This work has been supported by a project of the Principality of Asturias (FC-15-GRUPIN14-053) and a predoctoral grant from the Severo Ochoa Program (BP14-030). We would like to thank Anna Bujnowska for her contribution and Heather Marsh and Nigel Marsh for their help.

The present study was conducted according to the Declaration of Helsinki, which establishes the ethical principles for research involving human beings. The study&#39;s aims, scope and procedure were also approved by the Ethics Committee of the University of Oviedo and University Hospital of Asturias.
1. Parents Report
<ol>
	<li>Before starting the assessment protocol, obtain signed parental consent for the child to be assessed.</li>
	<li>Conduct a structured interview with families of the participants. 
	NOTE: The structured interview has been provided as an appendix.</li>
	<li>Have families and/or teachers of the participant complete the Scale for the Assessment of ADHD13. This scale comprises 20 items that provide information on the presence of symptoms related to attention deficit and hyperactivity/impulsivity that are referred in the Diagnostic and Statistical Manual of Mental Disorders.
	<ol>
		<li>In this section, have the therapist give the following instructions: &#34;Now, in order to get significant information about your child, you must complete this questionnaire referring to the symptoms that might be representative of your child&#39;s behavior. You must classify the frequency with which you observe these 20 symptoms&#34;.</li>
	</ol>
	</li>
</ol>
2. Cognitive Measures
<ol>
	<li>To measure the attentional variables, administer a cognitive scale.
	<ol>
		<li>Administer a Cognitive Scale14 (following the protocol in the manual) for assessing individual intelligence in children and adolescents between the ages of 6 years and 16 years 11 months.</li>
		<li>Analyze in depth the cognitive profile obtained by the scale. 
		NOTE: Considering the information included in the cognitive scales manual, the results from the Wechsler scale could be influenced by ADHD, leading to lower scores in processing speed and working memory in comparison with perceptual reasoning and verbal comprehension.</li>
	</ol>
	</li>
</ol>
3. Executive Measures Using Continuous Performance Tests
<ol>
	<li>Analyze the performance of the children using two CPTs: one based on a Virtual Reality CPT15 and the other consisting of a Traditional CPT16.</li>
	<li>Administer the virtual-reality based CPT15. This is a CPT based on a virtual reality environment that reproduces the conditions of a regular classroom. It evaluates attention, impulsivity, processing speed, and motor activity in children and adolescents between 6 and 16 years of age. The activities that make up the test are preset, and it is not possible to change any specific characteristic of the tasks.
	<ol>
		<li>Double-click on the Virtual Reality CPT app to start the program.</li>
		<li>Enter the username and password provided by the program license.</li>
		<li>Enter the participant information (name, surname and date of birth) and the click on the &#34;Go into the virtual classroom&#34; window.</li>
		<li>Ask the participant to wear the Head Mounted Display (HMD) glasses and headphones, and inform them of the importance of wearing it during the whole test. Have the therapist say: &#34;Now you need to wear these special glasses during the virtual reality tasks, if you don&#39;t see very well or have any issue about that, please tell me and I help you to resolve it&#34;. 
		NOTE: These glasses are connected to the PC, so the therapist can see the images the participants are looking at in order to ensure that they understand the test (Figure 2).</li>
		<li>Ask the participant to hold the button with their dominant hand before starting the virtual attentional tasks. Have the therapist say: &#34;You need to hold and press the button with the same hand you write with&#34;.</li>
		<li>Have the participant explore the virtual environment (a typical classroom) and take the perspective of a student sitting at one of the desks looking at the blackboard for 15 s (Figure 3). Conduct this part by the virtual teacher though these instructions: &#34;Hello, with the glasses that you are wearing, you can see the entire classroom: to the left, to your right, up, and down. You can see everything. Notice all the things in the room, look at the walls and the other people, look at whatever you want&#34;.</li>
		<li>Have the participant follow the virtual teacher, who guides them through the tasks. The first part the participant performs is training, which consists of visually locating balloons and popping them by pressing the button. Have the virtual teacher in this part instruct this part as follows: &#34;Now that you have inspected everything, you&#39;re going to start doing something fun. We have placed some shiny red balloons around you. You need to find the balloons by moving your head. When you find one, look at it closely, and press the button to make it pop. You have to find 4 balloons. Let&#39;s go! The challenge starts now!&#34;</li>
		<li>Have the participant continue to the next step which is the first exercise. This is based on the &#34;x-no&#34; paradigm (traditionally known as &#34;no-go&#34;), where the participant must press the button when he or she does not see or hear the stimulus &#34;apple&#34;. Have the virtual teacher say: &#34;Now, you will to see some drawings on the chalkboard. I will also say a few words to you. You have to pay attention and click the button, AS FAST AS POSSIBLE, when you see or hear anything THAT IS NOT AN APPLE. But be careful! You need to press the button every time you see or hear any other thing. For example, if you hear &#34;CLOUD&#34; or if a drawing of a cloud appears on the chalkboard, you have to press the button. Remember: Never press the button when you see or hear apple! The challenge starts... Now!&#34;. 
		Note: As the activities are preset by the program, it is not possible to change the target-stimulus (in this case, APPLE).</li>
		<li>Have the participant complete the last exercise by following the instructions to press the button whenever he/she sees or hears the number &#34;seven&#34;. This part consists of a &#34;X&#34; paradigm (or &#34;go&#34; task). Say these instructions: &#34;Now you are going to work with numbers. You will see numbers on the chalkboard and you will also hear numbers. You must press the button every time you hear or see the number &#34;SEVEN&#34;. DO NOT press the button for any other number. For example, if you hear or see the number &#34;THREE&#34; DO NOT press the button. Remember: Always press the button when you see or hear &#34;SEVEN&#34;! The test starts now!&#34; 
		NOTE: As the activities are preset by the program, is not possible to change the target-stimulus (in this case, SEVEN).</li>
		<li>Print the report of the test results via the test website adding the name and surname of the child assessed. This report compiles results for the following variables: omissions, commissions, response time, and variability and complement this information by differentiating these measures of sensory modality (visual vs. auditory), presence/ absence of distractors, and task type (go vs. no-go), thereby leading to different execution profiles.</li>
	</ol>
	</li>
	<li>Administer the Traditional CPT16. This is a CPT that can be used in a visual or auditory version, but in this protocol only the visual version is used. This test contains preset tasks. The visual norms for CPT go from 4 years to more than 80, by age and gender.
	<ol>
		<li>Make sure that the participant is relaxed, paying attention to their breathing and ensuring that it is controlled. If it appears that the participant is not controlling their breathing. Indicate as follows: &#34;You should focus on abdominal contraction and controlled breathing out&#34;.</li>
		<li>Open the CPT application and enter the name and birth date of the child, and press the okay button.</li>
		<li>Click on visual tasks window and click the launch button, the computer will start to load the visual tasks.</li>
		<li>Ensure the participant is holding the button (which is connected to a PC) with his/her dominant hand. Repeat the instruction from step 5 of the Virtual Reality CPT: &#34;You need to hold and press the button with the same hand you write with&#34;.</li>
		<li>Inform the participant of the following instructions for the test: &#34;You have to press the button when you see a black square in the upper part which is the target stimulus. However, do not press the button when the black square is in the bottom part&#34; (Figure 4).</li>
		<li>Have the participant perform the training part (which lasts for approximately 3 min) until it is clear the participant understands the task. In the training part of the CPT, the target (stimulus to which the participant has to respond) and the non-target (stimulus to which the participant has to avoid responding) stimulus appear randomly in the screen; the therapist should observe whether the participant presses the button correctly or not. If the participant does not press the button according to the instructions, have the therapist explain the instructions again and then the participant will need to repeat the training section.</li>
		<li>Tell the participant that the task is very long and that she/he must keep still and concentrate on doing the tasks.</li>
		<li>Have the participant complete the CPT activities. It is divided into two sections. The first section of the test (the &#34;Infrequent&#34; or vigilance mode), is characterized by target: non-target ratio of 1:3.5. This is evidenced by a tendency to not press the button. In the second section (the &#34;Frequent&#34; or high response demand mode) presents a target:non-target ratio of 3.5:1, so the child must inhibit the tendency to respond.</li>
		<li>After completion of the CPT, generate the report which compiles the following variables: omissions, RT (response time between the presentation of target stimulus and subject response), commissions, variability, D prime (quality of concentration during the test based on the number of errors in the test) and ADHD Index, attributable to a presence or absence of attention deficit with hyperactivity. Calculate this ADHD Index from the sum of: TR of first half + D prime second half + total variability and is interpreted as attributable to ADHD when the score is lower than -1.80.</li>
	</ol>
	</li>
</ol>
4. Cortical Activation Measures in Fp1 and Fpz/Cz Regions (Hemoencephalography, and Quantified Electroencephalography) 17
<ol>
	<li>Open the Bioexplorer software and choose the design POCKET_HEG_video_1ch_AI_HEGRatio.bxd.
	<ol>
		<li>Press time on button to see the time during the assessment (35 s).</li>
		<li>Inform the participant that he/she cannot move and must be still. The therapist informs the participant about the importance of remaining still during the assessment of blood oxygenation: &#34;Now it is very important that you are still during the assessment of blood oxygenation because if you move, the system will fail, and it will be necessary to measure again. Are you ready?&#34;</li>
		<li>Click on the red button and enter the name of the subject and the cortical point which is going to be measured (Fp1 region for assessment of inhibition capacity and Fpz for the assessment of attention capacity).</li>
		<li>Put the band on the participant&#39;s forehead in a specific area: Fp1 (for assessment of inhibition capacity) and Fpz (for the assessment of attention capacity) as can be seen in Figure 5.</li>
		<li>Make sure that no external light enters the band. Properly adjust the band to the participant&#39;s head in such a way that it is not loose and does not fall off during the evaluation.</li>
		<li>Put the Hemoencephalography-nirHEG (which is connected to the headband) around the participant&#39;s neck, like a necklace.</li>
		<li>Turn on the hardware to connect the band to the PC.</li>
		<li>Measure the blood oxygenation of a specific area through the nir-HEG program for approximately 35 s. Press the green button to start to measure blood oxygenation and 35 s later press the white button to end the assessment.</li>
		<li>Print the output provided by the program and analyze the nir-HEG Ratio of the participant provided by the program. The mean of this tool is 100 (SD = 20) which is used to calibrate the spectrophotometers. An Attention Index (AI) is also obtained through the nir-HEG , which shows the incapability of the participant to increase the ratio and brain activation. This is useful to identify attentional problems and measure capacity of concentration.</li>
	</ol>
	</li>
	<li>Measure the cortical activity levels of the participant using the quantified electroencephalography (Q-EEG), a computerized EEG system that provides levels of cortical activation through the beta/theta ratio. It is used to measure general levels of attention, not taking the performed task. 
	NOTE: The analysis of beta/theta ratio have been carried out according to previous studies12.
	<ol>
		<li>Inform the participant of the correct abdominal breathing required during the test.</li>
		<li>Open the Bioexplorer software and choose the design combined_Theta_Beta.bxd. The screen will appear divided into two windows (the left side shows the cortical activity of FP1 and the right side shows the cortical activity of the CZ)</li>
		<li>Click on the red button and enter the name of the subject and the cortical point which is going to be measured (Fp1 region for assessment of inhibition capacity and Cz region for the assessment of attention capacity).</li>
		<li>Put the red electrode in the Fp1 region and the blue electrode in the Cz region, applying electrode gel (using the information found at: &#8220;http://neurologiclabs.com/neuromonitoring/eeg/&#8221;, to ensure that the electrodes are correctly placed) (see Figure 6).</li>
		<li>Put two more control electrodes (the black and white electrodes) on the participant&#180;s left and right earlobes, applying electrode gel.</li>
		<li>Ensure the participant has her or his eyes open during the assessment. The therapist must watch the participant throughout the assessment. If the participant closes their eyes at any time, end the assessment process and repeat it once the participant is rested.</li>
		<li>Place an electromyography (EMG) sensor on the right forearm to identify the degree of movement.</li>
		<li>Inform the participant that he/she must remain relaxed, trying not to move during the task an breathing slowly. The participant must be concentrated on the computer screen while the theta and beta waves are being emitted. The therapist says: &#34;You must be still and relaxed for 35 s, the only thing you need to do is to watch the theta/beta waves that will be appearing on the computer screen&#34;.</li>
		<li>Press the green button to start to measure cortical activity, and 35 s later press the white button to end the assessment.</li>
		<li>Print the results generated from program and analyze them. If the beta/theta ratio is lower than 50% at Cz, this evidences a deficit of sustained attention. On the other hand, if the ratio is also lower at Fp1, this result is related to an impairment of executive control, attributable to hyperactivity.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>American Psychiatric Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diagnostic and statistical manual of mental disorders. , (2013).</li><li>Steinau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+criteria+in+attention+deficit+hyperactivity+disorder-Changes+in+DSM+5.">Diagnostic criteria in attention deficit hyperactivity disorder-Changes in DSM 5.</a> Front Psychol. 4 (49), (2013).</li><li>Polanczyk, G., de Lima, M. S., Horta, B. L., Biederman, J., Rohde, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+worldwide+prevalence+of+ADHD:+a+systematic+review+and+metaregression+analysis.">The worldwide prevalence of ADHD: a systematic review and metaregression analysis.</a> Am J Psychiat. 4 (6), 942-948 (2007).</li><li>Willcutt, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+DSM-IV+attention-deficit/hyperactivity+disorder:+a+meta-analytic+review.">The prevalence of DSM-IV attention-deficit/hyperactivity disorder: a meta-analytic review.</a> Neurotherapeutics. 9 (3), 490-499 (2012).</li><li>Catal&#225;-L&#243;pez, F., 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=Prevalence+of+attention+deficit+hyperactivity+disorder+among+children+and+adolescents+in+Spain:+a+systematic+review+and+meta-analysis+of+epidemiological+studies.">Prevalence of attention deficit hyperactivity disorder among children and adolescents in Spain: a systematic review and meta-analysis of epidemiological studies.</a> BMC psychiatry. 12 (1), 168 (2012).</li><li>Cubillo, A., Halari, R., Smith, A., Taylor, E., Rubia, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+fronto-striatal+and+fronto-cortical+brain+abnormalities+in+children+and+adults+with+Attention+Deficit+Hyperactivity+Disorder+(ADHD)+and+new+evidence+for+dysfunction+in+adults+with+ADHD+during+motivation+and+attention.">Review of fronto-striatal and fronto-cortical brain abnormalities in children and adults with Attention Deficit Hyperactivity Disorder (ADHD) and new evidence for dysfunction in adults with ADHD during motivation and attention.</a> Cortex. 48 (2), 194-215 (2012).</li><li>Lubar, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discourse+on+the+development+of+EEG+diagnostics+and+biofeedback+for+attention-deficit/hyperactivity+disorders.">Discourse on the development of EEG diagnostics and biofeedback for attention-deficit/hyperactivity disorders.</a> Appl Psychophys Biof. 16 (3), 201-225 (1991).</li><li>Monastra, V. J., 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=Assessing+attention+deficit+hyperactivity+disorder+via+Quantified+Electroencephalography:+An+initial+validation+study.">Assessing attention deficit hyperactivity disorder via Quantified Electroencephalography: An initial validation study.</a> Neuropsychol. 13 (3), 424-433 (1999).</li><li>Hall, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+utility+of+the+continuous+performance+test+and+objective+measures+of+activity+for+diagnosing+and+monitoring+ADHD+in+children:+a+systematic+review.">The clinical utility of the continuous performance test and objective measures of activity for diagnosing and monitoring ADHD in children: a systematic review.</a> Eur Child Adolesc Psych. 25 (7), 677-699 (2016).</li><li>Barbaresi, W. J., Katusic, S. K., Colligan, R. C., Weaver, A. L., Jacobsen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifiers+of+long-term+school+outcomes+for+children+with+attention-deficit/hyperactivity+disorder:+does+treatment+with+stimulant+medication+make+a+difference?+Results+from+a+population-based+study.">Modifiers of long-term school outcomes for children with attention-deficit/hyperactivity disorder: does treatment with stimulant medication make a difference? Results from a population-based study.</a> J Dev Behav Pediatr. 28 (4), 274-287 (2007).</li><li>Rodr&#237;guez, C., Gonz&#225;lez-Castro, P., Cueli, M., Areces, D., Gonz&#225;lez-Pienda, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attention+deficit/hyperactivity+disorder+(ADHD)+diagnosis:+an+activation-executive+model.">Attention deficit/hyperactivity disorder (ADHD) diagnosis: an activation-executive model.</a> Front Psychol. 7, (2016).</li><li>Gonz&#225;lez-Castro, P., Rodr&#237;guez, C., L&#243;pez, &. #. 1. 9. 3. ;., Cueli, M., &#193;lvarez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attention+deficit+hyperactivity+disorder,+differential+diagnosis+with+blood+oxygenation,+beta/theta+ratio,+and+attention+measures.">Attention deficit hyperactivity disorder, differential diagnosis with blood oxygenation, beta/theta ratio, and attention measures.</a> Int J Clinical and Health Psycho. 13 (2), 101-109 (2013).</li><li>Farr&#233;, A., Narbona, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EDAH: Scale for the assessment of attention deficit hyperactivity disorder. , (2001).</li><li>Wechsler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Wechsler Intelligence Scale for Children- 4th edition. , (2005).</li><li>Climent, G., Banterla, F., Iriarte, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AULA: Theoretical manual. , (2011).</li><li>Greenberg, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+normative+data+on+the+Test+of+Variables+of+Attention+(TOVA).">Developmental normative data on the Test of Variables of Attention (TOVA).</a> J Child Psychol Psyc. 34, 1019-1030 (1993).</li><li>Toomim, H., 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=Intentional+increase+of+cerebral+blood+oxygenation+using+hemoencephalography:+an+efficient+brain+exercise+therapy.">Intentional increase of cerebral blood oxygenation using hemoencephalography: an efficient brain exercise therapy.</a> J Neurother. 8, 5-21 (2005).</li><li>Gonz&#225;lez-Castro, P., 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=Cortical+activation+and+attentional+control+in+ADHD+subtypes.">Cortical activation and attentional control in ADHD subtypes.</a> Int J Clin Health Psycho. 10 (1), 23-39 (2010).</li><li>Garc&#237;a, T., Gonz&#225;lez-Castro, P., Areces, D., Cueli, M., Rodr&#237;guez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+functions+in+children+and+adolescents:+The+types+of+assessment+measures+used+and+implications+for+their+validity+in+clinical+and+educational+contexts.">Executive functions in children and adolescents: The types of assessment measures used and implications for their validity in clinical and educational contexts.</a> Papeles del Psic&#243;logo. 35, 215-233 (2014).</li></ol>

The authors who are listed above certify there are no financial interests or other conflicts of interest regarding the present study.

Here we present an effective protocol for assessing ADHD from 6-16 years of ages. Given the symptomatic complexity of ADHD and its high prevalence rates, professionals must have reliable and valid instruments to diagnose this disorder. Generally, questionnaires based on behavioral observations are widely used. However, the use of these instruments as the sole assessment measure has certain limitations, including potential subjectivity on the part of the observer18.
As the results show, this protocol placed emphasis on highlighting the differences between a participant with ADHD against a participant without ADHD. More specifically, it could be seen that a participant with ADHD had lower scores in attentional variables from both CPTs (omissions, commissions, response times and motor activity) as well as lower cortical activation and blood oxygenation in Fp1 and Fpz/Cz brain regions. For this reason, it is very relevant to contrast the information obtained by observation scales with the cognitive profile of the children and the performance in CPTs. Professionals can then perform a more realistic and reliable assessment and, thus provide recommendations for parents and teachers that are more specifically adapted to the individual needs of each child.
However, a critical step of this protocol is the management of exclusion criteria; professionals must ensure the ADHD symptoms are not due to another cause such as perceptive, emotional or social problems19.
A minor limitation of this protocol is the time required to complete each assessment. Generally, this protocol is best divided into several sessions to ensure participant well-being. The applicability of this method has been presented in previous studies11,12 which showed the effectiveness of the assessment model for getting an accurate diagnosis of ADHD according to the Diagnostic and Statistical Manual of Mental Disorders criteria.
Future lines of research would be based on the inclusion of neurological tools for getting an objective assessment of ADHD, considering functional and structural neuroimaging methods. Although, this research used quantitative analysis from Quantified Electroencephalography and Hemoencephalography (nir-HEG), it would be positive the inclusion of other instruments to contrast neuropsychological measures. Moreover, this type of study helps the clinicians to detect specific profiles of cortical activation (Q-EEG) and CPT (executive control) to differentiate the three types of ADHD presentations.

The overall goal of the present protocol is to develop a complete procedure or model of assessment for the diagnosis of Attention Deficit Hyperactivity Disorder, otherwise known as ADHD. ADHD is one of the problems that impacts academic performance. It is understood to be a disorder characterized by problems with attention, inhibitory control and hyperactivity, whose performance is significantly lower than their peers1,2. The latest version of the Diagnostic and Statistical Manual of Mental Disorders (DSM)1 includes different updates from the previous version: ADHD has been categorized as a neurodevelopmental disorder; the age of appearance of the symptoms has been increased so now the symptoms can manifest before 12 years of age; the term ADHD presentation should be used instead of subtype (predominantly hyperactive/impulsive; predominantly inattentive; and combined presentation, and); finally, it has been accepted as a comorbidity with autism spectrum disorders.
There are different estimations of prevalence rates depending on the country or region analyzed3,4,5. An international global systematic3 review observed an average prevalence rate of 5.29%. However, applying criteria from the Diagnostic and Statistical Manual of Mental Disorders4, the percentage ranges from 5.9 to 7.1%. Similarly, a meta-analysis of ADHD in a Spanish population provided an average of 6.8%5. The variations in prevalence rates could be due to the different assessment protocols used.
Although there is a considerable body of research suggesting a neurological basis for ADHD, the origins of this disorder remain unclear. Several studies have associated the ADHD symptomatology to brain cortical hypoactivation, which is related to a deficit in the dopaminergic and noradrenergic systems6. The noradrenergic system modules the selective attention and the activation levels needed to carry out a task. On the other hand, the dopaminergic system is responsible for inhibitory control, both at an executive and motivational level. The low cortical activation related to the dopaminergic and noradrenergic systems is presumed to be the basis for the inhibitory and attentional deficits presented in the ADHD. In this sense, children with ADHD show low cortical activation in the dopaminergic and noradrenergic systems, which is manifested by different profiles of electro cortical activity in a state of rest, evidenced by increased theta -and decreased beta- activity7,8 as well as low levels of blood oxygenation in the Fp1 (front left side of the frontal lobe), and FPz/Cz (central zone of the pre-frontal cortex) regions.
Blood oxygenation is measured using nir-HEG, which uses functional near-infrared spectroscopy to measure color changes in the blood in the brain to indicate oxygen saturation areas; oxygenated blood is bright red whereas de-oxygenated blood is a deep, almost purplish crimson. Cortical activation is measured using quantified electroencephalography (Q-EEG). This is a computerized EEG system that records electrical activity in the brain to provide levels of cortical activation through the beta/theta ratio. It measures attention in general, independently of the task being performed. Other studies have focused on the existence of an executive function (EF) impairment in the ADHD population9, which would explain the difficulty children with ADHD have controlling impulsive responses, resisting interference or distraction, organizing activities in a sequential manner, and sustaining cognitive effort while performing an activity.
Generally, these characteristic symptoms of ADHD have serious consequences which result in difficulties in scholastic, social and familial contexts. Children with ADHD have a higher probability of repeating a grade and/or completing fewer grades at school than children without ADHD. Moreover, dropping out of high school is three times more likely among youth with ADHD10,11.
Considering the modifications and the new categorization of ADHD in the current version of the Diagnostic and Statistical Manual of Mental Disorders 1, results relevant to establish the relationship between cortical activation levels in specific brain areas, executive functions, and diagnosis-related variables7,8,11,12 (i.e., differences between the three types of ADHD presentations).
One of the most common problems in the identification of ADHD is the over diagnosis of the disorder due to the absence of global protocols for assessment. The fact that professionals do not have a general protocol based on objective variables is causing a large percentage of false positive and false negative cases of ADHD. This situation highlights the need for professionals and clinicians to have a clear protocol that considers not only the relevant variables but also the relationships between them.
For this reason, the research group of School Learning, Difficulties and Academic Performance (ADIR) from the University of Oviedo has been working on developing a complete protocol to identifyprofiles of cortical activation and executive control to provide a more objective diagnosis of ADHD than what is currently in use. This protocol is particularly important because it takes into account the fact that cortical activation in the frontal and prefrontal cortex impacts the executive function. The current protocol will be useful for clinicians who are interested in performing a complete assessment that considers the interaction between relevant variables in the diagnosis. To that end, the protocol is based on the assessment model from a recent study proposed by Rodriguez et al. which takes into consideration the interaction between cortical activation and executive variables (Figure 1).
In summary, the purpose of this protocol is to provide a more objective diagnostic procedure for this developmental disorder than is currently available, and to analyze in depth the relationship between activation measures and executive function measures. The procedure will also take into consideration some of the hypothetical determinants of ADHD, both in the relationship between activation of selected areas of the brain and differences in performance on various aspects of executive functioning such as omissions, commissions or response times.

<table border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="160">
			<td height="160" style="height:160px;width:216px;">EDAH&#160;</td>
			<td style="width:163px;">TEA Company</td>
			<td style="width:344px;"></td>
			<td style="width:352px;">It is a Scale for the Assessment of ADHD that was administered to families and/or teachers. 
			&#160;It comprises 20 items that provide information on the presence of symptoms relating to attention deficit and hyperactivity/impulsivity, and helps differentiate between the threes subtypes of ADHD. A score above 90% in its subscales indicates attention deficit, hyperactivity/ impulsivity problems, or both.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Wisc-IV R</td>
			<td>TEA Company</td>
			<td></td>
			<td style="width:352px;">The WISC-IV by Wechsler (2005) is a tool that assesses 
			individual intelligence in children and adolescents between 
			the ages of 6 years and 16 years 11 months.</td>
		</tr>
		<tr height="123">
			<td height="123" style="height:123px;">QEEG</td>
			<td>Neurobics pocket</td>
			<td></td>
			<td style="width:352px;">Q-EEG (quantified electroencephalogram) is a computerized EEG system, 
			adapted by Toomin, which provides levels of cortical activation through 
			the beta/theta ratio. It measures attention in general, independently of the task to be performed.</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;">NIR-HEG</td>
			<td>Brain trainer company</td>
			<td></td>
			<td style="width:352px;">it is a tool used to measure blood oxygenation in expressly selected areas. The nir-HEG employs the translucent property of biological tissue, and low-frequency red and infrared lights with light emitting diodes (LED optodes).&#160;</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">AULA Nesplora</td>
			<td>Nesplora Company</td>
			<td></td>
			<td style="width:352px;">It is a Continuous Performance Test that evaluates attention, impulsivity, processing speed, and motor activity in participants between 6 and 16 years of age. 
			The task is performed in a virtual reality environment, 
			which is shown through three-dimensional (3D) glasses 
			(Head Mounted Display, HMD) equipped with motion 
			sensors and headphones.&#160;</td>
		</tr>
		<tr height="172">
			<td height="172" style="height:172px;">T.O.V.A (Test of Variables of Attention)</td>
			<td>The T.O.V.A.&#160; Company</td>
			<td>T.O.V.A. 8 kit</td>
			<td style="width:352px;">T.O.V.A. is an objective, accurate, and FDA cleared continuous performance test&#160; (CPT) that measures the key components of attention and inhibitory control. 
			The T.O.V.A. is used by qualified healthcare professionals as an aid in the assessment 
			of attention deficits, including attention-deficit/hyperactivity disorder (ADHD), in children and adults.</td>
		</tr>
	</tbody>
</table>

using brain activation (nir-heg/q-eeg) and execution measures (cpts) in a adhd assessment protocol

Attention Deficit Hyperactivity Disorder (ADHD) is a problem that impacts academic performance and has serious consequences that result in difficulties in scholastic, social and familial contexts. One of the most common problems in the identification of this disorder relates to the apparent over diagnosis of the disorder due to the absence of global protocols for assessment. The research group of School Learning, Difficulties and Academic Performance (ADIR) from the University of Oviedo, has developed a complete protocol that suggests the existence of certain patterns of cortical activation and executive control for identifying ADHD more objectively. This protocol takes into consideration some of the hypothetical determinants of ADHD, including the relationship between activation of selected areas of the brain, and differences in performance on various aspects of executive functioning such as omissions, commissions or response times, using innovative tools of Continuous Performance Testing (based on Virtual Reality CPT and Traditional CPT) and brain activation measures (two different tools, based on Hemoencephalography- nirHEG; and Quantified Electroencephalography --Q-EEG, respectively). This model of assessment aims to provide an effective assessment of ADHD symptomatology in order to design an accurate intervention and make appropriate recommendations for parents and teachers.

Using the assessment procedure presented here, it is possible to carry out an effective assessment about ADHD symptomatology in order to design an accurate intervention and make recommendations for parents and teachers. Below are a series of representative results of a participant with ADHD and a participant without ADHD, which will allow professionals to see the differences between the two profiles.
Once clinicians had the informed consent from the families, a cognitive scale (WISC IV) was administered to the children in order to exclude those participants who present low or high capacities. The following steps then compiled an attentional profile of the children using the Continuous Performance Test and Activation Cortical Techniques (Q-EEG and nir-HEG). Figure 7 shows the results of children with and without ADHD in the Virtual Reality CPT. These results show the children with ADHD have more omissions and commissions errors, as well as higher motor activity and larger response times.
Similarly, Figure 8 shows the results obtained by the Traditional CPT showing how the child with ADHD presented larger percentages in omissions, commissions, response times and in the variability of the response. While the child without ADHD showed the best scores at the end of the tasks, the child with ADHD did not show improvement in any of the four blocks.
Figure 9 shows an example of the Cortical Activation collected by nir-HEG in a child affected by ADHD who obtained 24.5 percentage points below the average in Fp1 region.
Similarly, an example of the measures collected by Q-EEG in a child with ADHD (FP1 region) are shown in Figure 10 that evidences how the symptoms of ADHD cause a decreased in cortical activation (ratio beta/theta under 0.5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56796/56796fig1.jpg" /> 
Figure 1. Model of ADHD assessment 
Based on Hemoencephalograpy (nir-HEG), Quantified Electroencephalography (Q-EEG), and Traditional CPT (CPT). Variables included: HEG-Fp1(nir-HEG ratio from left pre-frontal cortex); HEG-FpZ (nir-HEG ratio from central pre-frontal cortex); Q-EEG-Fp1(beta-theta ratio from left pre-frontal cortex); Q-EEG-CZ (beta-theta ratio from central cortex); CPT-OMIS (omissions committed in CPT ); CPT-COMIS (commissions committed in CPT); CPT-VAR (Response Variability during CPT tasks); CPT-RT (Response Time obtained in CPT); CPT-DPR (D prime Index provided by CPT); ADHD-INDEX (ADHD Index provided by CPT). This model reflects a stronger association between activation (central and left prefrontal) and execution in ADHD children than their peers without the disorder. Low cortical activation (by QEEF) and blood oxygenation (by nir-HEG) in Fp1 region, are related to low performance in CPT. Similarly, normal levels of electrical activation and blood oxygenation are associated to normal results from CPT. Provided by Rodr&#237;guez et al.11. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56796/56796fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56796/56796fig2.jpg" /> 
Figure 2. Head Mounted Display(HMD) glasses of the Virtual Reality CPT. 
The image shows the Head Mounted Display (HMD) glasses, the headphones and the button which must be held with the dominant hand. The image also shows the virtual classroom which is shown to the participant via the glasses and to the therapist via the computer screen. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56796/56796fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56796/56796fig3.jpg" /> 
Figure 3. Virtual classroom provided by the Virtual Reality CPT. 
Virtual classroom environment where the participant does the tasks which are explained by a virtual teacher. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56796/56796fig3large.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/56796/56796fig4.jpg" /> 
Figure 4. Images present in the Traditional CPT. 
The right-hand picture shows the non-target stimulus and the left-hand picture shows the target stimulus. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56796/56796fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56796/56796fig5.jpg" /> 
Figure 5. Participant wearing the nir-HEG band. 
The image shows the nir-HEG band placed in the Fp1 region and the hardware connected.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56796/56796fig6.jpg" /> 
Figure 6. Participant with electrodes of Q-EEG. 
The image shows the blue electrode in the Cz region and the red one in the Fp1 region. The control electrodes: black and white, have been placed on the participant&#180;s left and right earlobes, applying electrode gel. Finally, an electromyogram was placed on the right forearm.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56796/56796fig7.jpg" /> 
Figure 7. Performance and motor activity during the Virtual Reality CPT. 
The Y axis shows the standardized scores from the Virtual Reality CPT (scores above 60 mean low performance in the variable studied and scores below 60 mean good performance of the variables studied. The X axis shows the different variables provided by the Virtual Reality test. The colors: yellow, orange and red, represent the grade of severity: low, medium and high in the performance of different variables. A and C illustrate the performance of a participant without ADHD (A) and a participant with ADHD (C). 
Images B and D represent the subject&#180;s head movement (motor activity) throughout the test. The green square refers to the blackboard and the yellow square represents the area in which the virtual blackboard can be inside the visual range for detecting the stimulus. If child move out of the yellow square, it makes impossible to perform the visual task properly. The dot diagram provides a graphic which represents the child&#39;s attention towards the blackboard and to the general task. Image B represents the motor activity in a participant without ADHD and image D represents the motor activity of a participant with ADHD.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56796/56796fig8v2.jpg" /> 
Figure 8. Profile provided by the Traditional CPT in the main variables. 
Figure A represents the profile of a student with ADHD while Figure B shows the execution of a child with a performance similar to the normative group. The term &#34;SS&#34; refers to Standardized scores. Standardized scores below 80 represent low performance. &#34;Q1&#34;(25%), &#34;Q2&#34;(50%), &#34;Q3&#34;(75%) and &#34;Q4&#34;(100%) represent the four quartiles of the CPT tasks. This division is useful in order to see whether the subject&#39;s attention decreases during the activities or not.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56796/56796fig9.jpg" /> 
Figure 9. Activation measure with the nir-HEG. 
This Figure shows the ratio indicator of the oxygenation of the blood at specific point (Fp1/Fpz) and an attention index which is expressed as percentage (a percentage below 50% represents a low attentional index).
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56796/56796fig10.jpg" /> 
Figure 10. Cortical activation measures in FP1. 
The Figures show the output from Q-EEG. The top part shows beta/theta waves measured individually, and the bottom part shows the beta/theta ratio in the Cz region and electromyography results from the right forearm. The CZ beta/theta ratio is shown on the left (in this case is 0.55), cortical activation is good when the ratio is above 0.50. The electromyography from the right forearm is used to ensure that the participant is relaxed at a muscular level, and is on the right (in this case the value of EMG is 4.22). Good values in electromyography are below 5.0. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56796/56796fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Brain Activation nir-HEG/Q-EEG and Execution Measures CPTs in a ADHD Assessment Protocol at JoVE.com

Using Brain Activation nir-HEG/Q-EEG and Execution Measures CPTs in a ADHD Assessment Protocol

This work presents a new protocol for the assessment of Attention Deficit Hyperactivity Disorder (ADHD) by providing a more objective diagnostic procedure for this developmental disorder based on the use of innovative tools. It also analyzes the relationship between activation measures and executive function measures.

Using Brain Activation (nir-HEG/Q-EEG) and Execution Measures (CPTs) in a ADHD Assessment Protocol

Attention Deficit Hyperactivity Disorder (ADHD) is a problem that impacts academic performance and has serious consequences that result in difficulties in ...

using-brain-activation-nir-hegq-eeg-execution-measures-cpts-adhd

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.1K Views.  Oviedo University. This work presents a new protocol for the assessment of Attention Deficit Hyperactivity Disorder (ADHD) by providing a more objective diagnostic procedure for this developmental disorder based on the use of innovative tools. It also analyzes the relationship between activation measures and executive function measures.

Video: Using Brain Activation nir-HEG/Q-EEG and Execution Measures CPTs in a ADHD Assessment Protocol

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

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 ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Using Brain Activation (nir-HEG/Q-EEG) and Execution Measures (CPTs) in a ADHD Assessment Protocol

Summary

Explore More Videos

Chapters in this video

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

Studying Brain Function in Children Using Magnetoencephalography

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons