1. EEG Acquisition
<ol>
	<li>Explain the experimental procedures to the participant and obtain informed consent.</li>
	<li>Apply drop-down electrodes. Clean area on the face where drop-down electrodes will be located using an alcohol swab.</li>
	<li>Place electrode cap on the participant's head. Measure participant's head circumference and choose the appropriate cap size. Following the internationally recognized 10-20 system for electrode placement, measure the distance from nasion to inion along the midline and divide by 10%. Using that number, measure up from nasion and mark. Align the electrode cap position Fp with this mark and pull the cap back. Make sure that the center of the cap is in line with the nose. Measure nasion to Cz, and confirm that this distance is half the distance from nasion to inion. Tighten the chinstrap.</li>
	<li>Place gel-filled blunt-point syringe in the electrode holders. To create a conductive column of gel, start in contact with the scalp, then squeeze and pull back. Note that the application of too much gel may bridge the signals of neighboring electrodes.</li>
	<li>Fix active electrodes into the electrode holders.</li>
	<li>Position the subject in front of the monitor at the appropriate distance for the experiment. Ask the participant to remain still, emphasizing the importance of minimizing eye movements and blinks for a clean recording.</li>
	<li>Examine the electrode connections and EEG signal quality on the acquisition computer. Verify that all electrode offsets are low (&lt; 40 mV) and stable. If there is a problem with a particular electrode, take out that electrode and reapply gel to adjust impedances at that site.</li>
	<li>Save the file and start the experiment.</li>
</ol>
2. EEG Analysis
<ol>
	<li>After experimentation, but before extracting the particular statistic of interest, preprocess the continuous EEG data to remove artifacts using standard procedures of filtering and artifact rejection. Cut the continuous EEG into epochs corresponding to each discrete event, such as the presentation of photograph. In each epoch, include a 100 msec pre-stimulus window as a baseline.</li>
	<li>Event-related potentials (ERP) analysis captures synchronous brain activity that is phase-locked to the onset of the event. Average across trials to separate the evoked responses from the "noisy" (i.e. non-phase locked) background activity. The variability across trials and between-subjects presents a major challenge for the ERP method of analysis. To achieve a good signal-to-noise ratio the experimental protocol should include many discrete events with definable onsets. Time locking the brain's response to the onset of a salient event and then averaging over many like events helps to reduce some of this noise; however, the temporal synchrony created by this procedure typically dissolves within 1 sec. Identify ERP component peak amplitudes and latencies for each subject (for more detailed guidelines on ERP analysis, see Picton et al., 2000).</li>
	<li>Using Fourier analysis, transform the EEG signal from the time domain to the frequency domain and decompose the signal into its composite sine waves of varying frequencies 6.</li>
	<li>Multiscale entropy (MSE) is an information theoretic metric that estimates the variability of the neuroelectical signals over time and across multiple timescales. To provide a conceptual depiction of MSE analysis, consider two simulated waveforms, a regular waveform and a more stochastic one. Sample entropy values are near zero for the regular waveform and ~2.5 for the more variable waveform. An increase in sample entropy corresponds to an increase in signal complexity, which, according to information theory, can be interpreted as an increase in the amount of information processing capacity of the underlying system 7,16. Remember that the capacity of a brain is not fixed but changes depending on the neural context 2, i.e. the brain regions that happen to be functionally connected at a particular point in time.</li>
	<li>To calculate MSE, use the algorithm available at <a href="http://www.physionet.org/physiotools/mse/">www.physionet.org/physiotools/mse/</a>, which computes MSE in two steps.</li>
	<li>First, the algorithm progressively down-samples the EEG time series per trial and per condition. Down-sample the original time series to generate multiple time series of varying timescales. Time series 1 is the original time series. To create the time series of subsequent timescales, divide the original time series into non-overlapping windows of the timescale length and average the data points within each window. Down-sampling is similar to low-pass filtering; dividing the sampling frequency by the timescale will approximate the frequency at which the signal is low-pass filtered for that particular timescale. The application of MSE to a particular frequency range (e.g., alpha: 9 Hz to 12 Hz) can be interpreted as representing the composition of rhythms within that range as well as the interaction between those frequencies.</li>
	<li>Second, the algorithm calculates the sample entropy for each coarse-grained time series 14. Sample entropy estimates the complexity of a time series. In a nonlinear analysis of EEG, one assumes that an individual time series represents the manifestation of an underlying multi-dimensional non-linear dynamic model (see Stam, 2005 for a review). In this example, m (the pattern length) is set to two, which means that the variability of the amplitude pattern of each time series will be represented in two-dimensional versus three-dimensional space by considering the sequence pattern of two versus three consecutive data points, respectively. Parameter r (the similarity criterion), reflects the amplitude range (denoted by the height of the colored bands) within which data points are considered to "match". For a typical EEG time series with more than 100 data points, set the parameter m equal to 2 and the parameter r equal to a value between 0.5 and 1 (see Richman and Moorman, 2000; for a detailed procedure on selecting parameters refer to Lake et al., 2002). 
	To calculate sample entropy for this simulated time series, begin with the first two-component sequence pattern, red-orange. First, count the number of times the red-orange sequence pattern occurs in the time series; there are 10 matches for this two-component sequence. Second, count the number of times first three-component sequence pattern, red-orange-yellow, occurs in the time series; there are 5 matches for this three-component sequence. Continue with the same operations for the next two-component sequence (orange-yellow) and the next three-component sequence (orange-yellow-green) of the time series. The number of two-component matches (5) and three-component matches (3) for these sequences are added to the previous values (total two-component matches = 15; total three-component matches = 8). Repeat for all other sequence matches in the time series (up to N - m) to determine the total ratio of two-component matches to three-component matches. Sample entropy is the natural logarithm of this ratio. For each subject, compute the channel specific MSE estimate as the mean across single trial entropy measures for each timescale.</li>
</ol>

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	<li>Bressler, S. L., Arbib, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Event-related+potentials.">Event-related potentials.</a> The Handbook of Brain Theory and Neural Networks. , 412-415 (2002).</li><li>Bressler, S. L., McIntosh, A. R., Jirsa, V. K., McIntosh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neural+context+in+large-scale+neurocognitive+network+operations.">The role of neural context in large-scale neurocognitive network operations.</a> Springer Handbook on Brain Connectivity. , 403-419 (2007).</li><li>Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rhythms of the brain. , (2006).</li><li>Costa, M., Goldberger, A., Peng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+entropy+analysis+of+biological+signals.">Multiscale entropy analysis of biological signals.</a> Phys. Rev. E. 712, 1-18 (2005).</li><li>Deco, G., Jirsa, V., McIntosh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+concepts+for+the+dynamical+organization+of+resting-state+activity+in+the+brain.">Emerging concepts for the dynamical organization of resting-state activity in the brain.</a> Nat. Rev. Neurosci. 12, 43-56 (2011).</li><li>Friston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+labile+brain.+I.+Neuronal+transients+and+nonlinear+coupling.">The labile brain. I. Neuronal transients and nonlinear coupling.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355, 215-236 (2001).</li><li>Gatlin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Information Theory and the Living System. , (1972).</li><li>Heisz, J. J., Shedden, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semantic+learning+modifies+perceptual+face+processing.">Semantic learning modifies perceptual face processing.</a> Journal of Cognitive Neuroscience. 21, 1127-1134 (2009).</li><li>Heisz, J. J., Shedden, J. M., McIntosh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+brain+signal+variability+to+knowledge+representation.">Relating brain signal variability to knowledge representation.</a> NeuroImage. 63, 1384-13 (2012).</li><li>Lake, D. E., Richman, J. S., Griffin, P., Moorman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+entropy+analysis+of+neonatal+heart+rate+variability.">Sample entropy analysis of neonatal heart rate variability.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R789-R797 (2002).</li><li>Lobaugh, N. J., West, R., McIntosh, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+analysis+of+experimental+differences+in+event-related+potential+data+with+partial+least+squares.">Spatiotemporal analysis of experimental differences in event-related potential data with partial least squares.</a> Psychophysio. 38, 517-530 (2001).</li><li>McIntosh, A. R., Kovacevic, N., Itier, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+brain+signal+variability+accompanies+behavioral+variability+in+development.">Increased brain signal variability accompanies behavioral variability in development.</a> PLoS Computational Biology. 4, 7 (2008).</li><li>Picton, T. W., Bentin, S., Berg, P., Donchin, E., Hillyard, S. A., Johnson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+using+human+event-related+potentials+to+study+cognition:+Recording+standards+and+publication+criteria.">Guidelines for using human event-related potentials to study cognition: Recording standards and publication criteria.</a> Psychophysiology. 37, 127-152 (2000).</li><li>Richman, J. S., Moorman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+time+series+analysis+using+approximate+entropy+and+sample+entropy.">Physiological time series analysis using approximate entropy and sample entropy.</a> Am. J. Physiol. Heart Circ Physiol. 278, H2039-H2049 (2000).</li><li>Rossion, B., Jacques, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+physical+interstimulus+variance+account+for+early+electrophysiological+face+sensitivity+responses+in+the+human+brain?+Ten+lessons+on+the+N170.">Does physical interstimulus variance account for early electrophysiological face sensitivity responses in the human brain? Ten lessons on the N170.</a> NeuroImage. 39, 1959-1979 (2008).</li><li>Shannon, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mathematical+Theory+of+Communication.">A Mathematical Theory of Communication.</a> The Bell System Technical Journal. 27, 379-423 (1948).</li><li>Stam, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+dynamical+analysis+of+EEG+and+MEG:+review+of+an+emerging+field.">Nonlinear dynamical analysis of EEG and MEG: review of an emerging field.</a> Clinical Neurophysiology. 116, 2266-2301 (2005).</li><li>Vakorin, V. A., McIntosh, A. R., Rabinovich, M. I., Friston, K. 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No conflicts of interest declared.

The goal of the present article was to provide a conceptual and methodological description of multiscale entropy (MSE) as it applies to EEG neuroimaging data. EEG is a powerful non-invasive neuroimaging technique that measures neural network activity with the high temporal resolution. The EEG signal reflects post-synaptic activity of populations of pyramidal cells in the cortex, whose collective responses are modified by various excitatory and inhibitory reentrant connections. Accordingly, there are multiple ways to analyze EEG data and each method extracts a unique aspect of the data.
We discussed two common methods of analysis: event-related potential (ERP) analysis and spectral power analysis. ERP analysis captures the synchronous neuronal activity in the EEG signal that is phase-locked to the onset of a discrete event. ERPs reflect specific perceptual, motor, or cognitive operations, making this statistic ideal for examining specific processing stages. Spectral power analysis quantifies the relative contribution of a particular frequency to the EEG signal. Various excitatory and inhibitory feedback loops interact to entrain the activity of neuronal populations at a particular frequency 1,3. Such synchrony between disparate brain regions is thought to promote the binding of information across widespread neural networks. There is a rich literature supporting the link between the power within a particular frequency range and a specific emotional or cognitive state of function 3.
When analyzing EEG it is also important to keep in mind that neural networks are complex systems with non-linear dynamics. Such complexity is reflected in the EEG signal as irregular oscillations that are not the consequence of meaningless background noise. Like synchronous oscillatory activity, the interactions between various excitatory and inhibitory reentrant loops cause transient fluctuations in the brain signal over time 6. Such transients are believed to reflect transitions or bifurcations between network microstates that can be used to estimate the degrees of freedom or complexity of the underlying network; greater variability in the amplitude pattern of the signal over time is indicative of a more complex system 5. Critically, ERP or spectral power analyses are not sensitive to such irregular activity, whereas MSE is. Moreover, an index of network complexity cannot be obtained by simply counting the number of active brain regions as such a method is blind to the transient and dynamic recurrent interactions between brain regions.
Complementary methods for neuroimaging analysis combine to create a complete picture of the underlying neural activity. The interpretation of results from more traditional applications of neuroimaging data, such as ERP and spectral power, are augmented by measures of complexity like MSE; MSE provides a way to capture the sequence of changes in the spatiotemporal patterns of brain activity across multiple timescales that contributes to a specific cognitive operation. Applying MSE to new and existing data sets may provide further insight into how cognition emerges from neural network dynamics.

Recent advances in neuroimaging have dramatically augmented our understanding of brain function. However, many of the applications of neuroimaging data tend to reinforce the view of the brain in static states rather than emphasizing cognitive operations as they unfold in real time. Consequently, little is known about the space-time structure of brain networks and how the sequence of changes in spatiotemporal patterns across multiple timescales contributes to a specific cognitive operation. The present article describes multiscale entropy (MSE) 5, a new analytic tool for neuroimaging data that examines the complexity of the spatiotemporal pattern underlying specific cognition operations by providing information about how different neural generators in a functional brain network communicate across multiple timescales.
Derived from information theory, an applied branch of mathematics 7,16, MSE was originally designed to examine the complexity of electrocardiograms 4. In theory, MSE could be used to analyze the complexity of any time series; the primary requisite is that the signal time series contains at least 50 data points of continuous time. However, the timescale dependence and sensitivity to linear and nonlinear dynamics in the data may make MSE particularly informative of neural network dynamics.
Here, we focus on the application of MSE to electroencephalogram (EEG) neuroimaging data 9,12. EEG is a noninvasive neuroimaging technique whereby electrodes that are placed on the scalp capture the postsynaptic responses of populations of neurons in neocortex 1. With high temporal resolution, EEG easily meets the time series length requisite of MSE without altering the typical acquisition protocol. To emphasize the utility of the application of MSE to EEG data, we compare this novel method with more traditional approaches including event-related potential and spectral power. When used together, these complementary methods of analysis provide a more complete description of the data that may lead to further insight into neural network operations that give rise to cognition.

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applications of eeg neuroimaging data: event-related potentials, spectral power, and multiscale entropy

When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain signal. Typically, researchers represent the brain's response as the mean across repeated experimental trials and disregard signal fluctuations over time as "noise". However, it is becoming clear that brain signal variability conveys meaningful functional information about neural network dynamics. This article describes the novel method of multiscale entropy (MSE) for quantifying brain signal variability. MSE may be particularly informative of neural network dynamics because it shows timescale dependence and sensitivity to linear and nonlinear dynamics in the data.

Figures 1A and 2A represent the EEG signal in response to the presentation of a face image. Averaging across like trials produces an ERP waveform that consists of a series of positive and negative deflections called ERP components. Figure 1B illustrates an averaged waveform for a single subject and Figure 6A illustrates a grand average waveform for a group of subjects. There is a rich literature that relates each ERP component to a specific perceptual, motor, or cognitive operation. For example, the N170 is a negative deflection that peaks at approximately 170 msec post-stimulus onset and is implicated in face processing 8,15.
Figure 2B illustrates the decomposition of that same EEG signal into component frequency bands. The results from spectral power analysis reveal the frequency content of the signal (Figure 2C), whereby an increase in power at a particular frequency reflects an increase in the presence of that rhythm within the EEG signal.
Like spectral power, MSE is sensitive to the complexity of the oscillatory components contributing to the signal. However, unlike spectral power, MSE is also sensitive to the interactions between frequency components (i.e. nonlinear dynamics 18). The complexity of an EEG signal is represented as a function of sample entropy (Figure 5) over multiple timescales (Figure 4). As illustrated in Figure 3, sample entropy is low for regular signals and increases with the degree of signal randomness. Unlike traditional entropy measures that increase with degree of randomness, multiscale entropy is able to differentiate complex signals from white noise by considering entropy across multiple timescales. For example, Costa et al., 2005 compared multiscale entropy values for uncorrelated (white) noise versus correlated (pink) noise. While sample entropy was greater for white noise than pink noise at fine timescale, the opposite was observed at coarser timescales 5-20. In other words, when entropy was considered across multiple timescales, the true complexity of the signals was more accurately represented than would be if only a single timescale was considered. Depending of the temporal dynamics of a specific contrast, condition effects may be expressed: 1) in the same way across all timescales, 2) at some timescales but not other, or 3) as crossover effects whereby the contrast is different at fine versus coarser timescales.
Figure 6 depicts condition differences in ERP (Figure 6A), spectral power (Figure 6B), MSE (Figure 6C) contrasting the initial versus repeat presentations of face photographs 9. In this example all measures converged to reveal the same effect; however, the observed decrease in sample entropy that accompanies face repetition is important as it constrains the interpretation of the results. A decrease in complexity suggests that the underlying functional network is simpler and capable of processing less information.
Figure 7 depicts statistical results from the multivariate analysis of partial-least squares 11 applied to ERP, spectral power and MSE. The experiment manipulated the familiarity associated with different faces (Heisz et al., 2012). The contrast (bar graph) shows that ERP amplitude distinguished new faces from familiar faces but not among the familiar faces that varied in amount of prior exposure. Spectral power distinguished faces according to acquired familiarity but did not accurately distinguish between the faces of medium and low familiarity. MSE was most sensitive to the condition differences in that sample entropy values increased with increasing face familiarity. The image plots capture the spatiotemporal distribution of the condition effect across all electrodes and time/frequency/timescale. This example demonstrates a situation in which the analysis of EEG by MSE produced unique information that was not obtained using traditional methods of ERP or spectral power. This divergence of MSE suggests that the conditions differ with respect to nonlinear aspects of their network dynamics, possibly involving the interactions between various frequency components.
<img alt="Figure 1" src="/files/ftp_upload/50131/50131fig1.jpg" /> 
Figure 1. A) The EEG responses of a single subject as a function of amplitude deflection from baseline for each trial plotted against time from the onset of the trial. Each trial consisted of the presentation of a photograph of a face image. Positive amplitude deflections are depicted in red; negative amplitude deflections are depicted in blue. All trials show a positive deflection around 100 msec and 250 msec, indicating event-related phase-locked activity. B) Averaging across all trials depicted in Figure 1A produces an averaged ERP waveform with distinct positive and negative deflections called event-related components and named according to a standard nomenclature. For example, P1 is the first positive going component, and N170 is a negative component that peaks at approximately 170 msec post-stimulus onset.
<img alt="Figure 2" src="/files/ftp_upload/50131/50131fig2.jpg" /> 
Figure 2. A) The EEG response of a single subject for a single trial plotting amplitude by time (in data points, sampling rate 512 Hz). B) The EEG response of Figure 2A bandpass filtered to isolate frequency bands of delta (0-4 Hz), theta (5-8 Hz), alpha (9-12 Hz), beta (13-30 Hz) and gamma (&gt; 30 Hz). C) Spectral power density of the EEG response depicted in Figure 2A representing frequency composition of the signal as a function of power by frequency. An increase in spectral power at a particular frequency reflects an increase in the number of synchronously active neurons entrained within that particular frequency band. <a href="https://www.jove.com/files/ftp_upload/50131/50131fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/50131/50131fig3.jpg" /> 
Figure 3. A) Two simulated waveforms: a regular or predictable waveform depicted in purple, and a more stochastic waveform depicted in black. B) Sample entropy values of the two simulated waveforms for the first three timescales. Sample entropy is low for highly predictable signals than more stochastic signals. <a href="https://www.jove.com/files/ftp_upload/50131/50131fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/50131/50131fig4.jpg" /> 
Figure 4. Down-sampling the original time series generates multiple time series of varying timescales. Timescale 1 is the original time series. The time series of timescale 2 is created by dividing the original time series into non-overlapping windows of length 2 and average the data points within each window. To generate the time series of subsequent timescales, divide the original time series into non-overlapping windows of the timescale length and average the data points within each window.
<img alt="Figure 5" src="/files/ftp_upload/50131/50131fig5.jpg" /> 
Figure 5. A simulated waveform where each rectangle represents a single data point in the time series. Sample entropy estimates the variability of a time series. In this example, m (the pattern length) is set to two, which means that the variance of the amplitude pattern of each time series will be represented in two-dimensional versus three-dimensional space by considering the sequence pattern of two versus three consecutive data points, respectively; r (the similarity criterion), reflects the amplitude range (denoted by the height of the colored bands) within which data points are considered to "match". To calculate sample entropy for this simulated time series, begin with the first two-component sequence pattern, red-orange. First, count the number of times the red-orange sequence pattern occurs in the time series; there are 10 matches for this two-component sequence. Second, count the number of times first three-component sequence pattern, red-orange-yellow, occurs in the time series; there are 5 matches for this three-component sequence. Continue in this manner for the next two-component sequence (orange-yellow) and three-component sequence (orange-yellow-green). The number of two-component matches (5) and three-component matches (3) for these sequences are added to the previous values (total two-component matches =15; total three-component matches = 8). Repeat for all other sequence matches in the time series (up to N - m) to determine the total ratio of two-component matches to three-component matches. Sample entropy is the natural logarithm of this ratio. For each subject, compute the channel specific MSE estimate as the mean across single trial entropy measures for each timescale.
<img alt="Figure 6" src="/files/ftp_upload/50131/50131fig6.jpg" /> 
Figure 6. Condition differences in ERP (A), spectral power (B), MSE (C) contrasting the initial versus repeated presentations of facial photographs. <a href="https://www.jove.com/files/ftp_upload/50131/50131fig6large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 7" src="/files/ftp_upload/50131/50131fig7.jpg" /> 
Figure 7. Contrasting the EEG response to learned faces across measures of ERP, spectral power, and multiscale entropy. The bar graphs depict the contrast between conditions as determined by partial least squares analysis 11. The image plot highlights the spatial-temporal distribution at which this contrast was most stable as determined by bootstrapping. Values represent ~z scores and negative values denote significance for the inverse condition effect. <a href="https://www.jove.com/files/ftp_upload/50131/50131fig7large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Applications of EEG Neuroimaging Data: Event-related Potentials, Spectral Power, and Multiscale Entropy at JoVE.com

Applications of EEG Neuroimaging Data: Event-related Potentials, Spectral Power, and Multiscale Entropy

Neuroimaging researchers typically consider the brain's response as the mean activity across repeated experimental trials and disregard signal variability over time as "noise". However, it is becoming clear that there is signal in that noise. This article describes the novel method of multiscale entropy for quantifying brain signal variability in the time domain.

When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain ...

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33.6K Views.  Baycrest. Neuroimaging researchers typically consider the brain's response as the mean activity across repeated experimental trials and disregard signal variability over time as "noise". However, it is becoming clear that there is signal in that noise. This article describes the novel method of multiscale entropy for quantifying brain signal variability in the time domain.

Video: Applications of EEG Neuroimaging Data: Event-related Potentials, Spectral Power, and Multiscale Entropy

High Density Event-related Potential  Data Acquisition in Cognitive Neuroscience

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part because fMRI data acquisition and analysis techniques are readily available. Because fMRI has excellent spatial resolution but poor temporal resolution, this method can only be used to identify the spatial location of brain activity associated with a given cognitive process (and reveals virtually nothing about the time course of brain activity). By contrast, event-related potential (ERP) recording, a method that is used much less frequently than fMRI, has excellent temporal resolution and thus can track rapid temporal modulations in neural activity. Unfortunately, ERPs are under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and low density ERP recording has poor spatial resolution. In an effort to foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition. Critically, high density ERPs offer the promise of excellent temporal resolution and good spatial resolution (or excellent spatial resolution if coupled with fMRI), which is necessary to capture the spatial-temporal dynamics of human brain function.

Event-related potential (ERP) recording is under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and this method often has poor spatial resolution. To foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition.

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part ...

Basics of Multivariate Analysis in Neuroimaging Data

Multivariate analysis techniques for neuroimaging data have recently received increasing attention as they have many attractive features that cannot be easily realized by the more commonly used univariate, voxel-wise, techniques1,5,6,7,8,9. Multivariate approaches evaluate correlation/covariance of activation across brain regions, rather than proceeding on a voxel-by-voxel basis. Thus, their results can be more easily interpreted as a signature of neural networks. Univariate approaches, on the other hand, cannot directly address interregional correlation in the brain. Multivariate approaches can also result in greater statistical power when compared with univariate techniques, which are forced to employ very stringent corrections for voxel-wise multiple comparisons. Further, multivariate techniques also lend themselves much better to prospective application of results from the analysis of one dataset to entirely new datasets. Multivariate techniques are thus well placed to provide information about mean differences and correlations with behavior, similarly to univariate approaches, with potentially greater statistical power and better reproducibility checks. In contrast to these advantages is the high barrier of entry to the use of multivariate approaches, preventing more widespread application in the community. To the neuroscientist becoming familiar with multivariate analysis techniques, an initial survey of the field might present a bewildering variety of approaches that, although algorithmically similar, are presented with different emphases, typically by people with mathematics backgrounds. We believe that multivariate analysis techniques have sufficient potential to warrant better dissemination. Researchers should be able to employ them in an informed and accessible manner. The current article is an attempt at a didactic introduction of multivariate techniques for the novice. A conceptual introduction is followed with a very simple application to a diagnostic data set from the Alzheimer s Disease Neuroimaging Initiative (ADNI), clearly demonstrating the superior performance of the multivariate approach.

The current article describes the basics of multivariate analysis and contrasts it to the more commonly used voxel-wise univariate analysis. Both types of analysis are applied to a clinical-neuroscience data set. Supplementary split-half simulations show better replication of the multivariate results in independent data sets.

Multivariate analysis techniques for neuroimaging data have recently received increasing attention as they have many attractive features that cannot be ...

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Extracting Visual Evoked Potentials from EEG Data Recorded During fMRI-guided Transcranial Magnetic Stimulation

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological effects. Specifically, by creating a transient interference with the normal activity of a target region and measuring changes in an electrophysiological signal, we can establish a causal link between the stimulated brain area or network and the electrophysiological signal that we record. If target brain areas are functionally defined with prior fMRI scan, TMS could be used to link the fMRI activations with evoked potentials recorded. However, conducting such experiments presents significant technical challenges given the high amplitude artifacts introduced into the EEG signal by the magnetic pulse, and the difficulty to successfully target areas that were functionally defined by fMRI. Here we describe a methodology for combining these three common tools: TMS, EEG, and fMRI. We explain how to guide the stimulator&#39;s coil to the desired target area using anatomical or functional MRI data, how to record EEG during concurrent TMS, how to design an ERP study suitable for EEG-TMS combination and how to extract reliable ERP from the recorded data. We will provide representative results from a previously published study, in which fMRI-guided TMS was used concurrently with EEG to show that the face-selective N1 and the body-selective N1 component of the ERP are associated with distinct neural networks in extrastriate cortex. This method allows us to combine the high spatial resolution of fMRI with the high temporal resolution of TMS and EEG and therefore obtain a comprehensive understanding of the neural basis of various cognitive processes.

This paper describes a method for collecting and analyzing electroencephalography (EEG) data during concurrent transcranial magnetic stimulation (TMS) guided by activations revealed with functional magnetic resonance imaging (fMRI). A method for TMS artifact removal and extraction of event related potentials is described as well as considerations in paradigm design and experimental setup.

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological ...

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of providing precise and controlled stimulation. This article introduces a technique for recording somatosensory event-related potentials (ERP) that uses a novel mechanical stimulation method involving skin deformation using a robotic device. Controlled deformation of the facial skin is used to modulate kinesthetic inputs through excitation of cutaneous mechanoreceptors. By combining somatosensory stimulation with electroencephalographic recording, somatosensory evoked responses can be successfully measured at the level of the cortex. Somatosensory stimulation can be combined with the stimulation of other sensory modalities to assess multisensory interactions. For speech, orofacial stimulation is combined with speech sound stimulation to assess the contribution of multi-sensory processing including the effects of timing differences. The ability to precisely control orofacial somatosensory stimulation during speech perception and speech production with ERP recording is an important tool that provides new insight into the neural organization and neural representations for speech.

This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

How to Find Effects of Stimulus Processing on Event Related Brain Potentials of Close Others when Hyperscanning Partners

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen in one of two adjacent rooms. These rooms are separated by a glass window through which participants communicate to maintain feelings of togetherness while being fitted with the EEG cap. After checking for adequate EEG signals, the glass is covered by a curtain to prevent visual communication. Then, partners must be silent but are instructed to try to feel in the presence of their partner during the entire experiment. Just before it starts, participants are told that each of them will be presented with one image at a time and that these images will occur at the same time for both of them on their own screen. They are also instructed that, for each trial, the simultaneous images will always be different. However, unbeknownst to them, trials are randomized: only half of them are consistent with this instruction and actually include two different images. These trials form the DSC, that is, the different-stimuli condition. The other half of the trials are inconsistent with the instruction. They include two identical images and form the ISC (identical-stimuli condition). After the experiment, participants are sorted into two groups: those who reported that they felt in the presence of their partner during the majority of the trials and those who reported they did not. The impact of the stimulus processing of the partner is found by subtracting the mean voltages of the ERPs of the ISC (inconsistent with the instructions) from the ERPs of the DSC (consistent with the instructions) in at least two time windows (TWs): firstly, in the 75 to 150 ms TW, where the absolute values of these subtractions are greater, especially at right frontal sites, in those who felt in the presence of their partner than in those who did not; secondly, in the LPP time window (i.e., from 650 to 950 ms post onset), where ERPs are significantly less positive in the DSC than in the ISC in those in whom the raw results of the early (75-150ms) subtractions are negative.

This protocol describes key steps involved in assessing the sensitivity of the brain of one person to the stimulus processing of a close other by selecting pairs of partners, recording their electroencephalogram (EEG) simultaneously and computing their event-related brain potentials (ERPs).

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen ...

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in remote locations. This paper shows the development of field functional Near Infrared Spectroscopy (fNIRS) imaging to the study of child language, reading, and cognitive development in a rural village setting of C&#244;te d&#39;Ivoire. Innovation in methods and the development of culturally appropriate neuroimaging protocols allow&#160;a first-time look into the brain&#39;s development and children&#39;s learning outcomes in understudied environments. This paper demonstrates protocols for transporting and setting up a mobile laboratory, discusses considerations for field versus laboratory neuroimaging, and presents a guide for developing neuroimaging consent procedures and building meaningful long-term collaborations with local government and science partners. Portable neuroimaging methods can be used to study complex child development contexts, including the impact of significant poverty and adversity on brain development. The protocol presented here has been developed for use in C&#244;te d&#39;Ivoire, the world&#39;s primary source of cocoa, and where reports of child labor in the cocoa sector are common. Yet, little is known about the impact of child labor on brain development and learning. Field neuroimaging methods have the potential to yield new insights into such urgent issues, and the development of children globally.

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural C&#244;te d&#39;Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain&#39;s development and children&#39;s learning outcomes in environments with significant poverty and adversity.

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...