examining bilateral hemispheres when required, and the ability to generate three-dimensional maps of seizure propagation. Furthermore, these electrodes enable the identification of deep epileptogenic foci that were previously difficult to identify with surface electrodes. This procedure also provides the opportunity to investigate the neurophysiology and function of deep cortical structures, such as the limbic system, the mesoparietal cortex, the mesotemporal cortex, and the orbitofrontal cortex, all of which were previously difficult to directly investigate in humans.
This paper demonstrates how SEEG can be utilized to investigate cognitive function in the dorsal anterior cingulate cortex (dACC). The dACC is a widely investigated brain region, but it is also one of the most poorly understood. Considered a significant region for human cognition, it is likely that the dACC is central to the dynamic neural processing of decisions in the context of continuously changing demands imposed by the environment 14. Studies in both primates 15,16 and humans 17 suggest that the dACC integrates potential risks and rewards of a given action, especially in situations of multiple simultaneous conflicting demands18-21, and modulates these decisions in the context of previous actions and their outcomes 14,22,23.
The Multi-Source Interference Task (MSIT), a Stroop-like behavioral task, is frequently used to investigate conflict processing in the dACC. The MSIT task activates the dACC by recruiting neurons involved in multiple domains of processing regulated by the dACC 24,25. This task specifically activates the dACC by testing features of decision-making, target detection, novelty detection, error detection, response selection, and stimulus/response competition. In addition, the MSIT task introduces multiple dimensions of cognitive interference, which are utilized in this study to investigate dACC neural responses to simultaneous conflicting stimuli using SEEG.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trigger I/O cable</td>
			<td>Natus Medical Inc.</td>
			<td>5029</td>
			<td>PS2 to BNC cable</td>
		</tr>
		<tr>
			<td>BNC cables for analog pulses</td>
			<td></td>
			<td></td>
			<td>Can be ordered from most electronics stores.</td>
		</tr>
		<tr>
			<td>Power strip with surge protection and battery backup</td>
			<td>Tripp Lite</td>
			<td>SMART500RT1U UPC</td>
			<td>Power source and backup</td>
		</tr>
		<tr>
			<td>National instruments multifunctional daq data acquisition box NI PCIe-6382 DAQ cards</td>
			<td>National Instruments</td>
			<td>PCIe-6382 w/ BNC 2090A</td>
			<td>PCI cards for behavioral control interface</td>
		</tr>
		<tr>
			<td>Custom made button box - human interface device</td>
			<td></td>
			<td></td>
			<td>Any human interface device with three buttons may be used. Alternatively, 3 keyboard buttons may be used.</td>
		</tr>
		<tr>
			<td>Xltek 128 channel clinical intracranial EEG monitoring system EMU128FS</td>
			<td>Natus Medical Inc.</td>
			<td>002047c</td>
			<td>Clinical recording system</td>
		</tr>
		<tr>
			<td>Subject monitor and associated cables for visual stimulus presentation</td>
			<td>Dell</td>
			<td>U2212HMc</td>
			<td>Most Monitors are adequate here.</td>
		</tr>
		<tr>
			<td>Personal comptuer running behavioral software with DAQ cards installed</td>
			<td>Superlogics</td>
			<td>SL-2U-PD-Q87SLQ-BA</td>
			<td>Computer for recording neural data</td>
		</tr>
		<tr>
			<td>Mains cable for monitor</td>
			<td></td>
			<td></td>
			<td>Usually comes with the monitor, can be purchased at any electronics store.</td>
		</tr>
		<tr>
			<td>Monkey Logic software which runs on Matlab 2010A</td>
			<td></td>
			<td></td>
			<td>Free from MonkeyLogic website</td>
		</tr>
		<tr>
			<td>MATLAB 2010a software with data acquisition toolbox</td>
			<td>Mathworks</td>
			<td></td>
			<td>Matlab software</td>
		</tr>
		<tr>
			<td>sEEG electrodes AD TECH or PMT</td>
			<td>AD TECH</td>
			<td>2102-##-101</td>
			<td>Platinum tip, diameter (0.89 mm, 1 mm, 1.1 mm), uninsulated length 2.3 mm; The ## in the catalog number indicates the number of contacts on the electrode (08, 10, 12, or 16)</td>
		</tr>
		<tr>
			<td>Cabrio connectors</td>
			<td>PMT</td>
			<td>2125-##-01</td>
			<td>The ## in the catalog number indicates the number of contacts on the electrode (08, 10, 12, or 16)</td>
		</tr>
		<tr>
			<td>Tucker Davis Technologies Amplifier</td>
			<td>Tucker Davs Technologies</td>
			<td>PZ5</td>
			<td>preamplifier for neural data</td>
		</tr>
		<tr>
			<td>Tucker Davis Technologies processor</td>
			<td>Tucker Davs Technologies</td>
			<td>RZ2</td>
			<td>Neural signal processor for neural data</td>
		</tr>
		<tr>
			<td>TuckerDavis Technologies data streamer</td>
			<td>Tucker Davs Technologies</td>
			<td>RS4</td>
			<td>Data streamer and storage</td>
		</tr>
		<tr>
			<td>Fiber optics cables to connect TDT systems</td>
			<td>Tucker Davs Technologies</td>
			<td>F05</td>
			<td>Fiber optic cables for connecting Tucker Davis Technologies&#39; prodcuts.</td>
		</tr>
		<tr>
			<td>ribbon cable and snap serial connector for digital markers</td>
			<td></td>
			<td></td>
			<td>Can be ordered from ost electronics stores.</td>
		</tr>
		<tr>
			<td>personal computer fro running TDT RPvdsEx and OpenEx software</td>
			<td>Superlogics</td>
			<td>SL-2U-PD-Q87SLQ-BA</td>
			<td>computer for behavioral control</td>
		</tr>
		<tr>
			<td>middle atlantics server cabinet with casters</td>
			<td>Middle Atlantic Products</td>
			<td>PTRK-21</td>
			<td>Server case to house all of the research items</td>
		</tr>
		<tr>
			<td>Tucker Davis Technologies splitter box to split clinical and research recrodings</td>
			<td>Tucker Davs Technologies</td>
			<td></td>
			<td>This splitter box is a semi-custom device. Researchers should consult the attending neurologists about splitting the research and clinical recordings in a way that doesn&#39;t interfere with clinical care.</td>
		</tr>
		<tr>
			<td>Researcher monitor with requisite cables</td>
			<td>Dell</td>
			<td>U2212HMc</td>
			<td>Most Monitors are adequate here.</td>
		</tr>
		<tr>
			<td>button box power source - 5 volts, 2 amperes</td>
			<td></td>
			<td></td>
			<td>Can be purchased at any electronics store.</td>
		</tr>
		<tr>
			<td>TDT optical interface PCI card</td>
			<td>Tucker Davs Technologies</td>
			<td>P05</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the function of deep cortical and subcortical structures using stereotactic electroencephalography: lessons from the anterior cingulate cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Once a patient is selected for SEEG electrode placement, he/she undergoes a volumetric T2 and T1 contrast enhanced MRI. SEEG electrode trajectories are then planned using stereotactic navigation of the volumetric MRI sequences (Figure 1). This technique allows for the collection of local field potentials from structures deep within the cortex such as dorsal anterior cingulate cortex (light orange trajectory, Figure 1) that would not be possible with typical surface electrode placement. P...

Watch this Scientific Journal Video about Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex a

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

investigating-function-deep-cortical-subcortical-structures-using

Research

JoVE Journal

Neuroscience

12.1K Views.  Columbia University Medical Center, New York Presbyterian Hospital. Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Video: Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology1,2, is being used increasingly in the study of social cognitive functioning in infants and adults. While dEEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization3,4,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although several analytical software techniques can be used to establish indirect indices of source localization to improve the spatial resolution of dEEG, the HydroCel Geodesic Sensor Net (HCGSN) by Electrical Geodesics, Inc. (EGI), a dense sensory array that maintains equal distances among adjacent recording electrodes on all surfaces of the scalp, further enhances spatial resolution4,5,6 compared to standard dEEG systems. The sponge-based HCGSN can be applied rapidly and without scalp abrasion, making it ideal for use with adults7,8, children9,10,11, and infants12, in both research and clinical4,5,6,13,14,15 settings. This feature allows for considerable cost and time savings by decreasing the average net application time compared to other dEEG systems. Moreover, the HCGSN includes unified, seamless software applications for all phases of data, greatly simplifying the collection, processing, and analysis of dEEG data.
The HCGSN features a low-profile electrode pedestal, which, when filled with electrolyte solution, creates a sealed microenvironment and an electrode-scalp interface. In all Geodesic dEEG systems, EEG sensors detect changes in voltage originating from the participant's scalp, along with a small amount of electrical noise originating from the room environment. Electrical signals from all sensors of the Geodesic sensor net are received simultaneously by the amplifier, where they are automatically processed, packaged, and sent to the data-acquisition computer (DAC). Once received by the DAC, scalp electrical activity can be isolated from artifacts for analysis using the filtering and artifact detection tools included in the EGI software. Typically, the HCGSN can be used continuously for only up to two hours because the electrolyte solution dries out over time, gradually decreasing the quality of the scalp-electrode interface.
In the Parent-Infant Research Lab at the University of Toronto, we are using dEEG to study social cognitive processes including memory, emotion, goals, intentionality, anticipation, and executive functioning in both adult and infant participants.

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography dEEG

Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

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 ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...