Engineer H&#233;ctor Belmont, Dr. M&#243;nica Carlier, Dr. Yuria Cruz and Dr. Mar&#237;a Elena Ju&#225;rez collaborated in data collection. The authors thank Paul Kersey for revising English language use. The project was partially funded by PAPIIT grant IN2009/7 and CONACYT (National Council for Science and Technology, Mexico) grant 4971.

1. Preparation of the Newborns
NOTE: The procedure followed is innocuous and painless, so there are no counter-indications for evaluating full-term and preterm newborns, once they are clinically stable.
<ol>
	<li>Ensure two and half hour fasting and wakefulness before beginning the study, in neonates older than 40 weeks of postconceptional age.</li>
	<li>Make sure that the baby&#39;s head be washed with neutral soap the day before the study. Thus, his/her hair will be clean and dry. Do not apply conditioners.</li>
	<li>Allow the mother to start feeding the newborn 30 min before beginning the study. Allow him/her to burp and start the sleep wrapped in sheets. This will ensure that he/she sleeps easily and spontaneously.</li>
	<li>Wash hands carefully before handling the neonate.</li>
	<li>Use sanitary masks.</li>
	<li>Gently wipe the scalp of the newborn with a cotton ball or gauze soaked in alcohol to remove residual dirt and superficial grease, before the neonate falls asleep.</li>
	<li>Measure the distance between nasion and inion, and between both pre-auricular pits. Calculate 10% and 20% to ensure proper placement of the cranial electrodes according to the international 10-20 system of electrode placement.</li>
	<li>Cover the newborn&#39;s entire head with a tubular elastic mesh for the correct attachment of the electroencephalography (EEG) and VEP electrodes. Leave the face fully free and exposed, as shown in Figure 1.</li>
	<li>Mark on the mesh the location of the surface electrodes.</li>
	<li>Use a swab to perfectly separate the newborn&#39;s hair at the sites where each electrode will be placed, and lightly rub the skin with abrasive gel for neurophysiological studies. 
	NOTE: Reschedule the study if the neonate takes more than 2 h to fall asleep.</li>
</ol>
2. Placement of the Surface Electrodes for EEG and VEP Sleep Recording
NOTE: Before beginning, set the values of the instrument&#39;s frequency filters using the specifications in Table 1. It is advisable to connect all electrodes to the EEG and VEP instruments before placing them on the newborn.
<ol>
	<li>Place the elastic band sensor on the baby&#39;s chest to record thoracic respiratory expansion.</li>
	<li>Place the individual surface disc electrodes (standard silver-silver chloride, or gold disc electrodes) with conductive paste through the mesh to fix them in the cranial locations established by the international 10-20 EEG system, adapted for newborns.</li>
	<li>Locate the cranial electrodes for EEG at leads F3, F4, C3, C4, O1 and O2, or at least C3 and C4, referred to linked earlobes, to identify the stages of neonatal sleep.</li>
	<li>Fix the surface disc electrodes to the skin with medical adhesive tape. To record ocular movements (EOG), place one electrode 1 cm above the external canthus of the left eye and place another 1 cm below the external canthus of the right eye, also referred to linked earlobes.</li>
	<li>Similarly, attach the electrodes for surface electromyogram recording (EMG) on both sides of the chin, referenced against each other.</li>
	<li>Use two channels of the VEP equipment with the following leads: Oz (-) vs. Fz (+), and Oz (-) vs. A1 (+); the ground electrode is to be placed on the right mastoid.</li>
	<li>Set the analysis time for VEP registration in 600 ms. 
	NOTE: Table 1 shows the filter settings used for recording sleep EEGs and VEPs.</li>
	<li>Do not begin VEP recording until impedance values are below 5 k&#937;.</li>
</ol>
3. Sleep Recording
NOTE: VEPs are obtained while the newborn sleeps in hospital crib; the sleep stages are monitored simultaneously by polysomnography17,18.
<ol>
	<li>Prolong the EEG recording for 60&#8722;90 min or until AS is identified, to evaluate active (AS) and quiet sleep (QS) in newborns.</li>
	<li>Begin EEG recording while carefully observing the characteristics of neonatal sleep, to identify the active sleep stage, during which VEPs will be recorded.</li>
	<li>Identify neonatal sleep stages according to the criteria summarized in Table 2.</li>
</ol>
4. VEP Recording
NOTE: VEPs are registered according to established standards19,20.
<ol>
	<li>Allow one minute of EEG recording without visual stimulation when the neonate begins well-defined active sleep.</li>
	<li>Apply monocular light stimulation through handheld goggles with a LED matrix held manually 2 cm directly above each newborn&#39;s eyes.</li>
	<li>Observe if the infant has his eyes closed during VEP registration in AS and note if this does not occur.</li>
	<li>Begin the averaging of the VEPs in the equipment, presenting 20 to 40 luminous stimuli whose corresponding recordings are averaged to obtain an average curve or evoked response.</li>
	<li>Observe the reproducibility of the recorded averages. At least two reproducible evoked potentials are recommended.</li>
	<li>Visually recognize PII component of the VEPs during recording, since this peak is considered typical of neonatal VEPs. Identify the PII component as the maximal positive peak between 120 and 300 ms, preceded by a negative wave (NII) and followed by a maximal negativity between 200 and 400 ms, also called NIII.</li>
	<li>Stop the averaging of the VEPs if the newborn moves excessively, wakes up, or changes to another sleep stage, distinct from AS. Renew recording once the AS stage reestablishes. 
	NOTE: This point is critical, because VEPs obtained during QS or transitional sleep are less reliable than in AS.</li>
	<li>Finish registration after 2 averages with reproducible VEP are attained, or when 6 averages occur without a recognizable VEP. In the latter case, consider the result an absence of a replicable response.</li>
</ol>
5. Review and analysis of VEPs
NOTE: Figure 2 shows the main components of neonatal VEPs and their measurements.
<ol>
	<li>Evaluate the reproducibility of the VEPs by similar appearance and measurements between the two averaged curves. 
	NOTE: Some VEP recording systems offer a correlation measure between two averages.</li>
	<li>Measure the absolute latencies of the NII, PII and NIII waves using the device&#39;s cursors. Absolute latency is the time in ms elapsed from the onset of stimulation to the maximal or minimal peak of each component.</li>
	<li>Calculate the interpeak latencies in ms, including the differences between the absolute PII-NII, NII-NIII and PII-NIII latencies.</li>
	<li>Measure peak-to-peak amplitudes in &#181;V, for the NII-PII and PII-NIII components.</li>
	<li>Compare the latency and amplitude values obtained to the normal, or expected, values estimated for a population of healthy, similar-age newborns.</li>
</ol>

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+developmental+intervention+programs+provided+post+hospital+discharge+to+prevent+motor+and+cognitive+impairment+in+preterm+infants.">Early developmental intervention programs provided post hospital discharge to prevent motor and cognitive impairment in preterm infants.</a> Cochrane Database System Review. 24 (11), CD005495 (2015).</li><li>Huang, X., 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=Volume+Changes+and+Correlation+with+Visual+Evoked+Potential+in+Patients+with+Optic+Neuritis:+A+Voxel-Based+Morphometry+Study.">Volume Changes and Correlation with Visual Evoked Potential in Patients with Optic Neuritis: A Voxel-Based Morphometry Study.</a> Medical Science Monitor. 22, 1115-1123 (2016).</li><li>McGlone, L., 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=Neonatal+Visual+Evoked+Potentials+in+Infants+Born+to+Mothers+Prescribed+Methadone.">Neonatal Visual Evoked Potentials in Infants Born to Mothers Prescribed Methadone.</a> Pediatrics. 131 (3), 857-863 (2013).</li><li>Cruz, S., Crego, A., Ribeiro, E., Goncalves, O., Sampaio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+VEP+study+in+sleeping+and+awake+one-month-old+infants+and+its+relation+with+social+behavior.">A VEP study in sleeping and awake one-month-old infants and its relation with social behavior.</a> International Journal of Developmental Neuroscience. 41, 37-43 (2015).</li><li>Kato, T., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+evoked+potential+in+the+newborn:+Does+it+have+predictive+value?.">Visual evoked potential in the newborn: Does it have predictive value?.</a> Seminars in Fetal &amp; Neonatal Medicine. 11, 459-463 (2006).</li><li>Shepherd, A., Saunders, K., McCulloch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sleep+state+on+the+&#64258;ash+visual+evoked+potential.+A+case+study.">Effect of sleep state on the &#64258;ash visual evoked potential. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+visually+evoked+potentials+in+the+newborn+and+their+maturation+during+the+first+six+months+of+life.">Flash visually evoked potentials in the newborn and their maturation during the first six months of life.</a> Documenta Ophthalmologica. 110, 255-263 (2005).</li><li>Tsuneishi, S., Casaer, P., Fock, J. M., Hirano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+normal+values+for+flash+visual+evoked+potentials+(VEPs)+in+preterm+infants:+a+longitudinal+study+with+special+reference+to+two+components+of+the+N1+wave.">Establishment of normal values for flash visual evoked potentials (VEPs) in preterm infants: a longitudinal study with special reference to two components of the N1 wave.</a> Electroencephalography and Clinical Neurophysiology. 96, 291-299 (1995).</li><li>Roy, M. S., Gosselin, J., Hanna, N., Orquin, J., Chemtob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In&#64258;uence+of+the+state+of+alertness+on+the+pattern+visual+evoked+potentials+(PVEP)+in+very+young+infant.">In&#64258;uence of the state of alertness on the pattern visual evoked potentials (PVEP) in very young infant.</a> Brain &amp; Development. 26, 197-202 (2004).</li><li>Kraemer, M., Abrahamsson, M., Sjostrom, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neonatal+development+of+the+light+&#64258;ash+visual+evoked+potential.">The neonatal development of the light &#64258;ash visual evoked potential.</a> Documenta Ophthalmologica. 99, 21-39 (1999).</li><li>Apkarian, P., Mirmiran, M., Tijssen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Behavioral+State+on+Visual+Processing+in+Neonates.">Effects of Behavioral State on Visual Processing in Neonates.</a> Neuropediatrics. 22, 85-91 (1991).</li><li>Cubero-Rego, L., Corsi-Cabrera, M., Ricardo-Garcell, J., Cruz-Mart&#237;nez, R., Harmony, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+evoked+potentials+are+similar+in+polysomnographically+de&#64257;ned+quiet+and+active+sleep+in+healthy+newborns.">Visual evoked potentials are similar in polysomnographically de&#64257;ned quiet and active sleep in healthy newborns.</a> International Journal of Developmental Neuroscience. 68, 26-34 (2018).</li><li>Mizrahi, E. M., Mosh&#233;, S. L., Hrachovy, R. A., Niedermeyer, E., Lopes da Silva, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+EEG+and+Sleep:+Preterm+and+Term+Neonates.">Normal EEG and Sleep: Preterm and Term Neonates.</a> Niedermeyer's Electroencephalography: Basic Principles, Clinical Applications, and Related. , 154-163 (2011).</li><li>Grigg-Damberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Visual+Scoring+of+Sleep+in+Infants+0+to+2+Months+of+Age.">The Visual Scoring of Sleep in Infants 0 to 2 Months of Age.</a> Journal of Clinical Sleep Medicine. 12 (3), 429-445 (2016).</li><li>Husain, A. M., Niedermeyer, E., Lopes da Silva, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evoked+Potentials+in+Children+and+Infants.">Evoked Potentials in Children and Infants.</a> In Niedermeyer's Electroencephalography: Basic Principles, Clinical Applications, and Related Fields. , 1057-1082 (2011).</li><li>Odom, J. V., 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=ISCEV+standard+for+clinical+visual+evoked+potentials:+2016+update.">ISCEV standard for clinical visual evoked potentials: 2016 update.</a> Documenta Ophthalmologica. 133 (1), 1-11 (2016).</li><li>Pojda-Wilczek, D., Maruszczyk, W., Sirek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+visual+evoked+potentials+(FVEP)+in+various+stimulation+conditions.">Flash visual evoked potentials (FVEP) in various stimulation conditions.</a> Documenta Ophthalmologica. 138, 35-42 (2019).</li><li>Tsuneishi, S., Casaer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+decrease+in+VEP+latencies+and+the+process+of+myelination+in+the+human+visual+pathway.">Stepwise decrease in VEP latencies and the process of myelination in the human visual pathway.</a> Brain &amp; Development. 19, 547-551 (1997).</li></ol>

The authors have nothing to disclose.

Three components of visual-evoked responses (NII, PII and NIII) were characterized in healthy, full-term newborns while doing stimulation with LED googles, and recorded during polygraphically-identified sleep states. The VEP morphology observed is consistent with previous results reported for fewer neonates11,15. The characterization of VEP responses was achieved by recording 20 healthy, full-term newborns at similar post-conceptional age16</sup...

Early detection of abnormal development of the central nervous system in at-risk newborns is crucial for successful early interventions1,2. Visual event-related potentials (VEPs) provide a useful means of evaluating visual cortical status because they do not require patient cooperation, which is not possible in the first month of life, are objective, and are sensitive to structural and functional brain damage3,4.
Though, some studies of newborns have shown that normal visual-evoked responses indicate adequate neural maturation of the cerebral cortex4,5, and that this has often been studied in newborns to assess neurodevelopment and identify abnormal development of the visual pathways4,5, the clinical use of VEPs has been limited by the variability observed in their morphology4,5,6,7. Therefore, it is important to obtain better, more reliable characterizations of VEPs in newborns.
One cause of the variability in VEP morphology is that earlier studies have mixed preterm and older babies (over one month)8,9,10. However, the most important source is the lack of attention paid to the infants&#39; behavioral state while recording VEPs; namely, awake, quiet (QS), active (AS), or transitional sleep. QS and AS have either not been analyzed separately5,11,12, or studies have relied exclusively on behavioral observation without using polysomnography to identify states7,8. Trac&#233; alternant, which consists in bursts of high amplitude slow activity alternating with inter-burst intervals of minimal amplitudes is present in QS, but has not been taken into account when averaging VEPs. Some studies with newborns have measured VEPs by recording during wakefulness13,14, but at this stage of development waking periods are brief and newborns are usually crying or moving, which makes it difficult to obtain high quality, reliable recordings.
Few studies have used light-emitting diodes (LEDs) googles6,9 to elicit VEPs, though this light source generates more consistent recordings than the usual strobe flashes of white light11,14,15, which are less reliable. Obtaining replicable VEPs in the same newborn is indispensable for clinical use4, but another cause of variability is the low reproducibility of VEP morphology, likely due to the lack of control of physiological states and of the stimuli used to elicit VEPs. Given these conditions, the high variability of VEP morphology is hardly surprising.
A previous study conducted with 20 healthy full-term newborns that considered several sources of variability: post-conceptional age, polysomnographically-identified sleep states, LED googles to elicit VEPs, and measures of reproducibility between two VEP averages found that a clearer, more reliable VEP morphology can be obtained during active sleep. During this sleep stage all infants generated clear VEPs with higher correlations between two averages than in QS. Also, fewer VEP averages were required to obtain reproducibility16.
Given the clinical usefulness of VEP studies to assess, as early as possible, the integrity of visual pathways, this study proposes a series of methodological steps designed to obtain reliable VEPs in preterm and older newborns, using LED goggles during AS unambiguously defined by simultaneous polysomnography.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digital Electroencephalograph</td>
			<td>Neuronic Mexicana, SA</td>
			<td>Medicid 3E</td>
			<td>Sleep electroencephalogram record</td>
		</tr>
		<tr>
			<td>Evoked Potentials equipment</td>
			<td>Neuronic Mexicana, SA</td>
			<td>Neuronic PE (N_N-SW-2.0)</td>
			<td>Visual evoked potentials record</td>
		</tr>
		<tr>
			<td>Nuprep Gel</td>
			<td colspan="2">WEAVER and Company</td>
			<td>Skin preparing abrasive gel (114 g)</td>
		</tr>
		<tr>
			<td>Ten20 Conductive Paste</td>
			<td colspan="2">WEAVER and Company</td>
			<td>Neurodiagnostic electrode paste (228 g)</td>
		</tr>
		<tr>
			<td>Tubular elastic mesh bandage</td>
			<td>Le Roy</td>
			<td></td>
			<td>Fixation of cranial surface electrodes, Size 4 or Small</td>
		</tr>
	</tbody>
</table>

how to obtain reliable visual event-related potentials in newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

To detect adequate maturation in the function of the visual pathway it is essential to obtain the PII component of the VEP, which can be seen in both term and preterm infants. The simultaneous recording of VEPs with polysomnography during AS makes it possible to obtain typical VEPs.
Reliable VEP studies require obtaining reproducible average waveforms that will be indispensable for clinical use. Figure 2</st...

Watch this Scientific Journal Video about How to Obtain Reliable Visual Event-related Potentials in Newborns at JoVE.com

How to Obtain Reliable Visual Event-related Potentials in Newborns

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable ...

how-to-obtain-reliable-visual-event-related-potentials-in-newborns

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

6.3K Views.  National Autonomous University of Mexico. Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

Video: How to Obtain Reliable Visual Event-related Potentials in Newborns

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 ...

How to Create and Use Binocular Rivalry

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent representation. However, when the eyes are presented with images that are sufficiently different from each other, an interesting thing happens: Rather than fusing the two images into a combined conscious percept, what transpires is a pattern of perceptual alternations where one image dominates awareness while the other is suppressed; dominance alternates between the two images, typically every few seconds. This perceptual phenomenon is known as binocular rivalry. Binocular rivalry is considered useful for studying perceptual selection and awareness in both human and animal models, because unchanging visual input to each eye leads to alternations in visual awareness and perception. To create a binocular rivalry stimulus, all that is necessary is to present each eye with a different image at the same perceived location. There are several ways of doing this, but newcomers to the field are often unsure which method would best suit their specific needs. The purpose of this article is to describe a number of inexpensive and straightforward ways to create and use binocular rivalry. We detail methods that do not require expensive specialized equipment and describe each method's advantages and disadvantages. The methods described include the use of red-blue goggles, mirror stereoscopes and prism goggles.

Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli.

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent ...

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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.

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 ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...

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 ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

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

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

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