Name: a familiarization protocol facilitates the participation of children with asd in electrophysiological research
Uploaded: 2017-07-31T13:00:00
Description: Watch this Scientific Journal Video about A Familiarization Protocol Facilitates the Participation of Children with ASD in Electrophysiological Research at JoVE.com

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 pr...

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L., Grela, B., Harold, O., Andrada, K., Landi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+correlates+of+speech+perception+in+toddlers:+Associations+among+ERPs,+nonword+repetition,+and+language.">Electrophysiological correlates of speech perception in toddlers: Associations among ERPs, nonword repetition, and language.</a> J Speech Lang Hearing Res. , (1995).</li><li>Hodgdon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Visual strategies for improving communication. , (1995).</li><li>Irwin, J., Avery, T., Brancazio, L., Turcios, J., Ryherd, K., Landi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Indices+of+Audiovisual+Speech+Perception:+Beyond+the+McGurk+Effect+and+Speech+in+Noise.">Electrophysiological Indices of Audiovisual Speech Perception: Beyond the McGurk Effect and Speech in Noise.</a> Multisens Res. , .</li><li>Irwin, J. R., Tornatore, L. A., Brancazio, L., Whalen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+children+with+autism+spectrum+disorders+"hear"+a+speaking+face.">Can children with autism spectrum disorders "hear" a speaking face.</a> Child Dev. 82 (5), 1397-1403 (2011).</li><li>Mancil, G. R., Pearl, C. 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M., Edwards, R. P., Rabian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreasing+disruptive+behaviors+of+children+with+autism+using+social+stories.">Decreasing disruptive behaviors of children with autism using social stories.</a> J Autism Dev Disord. 3 (26), 535-543 (2002).</li><li>Thiemann, K. S., Goldstein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+stories,+written+text+cues,+and+video+feedback;+Effects+on+social+communication+of+children+with+autism.">Social stories, written text cues, and video feedback; Effects on social communication of children with autism.</a> J Appl Behav Anal. 34 (4), 425-446 (2001).</li><li>Watson, D., Clark, L. A., Tellegen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+brief+measures+of+positive+and+negative+affect:+The+PANAS+scales.">Development and validation of brief measures of positive and negative affect: The PANAS scales.</a> J Pers Soc Psychol. 4 (6), 1063-1070 (1988).</li><li>Wong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+practices+for+children,+youth+and+young+adults+with+Autism+Spectrum+Disorders:+a+comprehensive+review.">Evidence-based practices for children, youth and young adults with Autism Spectrum Disorders: a comprehensive review.</a> J Autism Dev Disord. 45 (7), (2013).</li></ol>

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.
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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...

Watch this Scientific Journal Video about A Familiarization Protocol Facilitates the Participation of Children with ASD in Electrophysiological Research at JoVE.com

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 ...

The overall goal of this familiarization protocol is to increase understanding as well as potentially reduce anxiety for child participants, resulting in a greater likelihood of the successful completion of the research protocol for the collection of EEG data. This method can help support the ethical application of research for all fields of study, especially those that involve vulnerable participants such as individuals with linguistic and cognitive challenges. The main advantage of this technique is the use of evidence-based practices to increase knowledge and understanding of the activities of the research protocol, while ensuring ongoing participant consent.This protocol may extend the capability of other researchers to obtain reliable data from all participants as it provides a model that is easily adapted and replicated for use with different research protocols. Generally, individuals new to this protocol may struggle with lack of familiarity with EBP. However, resources exist to support development and implementation of these techniques.Visual demonstration of the key steps of this method is critical, as singular components, while relatively easy to implement, may be more challenging as part of a collective package. Begin by escorting the participant to the designated play area, containing various toys on the ground. Show the participant the five-point emotion rating scale, and ask them to point to the picture that best represents how they feel.If the participant selects a one or two, proceed by reading and reviewing a social narrative with them. Now we're gonna look over all the games we're gonna play today. Please let me know if you have any questions of anything you might not be sure of, alright?Following the review of the social narrative, assess the participant's emotional state and readiness to continue with the research by showing them the self-rating emotion scale and asking how they feel. At the same time, have the research assistant use a separate scale to rate the participant to ensure that some of the anxiety has been reduced. Next, hand a consent form to the parent and read it with the child participant if they are unable to read the form independently.Ask the participant if they would like to continue and complete the activities described in the social narrative. Then have the participant and parent sign the consent form. After the form is signed, introduce the participant to the visual schedule.These are all the games that we will be playing today. At the end of the game you can take down each card for completing the game. Then point to the take a break card and tell the participant that they may use this card if they need to take a break.Point to the stop card and tell them that they may use this card if they do not want to play any more games for the day. Finally, after the participant has completed half of the assessments, evaluate the participant's emotions using both emotion rating scales and ensure that they maintain a level one or two emotion rating prior to continuing activities. Begin by introducing the participant to a stuffed bear and model the use of the electroencephalography or EEG cap on it.Then read the section in the social narrative that describes use of the cap to the participant. Show the participant how the cap is placed on the bear's head. Allow the participant to touch the cap and interact with it by placing it on the bear's head.Next, apply a modified version of the Premack Principle by saying-Would you like to wear the net hat? If you wear the net hat, then you will get a gift card. If you choose not to, we can do something else.Then use a tape measure to measure the participant's head circumference to determine the cap size. Soak the appropriate cap in the warm electrolyte solution. After this initial measurement, determine the Cz electrode location by measuring from the inion, a location on the back of the skull, to the nasion, a location between the eyebrows above the nose, as well as from the right to left ear.Mark the intersection of these two measurements with a cross using a washable marker. Then place a towel around the participant's neck to prevent water from dripping on their clothing. Tell the participant to close their eyes while the cap is being placed on their head.Carefully line up the vertex electrode on the cap with the intersection mark to ensure that the cap is aligned properly. Once the cap is placed on the head and adjusted, let the participant know that they can open their eyes. After the final activity, assess the participant's emotions one last time to ensure that a one or two emotion level was maintained throughout the protocol, and that wearing the cap did not increase anxiety or decrease the participant's level of comfort.Finally, after the mock EEG experiment is complete, provide the participant with the promised compensation and thank them for their participation. On the next visit, begin by presenting the participant with a social narrative that describes the step of the second appointment. Ask the participant if they are willing to wear the cap in the same manner as the prior visit.Present the participant with a visual schedule that includes all the EEG data collection procedures. Prep the participant for the EEG procedure in the same manner as the familiarization visit. Once the EEG 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 EEG when they move. Next, start the stimulus presentation. Ask the child to sit very still like a statue while pointing to the visual sign that shows this behavior.Finally, after completing the EEG portion of the experiment, provide the participant with the promised compensation and thank them for their participation. As part of a series of studies on eye gaze and audio-visual speech perception, an initial cohort of 25 participants with ASD and average IQs was recruited using a similar EEG protocol. 72%of the children in this previous cohort completed the EEG protocol.In the current cohort, thus far, 15 participants with ASD were recruited, 12 of whom participated in the familiarization protocol. Of the 12 that completed the familiarization protocol, 100%completed the full EEG acquisition protocol. While attempting this procedure, it's important to remember to adhere to the protection of participants through the provision of ongoing consent.In addition, each EVP technique contains specific requirements and criteria that must be adhered to to ensure fidelity of development and implementation. Following this procedure, other EBP methods like video modeling can be developed and implemented to support participant engagement. After watching this video, you should have a good understanding of how to implement a familiarization protocol that uses EBP during research activities while adhering to NIH guidelines as well as your specific research protocol.Don't forget that engaging participants in research activities that require adherence to particular behavioral expectations may be challenging for individuals with linguistic and cognitive challenges.

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Neuroscience

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 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 ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...