This research was supported by the University of Texas at Austin Moody College of Communication Grant Preparation Award and the University of Texas at Austin Office of the Vice President of Research Special Research Grant.

All methods described here have been approved by the Institutional Review Board (IRB) for Human Research at the University of Texas at Austin.
1. Stimuli Characteristics
<ol>
	<li>Create object stimuli using open source images available through the Bank of Standardized Stimuli (BOSS). This database consists of standardized images used throughout visual cognitive experiments.&#160; Download four images (e.g., ball02, book01a, brick, button03) with a high rate of identification (above 75%)20,21.&#160;&#160;</li>
	<li>Create motion stimuli using a modified version of the DotDemo script, which is available through the open source Psychtoolbox-3 set of functions operated via MATLAB, as well as the movie function available in MATLAB (see the Supplementary File).
	<ol>
		<li>Configure the dot field parameters according to the size of the presentation screen and viewing distance.&#160;&#160;</li>
		<li>Enter 3600 for the number of movie frames.&#160;</li>
		<li>Enter 80 (in cm) for monitor width.</li>
		<li>Enter dot speed at 5&#176;/s.</li>
		<li>Enter a dot limited lifetime fraction of 0.05.</li>
		<li>Enter 200 for the number of dots.</li>
		<li>Enter the minimum radius of the field annulus as 1&#176; and the maximum as 15&#176;.&#160;&#160;</li>
		<li>Enter 0.2&#176; for the width of each dot.</li>
		<li>Enter 0.35&#176; for the radius of the fixation point.</li>
		<li>Specify that white dots are used on a black background.</li>
		<li>Export the movie in .avi format.</li>
	</ol>
	</li>
</ol>
2. Visual Paradigm Design
<ol>
	<li>Create paradigms via stimulus-presentation software. Generate fixation crosses with Courier New size 18 font, bold, and centered on the presentation screen.</li>
	<li>Design the visual object paradigm without temporal jitter (i.e., consistent ISI values) by creating a black fixation cross on a white background presented for 500 ms, followed by one of four objects presented in randomized order: ball, book, brick, or button.&#160;&#160;
	<ol>
		<li>Present each object for 600 ms (Figure 1A).&#160; Show all objects 75 times, for a total of 300 trials and a paradigm duration of 5.5 min.&#160;&#160;</li>
	</ol>
	</li>
	<li>Design the visual object paradigm with temporal jitter to consist of the same black fixation cross on a white background, shown for a period of 500 or 1,000 ms and followed by one of the four objects, lasting for 600 or 1000 ms (Figure 1B).&#160;&#160;
	<ol>
		<li>Create four trials using stimulus-presentation software: a fixation cross with a duration of 500 ms, followed by an object for 600 ms; a fixation cross with a duration of 500 ms, followed by an object for 1,000 ms; a fixation cross with a duration of 1,000 ms, followed by an object for 600 ms; and a fixation cross with a duration of 1,000 ms followed by an object for 1,000 ms.&#160;&#160;
		<ol>
			<li>Randomize these trials. Present each trial 19 times, culminating in 304 trials and resulting in a viewing time of approximately 7.85 minutes.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Create the visual motion paradigm without temporal jitter by generating a white fixation cross centered on a black background, lasting for 500 ms, followed by the visual motion movie, which is truncated to present for approximately 1,000 ms (Figure 2A).&#160;
	<ol>
		<li>Repeat this sequence a total of 300 times, for a viewing duration of approximately 7.5 min.&#160;&#160;</li>
	</ol>
	</li>
	<li>Create the visual motion paradigm with temporal jitter using the same fixation cross, lasting for intervals of 500, 750, or 1,000 ms.&#160;&#160;
	<ol>
		<li>After each fixation cross, present the visual motion movie with a duration of approximately 600 or 1,000 ms (Figure 2B).&#160;&#160;</li>
		<li>Create six trials: A fixation cross with a duration of 500 ms, followed by a movie for 600 ms, a fixation cross with a duration of 750 ms, followed by a movie for 600 ms, a fixation cross with a duration of 1,000 ms, followed by a movie for 600 ms, a fixation cross with a duration of 500 ms followed by a movie for 1000 ms, a fixation cross with a duration of 750 ms followed by a movie for 1,000 ms and a fixation cross with a duration of 1,000 ms followed by a movie for 1,000 ms.&#160;&#160;
		<ol>
			<li>Randomize these trials, with each shown 50 times.&#160; Present a total of 300 trials, for a viewing period of approximately 7.75 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Participant Consent, Case History, and Vision Screening
<ol>
	<li>Greet the participant on arrival. Obtain informed consent by presenting the participant with a consent for participation in research form. Explain the consent form to the participant and answer any questions that arise.</li>
	<li>Have the participant fill out a case history form that includes information on native language, handedness, hearing status, vision status, and other diagnoses the participant may have (e.g., psychological and neurological). Exclude participants who report hearing loss and/or neurological diagnoses, such as traumatic brain injury.&#160; Include all other participants.</li>
	<li>Escort the participant out of the lab to complete a vision screening using a Snellen chart to determine visual acuity. Have the participant stand 20 feet away from the chart and begin by covering his or her left eye to determine right eye visual acuity, and then switch eyes to determine left eye visual acuity. Calculate visual acuity based on the smallest line of text that the participant can repeat at least one more than half of the total number of letters.&#160; 
	NOTE: For example, if the participant repeats 5 of the 8 letters on the 20/20 line, visual acuity is calculated as 20/20 in that eye.</li>
	<li>Escort the participant into the EEG recording room. Have the participant sit in the designated chair in the center of a double-walled magnetic-shielded soundproof booth.&#160;&#160;</li>
</ol>
4. EEG Preparation
<ol>
	<li>Measure the head circumference of the participant in centimeters and select the appropriate EEG net size. Measure and mark the midpoint of the scalp (midway between nasion/inion and right and left mastoids) for the placement of the reference electrode.</li>
	<li>Prepare a solution of warm water (1 L) mixed with baby shampoo (5 mL) and potassium chloride (11 g/10 cc), which increases the electrical conductance between the electrodes and scalp, leading to lower voltage impedances and an increased signal-to-noise ratio.</li>
	<li>Place the EEG net in the solution. Allow the net to soak in the solution for 5 min before placing on the participant&#8217;s scalp.</li>
	<li>Turn on the stimulus-presentation computer and the EEG acquisition computer.</li>
	<li>Place a towel or other absorbent material around the participant&#8217;s neck to prevent the solution from dripping onto his or her clothes.&#160;</li>
	<li>Connect the EEG net to the amplifier. Instruct the participant to close his or her eyes when putting on the EEG net to prevent the solution from dripping into his or her eyes.</li>
	<li>Firmly grip the EEG net with both hands and spread into place onto the participant&#8217;s head. Ensure that the net is placed symmetrically on the scalp head, with the reference electrode at the scalp midline point that was measured. Tighten the chin and ocular net lines to ensure a secure connection between the scalp and electrodes. Ask the participant if he or she is comfortable and if anything needs to be adjusted.</li>
	<li>Check for the proper electrode impedance values, with an average target of 10 k&#937;.</li>
	<li>To reduce impedance values following the placement of the electrode net, use a 1 mL pipette to apply the potassium chloride solution onto the scalp/electrodes that have a high impedance. Continue this process until adequate impedances values across the electrodes are achieved.&#160;</li>
</ol>
5. EEG Recording
<ol>
	<li>Instruct the participant to focus on the visual stimuli that will appear on the monitor. The viewing distance is approximately 65 inches.</li>
	<li>Use a pseudorandom number generator to determine the order of presentation for the four visual paradigms.&#160;</li>
	<li>Begin the visual tasks and EEG recording.</li>
	<li>Monitor the EEG recording as necessary. If ongoing EEG shows high myogenic or 60 Hz activity, pause the experiment to recheck electrode-scalp connectivity.</li>
	<li>Repeat steps 5.3 and 5.4 for the visual object paradigm, the visual object with the temporal jitter paradigm, the visual motion paradigm, and the visual motion with temporal jitter paradigm.</li>
	<li>At the conclusion of the experiment, instruct the participant to close his or her eyes in order to prevent the solution from entering his or her eyes when removing the net. Begin by loosening the chin and ocular net lines, then remove the net by gently pulling the chin strap up and over the participant&#8217;s head, making sure to pull slowly to ensure the net will not get tangled in the participant&#8217;s hair.&#160;</li>
	<li>Disconnect the EEG net from the amplifier. Begin the disinfection process by placing the EEG cap in and out of a bucket filled with water and rinsing under a faucet. Then, create the disinfectant solution by adding approximately 2 l of water to the disinfectant bucket and mixing 15 ml of disinfectant with the water.&#160;</li>
	<li>Immerse the sensor end of the net in the disinfectant. Set a timer for 10 min; for the first 2 min, continuously plunge the net up and down. Leave the net soaking for the remainder of the 10 min.</li>
	<li>Remove EEG net from disinfectant solution. Place the EEG net in and out of the electrode bucket filled with water and under running water to rinse. Repeat four times.&#160; Allow the net to air dry.&#160;</li>
</ol>
6. EEG Analyses
<ol>
	<li>Export EEG files for analyses in MATLAB via the EEGLAB toolbox using a 1 Hz high-pass filter, segmentation around each trial (or event) of 100 ms pre-stimulus and 500 ms post-stimulus periods.&#160;&#160;</li>
	<li>Import data using the EEGLAB toolbox.&#160;&#160;
	<ol>
		<li>Choose the File option from the drop-down menu and click on Import data.&#160; Select using EEGLAB Functions and plugins from the menu.&#160; Next click on the appropriate export file format.</li>
	</ol>
	</li>
	<li>Re-assign channel locations based on the type of electrode montage used by choosing Edit from the drop-down menu and selecting Channel locations.&#160; Click on Look up locs and select the ellipses to locate the path of the electrode montage file of interest.&#160;</li>
	<li>Assign pre- and post-stimulus times to the epoch start and end times. Enter a value of -0.1 s in the Start time box.&#160;</li>
	<li>Baseline-correct data according to the pre-stimulus interval.</li>
	<li>Identify and remove bad channels using probability at a Z-score threshold of 2.5.&#160;
	<ol>
		<li>Verify successful identification and removal of bad channels by plotting all electrodes.&#160;Manually remove channels with mean voltage amplitudes outside of the range of +/- 30 &#181;V.</li>
	</ol>
	</li>
	<li>Perform artifact rejection by entering values of -100 &#181;V and +100 &#181;V. 
	NOTE: This method is effective in the removal of ocular activity recorded at ocular electrodes (126, 127). However, it may be necessary to manually remove trials with artifact occurring at small-voltage amplitude (i.e., within the +/- 100 &#181;V range) for certain participants.&#160;
	<ol>
		<li>Take note of channels that were bad for entire segments (i.e., with voltages outside of the +/-100 &#181;V range) and highlighted in red. Manually remove these bad channels if they constitute 60% or more of the rejected trials. Repeat this step as many times as necessary.&#160;</li>
		<li>Follow artifact removal steps as previously described. Ensure that a minimum of 100 sweeps are accepted. Remove trials marked for rejection.</li>
	</ol>
	</li>
	<li>Plot channel 75 (equivalent to Oz), or the channel(s) of interest, to categorize morphological patterns. Prior to plotting this channel, make sure to perform pre-stimulus baseline correction.&#160;&#160;</li>
	<li>Choose pattern A if CVEP morphology is characterized by a large positive peak at approximately 100-115 ms (P1), followed by a negative peak at approximately 140-180 ms (N1) and a positive peak at approximately 165-240 ms (P2).&#160;&#160;</li>
	<li>Choose pattern B if CVEP morphology is characterized by a large positive peak at approximately 100-115 ms (P1), followed by a negative peak at approximately 140-180 ms (N1a), a positive peak at approximately 180-240 ms (P2a), then a negative peak at approximately 230-280 ms (N1b) and positive peak at approximately 260-350 ms (P2b).&#160; &#160;&#160;</li>
	<li>Append individual datasets together according to the morphological pattern visually observed to create a group average. Name and save the newly merged dataset file.&#160;&#160;</li>
	<li>View appended files as an average by plotting the channel(s) of interest.</li>
</ol>

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The authors have nothing to disclose.

The goal of this methodological report was to evaluate the feasibility in recording differential CVEP morphology by using visual object and motion stimuli specifically designed to separately stimulate ventral and dorsal streams in passive viewing tasks6,7,8, both with and without variation of ISIs (jitter)19. Conditions were not designed to be directly compared, rather, observations were made as to whethe...

Electroencephalography (EEG) is a tool that offers an inexpensive and non-invasive approach to the study of cortical processing, especially when compared to cortical assessment methods such as functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and diffusion tensor imaging (DTI)1. EEG also provides high temporal resolution, which is not possible to attain when using measures such as fMRI, PET, or DTI2. High temporal resolution is critical when examining central temporal function in order to obtain millisecond-precision of neurophysiologic mechanisms related to the processing of specific input or events.&#160; In the central visual system, cortical visual evoked potentials (CVEPs) are a popular approach in studying time-locked neural processes in the cerebral cortex.&#160; CVEP responses are recorded and averaged over a number of event trials, resulting in peak components (e.g., P1, N1, P2) arising at specific millisecond intervals. The timing and amplitude of these peak neural responses can provide information concerning cortical processing speed and maturation, as well as deficits in cortical function3,4,5.&#160;&#160;
CVEPs are specific to the type of visual input presented to the viewer. Using certain stimuli in a CVEP paradigm, it is possible to observe the function of distinct visual networks such as the ventral stream, involved in processing form and color, or parvocellular and magnocellular input6,7,8, and the dorsal stream, which largely processes motion or magnocellular input9,10. CVEPs generated by these networks have been useful not only in better understanding typical neurophysiologic mechanisms underlying behavior but also in the targeted treatment of atypical behaviors in clinical populations. For example, delayed CVEP components in both dorsal and ventral networks have been reported in children with dyslexia, which suggests that visual function in both these networks should be targeted when designing an intervention plan11.&#160; Thus, CVEPs recorded via EEG offer a powerful clinical tool through which to assess both typical and atypical visual processes.&#160;
In a recent study, high-density EEG was used to measure the apparent motion-onset CVEPs in typically developing children, with the goal of examining variable CVEP responses and related visual cortical generators across development. Participants passively viewed apparent motion stimuli12,13,14,15, which consisted of both shape&#160;change and motion,&#160;designed to simultaneously stimulate dorsal and ventral streams. It was found that approximately half of the children responded with a CVEP waveform shape, or morphology, consisting of three peaks (P1-N1-P2, pattern A).&#160; This morphology is a classic CVEP response observed throughout the literature. In contrast, the other half of the children presented with a morphological pattern comprised of five peaks (P1-N1a-P2a-N1b-P2b, pattern B). To our knowledge, the robust occurrence and comparison of these morphological patterns have not previously been discussed in CVEP literature in either child or adult populations, although variable morphology has been noted in both apparent-motion and motion-onset CVEPs14,16. Furthermore, these morphological differences would not have been apparent in research using other cortical functional assessment methods, such as fMRI or PET, due to the low temporal resolution of these measures.
To determine the cortical generators of each peak in CVEP patterns A and B, source localization analyses were performed, which is a statistical approach used to estimate the most likely cortical regions involved in the CVEP response12,13. For each peak, regardless of the morphological pattern, primary and higher-order visual cortices were identified as sources of the CVEP signal.&#160; Thus, it appears that the main difference underlying CVEP morphology elicited by apparent motion is that those with pattern B activate visual cortical regions additional times during processing. Because these types of patterns have not been previously identified in the literature, the purpose of the additional visual processing in those with CVEP pattern B remains unclear.&#160; Therefore, the next aim in this line of research is to gain a better understanding of the cause of the differential CVEP morphology and whether such patterns may relate to visual behavior in both typical and clinical populations.&#160;
The first step in understanding why some individuals might demonstrate one CVEP morphology versus another is to determine whether these responses are intrinsic or extrinsic in nature.&#160; In other words, if an individual demonstrates one pattern in response to a visual stimulus, will they respond with a similar pattern to all stimuli?&#160; Or is this response stimulus-dependent, specific to the visual network or networks activated?
To answer this question, two passive visual paradigms were designed, intended to separately activate specific visual networks. The stimulus presented in the initial study was designed to stimulate both dorsal and ventral streams simultaneously; thus, it was unknown if one or both networks were involved in generating specific waveform morphology. In the current methodological approach, the paradigm designed to stimulate the ventral stream is composed of highly identifiable objects in basic shapes of squares and circles, eliciting object-onset CVEPs. The paradigm designed to stimulate the dorsal stream consists of visual motion via a radial field of coherent central dot motion dots at a fixed speed toward a fixation point, eliciting motion-onset CVEPs.&#160;
A second question that arose as a result of the initial study was whether differential VEP morphology could be due to participant anticipation of upcoming stimuli13. For instance, research has shown that top-down cortical oscillatory activity occurring prior to a target stimulus may predict subsequent CVEP and behavioral responses to some degree17,18,19. The apparent motion paradigm in the first study employed non-randomized frames of a radial star and circle with consistent inter-stimulus intervals (ISIs) of 600 ms. This design may have encouraged the expectation and prediction of the upcoming stimulus, with resulting oscillatory activity affecting subsequent CVEP morphology12,13,19.&#160;&#160;
To address this issue, the visual object and motion paradigms in the current protocol are designed with both consistent ISIs of the same temporal value and randomized ISIs with different temporal values (i.e., jitter).&#160; Using this approach, it may be possible to determine how temporal variation can affect VEP morphology within distinct visual networks. Altogether, the aim of the described protocol is to determine if the visual object and motion stimuli would be sensitive to variations in CVEP morphology and whether the temporal variation of stimuli presentation would affect characteristics of the CVEP response, including peak latency, amplitude, and morphology. For the purpose of the current paper, the goal is to determine the feasibility of the methodological approach. It is hypothesized that both visual objects and motion may elicit variable morphology (i.e., patterns A and B will be observed across subjects in response to both stimuli) and that temporal variation would affect object-onset and motion-onset CVEP components.&#160;

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			<td height="42" style="height:42px;width:216px;">Control III Disinfectant Germicide</td>
			<td style="width:240px;">Control III</td>
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			<td height="63" style="height:63px;width:216px;">EEGlab v14.1.2</td>
			<td style="width:240px;">Swartz Center for Computational Neuroscience, University of California, San Diego</td>
			<td style="width:269px;">https://sccn.ucsd.edu/eeglab/index.php</td>
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			<td>Used in generation of visual object stimuli&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Psychtoolbox-3</td>
			<td>Psychophysics Toolbox Version 3 (PTB-3)</td>
			<td style="width:269px;">http://psychtoolbox.org/</td>
			<td>Used in generation of visual motion stimuli</td>
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stimulus-specific cortical visual evoked potential morphological patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

Figure 3 and Figure 4 show the representative object-onset and motion-onset CVEP results of five participants, aged 19-24 years, who passively viewed each visual paradigm. This design allowed observation of CVEP responses elicited by visual objects (with and without jitter) and visual motion (with and without jitter) both within and across subjects according to each condition.&#160; Participant CVEPs were grouped according to the morphological pattern elicited b...

Watch this Scientific Journal Video about Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns at JoVE.com

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

stimulus-specific-cortical-visual-evoked-potential-morphological

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

5.9K Views.  The University of Texas at Austin. In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.  

Video: Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

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 ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

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 ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

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Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

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

Subtype-selective Electroporation of Cortical Interneurons

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

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

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor