The work was supported by NIH grants R15DC013864 and R21DC011342 (J. Irwin, PI). The parents and individuals that appear in the pictures gave permission for all pictures shown here to be used for publication purposes.

All procedures have been approved by the appropriate research ethics committee at Southern Connecticut State University and Yale University, with consent from participants.
1. Create a Social Narrative
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55941/55941fig1.jpg" /> 
Figure 1. Social Narrative. A sample social narrative, read to the participant prior to the first appointment and by the researcher prior to initiating the experiment and obtaining consent.
Note: Sections 1, 2, and 3 present the tools that were developed for the familiarization protocol.
<ol>
	<li>Create a social narrative that includes images and a short description of each step of the research protocol created by the researchers, starting with consent and ending with compensation (if applicable) and a short explanation. 
	NOTE: When possible, the image used should have high iconicity (iconicity refers to the degree to which a symbol represents its referent)3 to facilitate the understanding of the participant. Follow the steps for developing the social narrative (adapted from Myles, Trautman, and Schelvan)15.
	<ol>
		<li>In the social narrative, describe in sequential order the research activities from the participant&#39;s perspective. Write the story in first-person or second-person language and use either present or future tense.
		<ol>
			<li>In the description, include: a) where the activity will occur; b) what the participant will do; c) what the researchers will do; d) why the participant will complete each activity; and e) the possible thoughts or feelings of the researchers or the parents, if applicable (i.e.&#160;&#34;we&#39;re happy that you came;&#34; &#34;calm when you wait for directions;&#34; or &#34;Mom is happy that you can help.&#34;)</li>
		</ol>
		</li>
		<li>Select pictures that are iconic to use within the social narrative to increase the understanding of the participant (i.e., the building, the room, the EEG cap, and the researchers).</li>
		<li>Place the social narrative into a presentation software (e.g., PowerPoint), including the iconic pictures. Use the presentation mode to show the narrative to the child as a story on a computer, or present it as a printed version.</li>
	</ol>
	</li>
	<li>Send the social narrative to the parent/caregiver by mail or email before the participant comes to the research appointment.
	<ol>
		<li>Ask the parent or guardian to read the social narrative to the participant at least 3 times before the first visit of the research study.</li>
	</ol>
	</li>
</ol>
2. Create a Visual Schedule
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55941/55941fig2.jpg" /> 
Figure 2. Visual Schedule. A sample visual schedule, presented to the participant after obtaining consent and before initiating the first step of the experiment, as well as before and after the completion of each activity.
<ol>
	<li>Create a visual schedule that outlines each activity of the research protocol. Ensure that the visual schedule includes different cards, with pictures that represent each activity and a one- to three-word description printed under each picture (adapted from Hodgdon)10.
	<ol>
		<li>Within the visual schedule, include a card with a picture that represents a break, along with the written description, &#34;Take a break.&#34;</li>
		<li>Within the visual schedule, include a card with a picture that refers to being finished or all done; this card may include the word &#34;stop&#34; or &#34;finish&#34; printed in red font. 
		NOTE: The activities outlined in the visual schedule should be the same as those described in the social narrative. The written language and pictorial representations provided through these two tools should be consistent.</li>
	</ol>
	</li>
</ol>
3. Create Emotion-rating Scales
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55941/55941fig3.jpg" /> 
Figure 3. Self-rating Emotion Scale. A sample self-rating emotion scale, which is used by the participant to rate his/her state of emotion. The researcher uses a separate scale to rate the participant&#39;s emotion. <a href="http://cloudfront.jove.com/files/ftp_upload/55941/55941fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55941/55941fig4.jpg" /> 
Figure 4. Researcher&#39;s Rating Scale. A scale used to make sure that the participant&#39;s rating is consistent with outward observed behavior. This scale is to be completed by a researcher that is in the room with the child participant. <a href="http://cloudfront.jove.com/files/ftp_upload/55941/55941fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Create a scale that depicts a range of 5 emotions, with pictorial representations and/or written words. Select emotions that are relevant to supporting the participant with engaging in the activities in the given research protocol (i.e.&#160;emotions related to anxiety, pleasure, disgust, etc.). 
	NOTE: The emotion vocabulary and selected icons should match the developmental levels of the participant (adapted from Dunn-Buron and Curtis)7.
	<ol>
		<li>Include an action that the participant may complete to maintain positive emotion in one column and label it &#34;What I can do&#34; (see Figure 3).</li>
		<li>Include a smiley or picture of a face in a separate column and label it &#34;How I feel.&#34; 
		NOTE: All five emotions listed under the &#34;How I feel&#34; column should correspond with the &#34;What I can do&#34; column (i.e., on the &#34;How I feel&#34; column, &#34;I am confused and getting irritated&#34; would correspond with &#34;Ask to take a break&#34; under &#34;What I can do.&#34;)</li>
	</ol>
	</li>
	<li>Create a Likert-type scale for rating emotions to be used by the researcher (see Figure 4) (adapted from the Positive and Negative Affect Schedule (PANAS)21. 
	NOTE: Below are the steps to follow when implementing the familiarization tools over two visits to complete the research protocol.</li>
</ol>
4. Visit 1: Social, Cognitive, and Language Assessments
<ol>
	<li>Greet the participants and bring them to an area with various toys (see Figure 5) in a designated &#34;play area.&#34; Ensure that the participant is comfortable within the setting.</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55941/55941fig5.jpg" /> 
Figure 5. Welcoming the Participant to the Visit. Researcher playing with participants, familiarizing them to the environment and getting them accustomed to the context.
<ol start="2">
	<li>Show the participant the 5-point emotion-rating scale (see Figure 3) and ask them to point to the picture that best represents how they feel.</li>
	<li>Ask the participant how they feel and support the participant in saying &#34;I feel _____,&#34; while pointing to the corresponding emotion on the scale (see Figure 3).
	<ol>
		<li>If the participant selects a 1 or 2, move to step 4.4. If the participant selects a 3 or 4, ask the participant to follow the suggested action under the &#34;What I can do&#34; section (see Figure 3).</li>
		<li>Give the participant 5 min to follow the suggested action.</li>
		<li>After 5 min, ask the participant rate how they feel, again. If they select 1-3, continue the session. If they select 4 or 5, end the research appointment and ask the participant to decide to come back on a different day or not to come back at all. 
		NOTE: See step 3 for creating the emotion-rating scale. The researcher is to rate the participant&#39;s emotions on a separate scale at the same time (Figure 4) to ensure that the participant is appropriately rating their emotion state.</li>
	</ol>
	</li>
	<li>Sit next to the participant and read and review the social narrative with them (see Figure 1). Say &#34;we will look over all the games that we will be playing today; ask me questions if there is a game that you are not sure of.&#34;</li>
	<li>Following the review of the social narrative, assess the participant&#39;s emotional state and readiness to continue with the research by asking, &#34;How do you feel?&#34; while showing them the self-rating emotion scale (Figure 3).
	<ol>
		<li>At the same time, ask the research assistant to use a separate scale to rate the participant (see Figure 4) to ensure that some of the anxiety (if any) has been reduced.</li>
	</ol>
	</li>
	<li>Hand a consent form to the parent and read the assent form with the child participant (if they cannot read it independently).</li>
	<li>Ask the participant if they would like to continue and complete the activities described in the social narrative.</li>
	<li>Ask the participant to sign the consent form. 
	NOTE: The participant should only be asked to sign if they agree to participate. If the participant says no, offer to read the social narrative again to ensure that the participant understands the requests being made.</li>
	<li>Introduce the participant to the visual schedule, stating, &#34;These (pointing to visual schedule pictures) are all of the games that we will be playing today. At the end of each game, you can take down the card for that game.&#34;
	<ol>
		<li>Point to the &#34;Take a Break&#34; card and say, &#34;You can also use this card if you need to take a break;&#34; point to the &#34;STOP&#34; card and say &#34;or this card if you do not want to play any more games for today.&#34;</li>
	</ol>
	</li>
	<li>Arrange the schedule so that all social, cognitive, and language assessments are completed prior to the final activity.
	<ol>
		<li>Periodically, at the discretion of the researcher and based upon the attending behavior of the participant, implement a modified use of the Premack principle by stating, &#34;Remember, first (activity name), then you may take a break.&#34; 
		NOTE: This modified version of the Premack principle involved giving the child the preferred activity after the third attempt, even if the child did not complete the less preferred activity. Therefore, the participant is compensated for their time even when they do not complete the experiment.</li>
	</ol>
	</li>
	<li>Once the participant has completed half of the assessments, evaluate the participant&#39;s emotions using both emotion-rating scales (as described in step 4.5); ensure that the participant maintains a level 1 or 2 emotion rating prior to continuing activities.
	<ol>
		<li>If the participant is at a higher level on the self-rating emotion scale, follow the action on the scale under the &#34;What I can do&#34; section.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55941/55941fig6.jpg" /> 
Figure 6. Bear used as Sample for Wearing the Cap. Demonstration of the bear wearing the cap, allowing the participant to explore wearing the cap&#160;and holding the bear while it wears the cap, done before the participant is asked to wear the cap.
<ol start="12">
	<li>EEG data acquisition simulation.
	<ol>
		<li>Introduce the participant to a stuffed bear (or doll, or any other toy of choice), and model the use of the EEG cap (see Figure 6) on it. Read to the participant, the section in the social narrative that describes use of the cap&#160;to the participant.</li>
		<li>Show the participant how the cap is placed on the bear&#39;s head. Allow the participant to touch the cap and interact with it by placing it on the bear&#39;s head (if needed).</li>
		<li>Apply a modified version of the Premack principle by saying, &#34;First, try the&#160;cap. Then, you will be given a gift card.&#34; If the participant says no, suggest the &#34;First, then&#34; statement one more time. If the participant says yes, continue to step 4.12.4.
		<ol>
			<li>If the participant continues to say no, compensate the participant for their time (if applicable). If the participant agrees to wear the cap, continue to step 4.12.4.</li>
		</ol>
		</li>
		<li>With a tape measure, measure the participant&#39;s head circumference to determine the cap size. After this initial measurement, measure from a location on the back of the skull (inion) to a location between the eyebrows above the nose (nasion), and from the right to left ear (using the tragus). 
		NOTE: This is done to identify the scalp vertex reference point for the placement of the Cz electrode. These steps are designed to be as similar to the actual acquisition protocol, which uses this procedure to maximize similarity of electrode placement across the scalp across participants.
		<ol>
			<li>Mark the vertex reference point with a washable magic marker.</li>
			<li>Soak the appropriate-size cap&#160;in water with a potassium chloride solution and baby shampoo.</li>
		</ol>
		</li>
		<li>Place a towel around the participant&#39;s neck to prevent water from dripping on their clothing. Tell the participant to close their eyes while the cap&#160;is being placed on their head.</li>
		<li>Adjust the cap on the participants&#39; head so that it is comfortable for the participant (see Figure 7). Once the cap&#160;is placed on the head and adjusted, let the participant known that they can open their eyes.</li>
		<li>Have the participant practice wearing the cap and sit in front of a screen to watch a movie for at least 10 min (see Figure 8). If this step is completed successfully, invite the participant to come back for a second appointment. 
		NOTE: Participants had to wear the cap&#160;for approximately one hour during the actual research appointment. However, breaks were built into the experiment every 3 minutes.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55941/55941fig7.jpg" /> 
Figure 7. Placing the EEG Cap&#160;on the Participant. The researcher capping the participant to practice wearing the cap; this is the role-play portion of the protocol, when the participant wears the towel.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55941/55941fig8.jpg" /> 
Figure 8. Adjustment of the EEG Cap. The participant is wearing the cap&#160;while the researcher adjusts it to make sure that the participant is comfortable.
<ol start="13">
	<li>Following the final activity, assess the participant&#39;s emotions one last time (Figures 3 &#38; 4) to ensure that level-1 or -2 emotion has been maintained throughout the protocol and that wearing the cap did not increase anxiety and/or decrease the participant&#39;s level of comfort. 
	NOTE: If any anxiety is present, the researcher should assist the child in following the suggested actions in the self-rating emotion scale (Figure 3).</li>
</ol>
5. Visit 2: EEG Data Collection
Note: As an integral part of the familiarization protocol, throughout the span of the experiment, a research assistant should code the participant&#39;s state (i.e.&#160;whether they alert or appears anxious; see Figure 4), and participants should provide a self-rating of their emotional state by pointing to a visual representation on an emotion scale (Figure 3).
<ol>
	<li>Present the participant with a social narrative that describes the steps of the second appointment. Ask the participant if they are willing to wear the cap and mention, &#34;like you did last time;&#34; (see step 1.1.1 for creating the social narrative). 
	NOTE: If the participant says yes, proceed to step 5.2. If the participant says no, repeat step 5.1 with the parent in the room. If the participant continues to say no, end the experiment.</li>
	<li>Present the participant with a visual schedule that includes all the EEG data collection procedures (i.e., measuring the head, wearing a towel, and placing the cap&#160;on the head). 
	NOTE: See step 2.1 for creating a visual schedule; a picture depicting a break is included. Periodically offer the participant the opportunity to take a break to maintain comfort and compliance.
	<ol>
		<li>Measure the participant&#39;s head circumference and then identify and mark the vertex reference point for the Cz electrode, following the steps outlined in step 4.12, above.
		<ol>
			<li>Mark the vertex reference point with a washable magic marker.</li>
			<li>Soak the cap&#160;in water with a potassium chloride solution and baby shampoo.</li>
		</ol>
		</li>
		<li>Place a towel around the participant&#39;s neck to avoid water from dripping on their clothing. Tell the participant to close their eyes while the cap&#160;is being placed on their head.</li>
		<li>Position the appropriate-size cap on the participant and ask them to sit in a comfortable position.</li>
		<li>Once the ERP waves can be seen on a computer screen, ask the participant to blink and move their hands and feet. Then, have the child look at the distortion that is created in the ERP waves when they move.</li>
	</ol>
	</li>
	<li>Start the stimulus presentation and ask that the participant sit very still, &#34;like a statue;&#34; point to a visual sign that shows a child sitting still, along with the text, &#34;sit still, like a statue.&#34; Use the Premack principle as needed. 
	NOTE: See step 4.9 for the use of the Premack principle.</li>
	<li>After completing the EEG portion of the experiment, provide the participant with the promised compensation and thank them for their participation.</li>
</ol>

<ol>
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The authors have no disclosures to report.

The familiarization protocol outlined above applies EBPs&#160;to increase the likelihood that child participants with ASD will be able to complete multiple testing sessions and EEG data collection. Prior to the implementation of the steps in this familiarization process, participants were unable to complete all components of the EEG research acquisition protocol. Preliminary data from this study show that all participants who engage in the familiarization protocol were able to complete the full EEG data collection.
The critical steps that were implemented include the provision of a social narrative and visual schedule, the use of the Premack principle (reinforcement) and modeling/role playing, and the desensitization to the EEG cap. Social narrative and the visual schedule make the protocol expectations and steps clear to the child, from the beginning of study enrollment to the completion of the study. Reinforcement helps the child persist through the tasks and provides a sense of mastery. Role playing and desensitization allow the child to try out unfamiliar equipment and novel sensory experiences. Finally, through the use of emotion-rating measures, the researcher ensures ongoing consent while engaging child participants in assessments and challenging experimental tasks.
Given the tools used for this protocol, a number of modifications can be made to accommodate for the varied backgrounds of the participants, relating to chronological age, mental age, and level of communicative competence. To account for a wide range of emotions that may be experienced during the research protocol, investigators may utilize the researcher rating scale to adapt the self-rating scale used by the participant. The language of the social narrative and the language of the visual schedule should align with the desired research schedule and match the cognitive and/or chronological level of the participants. Changes to these tools can include adding higher-level vocabulary for older participants or images with higher iconicity for younger participants. The activities outlined in either the visual schedule or social narrative can be changed, if necessary. For example, if participants are required to sit still for long periods of time, practicing sitting still, in the form of a game, may be beneficial. The protocol can also be modified to include a toy reward at some point in the experiment, as young children may find this more rewarding than money. Lastly, parents can be asked to bring a preferred toy of the child, to be used as a reward and to apply the Premack principle. However, when using a preferred toy, researchers must be cautious that the toy does not become a distraction during the experiment.
A potential limitation of the familiarization protocol is that it is constrained by the understanding of the language embedded in the procedure, which may limit its use for individuals with very poor receptive language skills. The quality of implementation of the described tools (i.e.&#160;social narratives) can impact the accuracy or fidelity of the familiarization protocol. Finally, this paradigm uses a soft-sponge electrode cap, which is designed to be child-friendly; as such, the results of 100% participation after familiarization may not generalize fully to protocols that use gel-based EEG data collection systems, which require scalp abrasion, or other data collection methods that entail greater physical or emotional discomfort.
While children with ASD are highlighted here as the population of study in this project, these principles can be used with young children in general, including those with other developmental disabilities, as well as those with typical development. Furthermore, although this procedure was designed for combined behavioral and EEG data collection, it could be easily adapted for use in other experimental protocols that have multiple visits and methodologies and that require participants to adapt to different contexts. For example, social narratives, visual schedules, and emotion-rating scales could be included, along with mock scanning sessions, in combined behavioral and MRI protocols. Furthermore, these elements, in addition to the cap familiarization procedure, could be augmented to allow for familiarization with Near-Infrared Spectroscopy (NIRS) probe placement.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by a constellation of social communicative difficulties and restricted, repetitive patterns of behavior1,2. As the number of identified individuals who meet the criteria for ASD increases, so does the desire to better understand the neurological underpinnings associated with ASD. Experts have suggested that future research should include the development of processes for the early detection of atypical brain function, which may then lead to interventions to support the brain systems hypothesized to impact social behavior14. In particular, this research uses temporally sensitive neuroimaging approaches- namely, electroencephalography (EEG) and Event-Related Potentials (ERPs)- to assess Audio-Visual (AV) speech perception in children with typical development and those at-risk for ASD and other developmental disabilities. The use of electrophysiology techniques, both EEG and ERP, will provide a better understanding of the neural bases for both typical and atypical AV speech perception across development. This research approach includes the analysis of both continuous EEG data and ERPs. Because ERPs are time-locked, multi-trial-averaged portions of the EEG, this paper will discuss procedures for the collection of EEG data.
To ensure that this work is as representative of the population as possible, individuals with ASD who present with varying degrees of social, cognitive, and language ability are included as participants. At present, the systems available for the collection of high-quality EEG data make significant demands on children with developmental disabilities. Examples of behaviors that individuals with ASD may have difficulty with include adapting to novel situations, tolerating uncomfortable sensory stimuli (i.e.&#160;wearing an EEG cap), and sitting quietly. These difficulties may be linked to the individual&#39;s sensory processing challenges, his/her disconnect in social perspective-taking, potential cognitive difficulties, and insistence on sameness. Collectively, these characteristics may impact a child&#39;s ability to comply with the required tasks that comprise the research activity1. Therefore, it is important to develop research protocols that support the children&#39;s strengths and accommodate their limitations to facilitate the participation of children with ASD. The goal is to assist children with ASD in completing research activities, without duress and in a manner that leads to useable neurobiological data.
The current work is part of a larger study on eye gaze and neural response to audiovisual speech in children with language and social disabilities, including children with ASD11,12. The primary experimental protocol involves multiple behavioral assessment sessions, as well as one simulated and one real EEG data collection session. Details of the EEG and ERP processing and analysis will not be discussed here; see Harwood et al.9 for the ERP data processing protocols.
The focus of this paper is a familiarization protocol designed to address the challenges of getting children (particularly those with developmental disabilities such as ASD) to engage in neurobiological research. As in many research studies of this type, participants must complete a variety of standardized assessment tasks in addition to participating in EEG data collection. Due to the challenges that individuals with ASD face when coping with novel environments, tolerating uncomfortable sensory stimuli, and sitting still for extended periods of time, strategies must be developed to increase participant understanding of the research activities. Increased understanding and agency will increase the participant&#39;s ability to successfully complete all research activities. Importantly, this protocol provides multiple opportunities for each participant to make an active decision regarding his/her participation, with a clear ability to opt out and, by monitoring the emotional state throughout the experiment, continuing consent is&#160;ensured4.
The National Professional Development Center on Autism and the National Autism Center are two organizations that have developed clear criteria for the establishment of evidence-based practices for individuals with ASD16,22. The protocol for the current research project used these criteria to identify, select, develop, and implement specific interventions that are applicable to addressing the challenges individuals may face in successfully participating in the research activities. The specific interventions from these sources include social narratives, visual supports in the form of a visual schedule, role-playing, and modeling22. In addition, the protocol includes an application of the Premack principle18. The Premack principle involves following a low-preference activity with a high-preference activity13,18, providing a short, clear connection between the expected behaviors of the individual to the reward. For example, if a child answers questions for 10 min, he/she is then able to play with a preferred toy for 10 min.
Prior research suggests that social narratives are an effective intervention for children with ASD who exhibit disruptive behaviors19. Social narratives provide background and information about the context of a situation as a means of providing increased understanding related to social expectations. Most social narratives include expected activities, possible perspectives of others involved, and ways to interact with other people during structured activities (see Figure 1 for an example of the social narrative used here). Social narratives have been shown to reduce anxiety and support an individual with expectations in the new situation15.
A visual schedule can be effective in helping children with ASD to cope in novel social situations20. Visual supports in the frame of a schedule provide individuals with ASD a static reference to aid in predicting upcoming activities (see Figure 2 for an example of the visual schedule used here). In addition, the removal of activities as they occur supports an understanding of the passage of time and provides a concrete visual representation of the session. Without this visual representation, individuals with ASD may become anxious if they are unable to conceptualize how much time has passed and how much time is left in an activity or session. A visual support that allows the participant to express his or her emotions may also be useful in allowing the participant to more easily cope with different emotions (see Figure 3 for an example of an emotion-rating scale).
Role-playing and modeling provide an opportunity to view and practice new situations for individuals with ASD. The process of rehearsal provides clarity about the expected behaviors and further reduces anxiety for these novel scenarios8,22. Role-playing, including modeling, provides the individual with a clear opportunity to practice and make a decision regarding his/her choice of participating or opting out of a particular activity (see Figures 5 &#38; 6 for examples of role play and modeling with a toy bear and the child wearing the cap).
Some individuals with ASD and/or other developmental disabilities may not have developed the maturity or cognitive capability to clearly understand research tasks. Indeed, there is an ethical dilemma surrounding the participation of vulnerable groups due to their difficulties with comprehension in complex social situations and the impact this has on informed consent17. However, processes have been developed to better ensure informed consent without undue pressure4,17. For example, visual symbols and bullet points with simple explanations are provided here, and participants are asked if they agree to participate in the study as an ongoing process3. To facilitate the understanding of individuals with developmental disabilities, a protocol must be developed that both aligns with study goals yet adheres to ethical guidelines, including the subject choosing to participate without persuasion or undo pressure6.

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a familiarization protocol facilitates the participation of children with asd in electrophysiological research

This paper includes a detailed description of a familiarization protocol, which is used as an integral component of a larger research protocol to collect electroencephalography (EEG) data and Event-Related Potentials (ERPs). At present, the systems available for the collection of high-quality EEG/ERP data make significant demands on children with developmental disabilities, such as those with an Autism Spectrum Disorder (ASD). Children with ASD may have difficulty adapting to novel situations, tolerating uncomfortable sensory stimuli, and sitting quietly. This familiarization protocol uses Evidence-Based Practices (EBPs) to increase research participants&#39; knowledge and understanding of the specific activities and steps of the research protocol. The tools in this familiarization protocol are a social narrative, a visual schedule, the Premack principle, role-playing, and modeling. The goal of this familiarization protocol is to increase understanding and agency and to potentially reduce anxiety for child participants, resulting in a greater likelihood of the successful completion of the research protocol for the collection of EEG/ERP data.

As part of a series of studies on eye gaze and audiovisual speech perception, an initial cohort of 25 participants with ASD and average IQs (n = 25, 19 boys, 6 girls, mean age = 10.25 years) was recruited using a similar EEG protocol (Figure 9). 72% of the children in this previous cohort completed the EEG protocol. In the current cohort, thus far 15 participants with ASD were recruited &#160;(n = 15, 11 boys, 4 girls, mean age = 9.4 years), 12 of whom participated in the familiarization protocol. Of the 12 that completed the familiarization protocol, 100% completed the full EEG acquisition protocol (Figure 9).
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55941/55941fig9v2.jpg" /> 
Figure 9. Completion of EEG. Representation of the number of participants that completed the experiment, with and without the use of the familiarization protocol. <a href="http://cloudflare.jove.com/files/ftp_upload/55941/55941fig9v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Familiarization Protocol Facilitates the Participation of Children with ASD in Electrophysiological Research

Electrophysiological research is an important tool for identifying biomarkers of developmental disorders, including Autism Spectrum Disorders (ASD), but data collection in these populations remains challenging. This work presents a familiarization protocol to accompany research that includes electroencephalography (EEG) to improve the likelihood of collecting EEG data from children with ASD.

This paper includes a detailed description of a familiarization protocol, which is used as an integral component of a larger research protocol to collect ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.2K Views.  Southern Connecticut State University. Electrophysiological research is an important tool for identifying biomarkers of developmental disorders, including Autism Spectrum Disorders (ASD), but data collection in these populations remains challenging. This work presents a familiarization protocol to accompany research that includes electroencephalography (EEG) to improve the likelihood of collecting EEG data from children with ASD.

Video: A Familiarization Protocol Facilitates the Participation of Children with ASD in Electrophysiological Research

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...