We thank Hubertus Lohmann for sharing his expertise in fTCD techniques. This work was funded by Wellcome Trust programme grant no. 082498/Z/07/2.

1. Using fTCD to Assess Language Lateralization in Children
<ol>
 <li> Where possible, we familiarise the child with pictures showing characters from the video stories in a separate session before introducing them to Doppler system.</li>
 <li> It is important that the procedure includes sufficient trials to get a reliable measure: ideally 20 or more, though we have obtained useful data with as few as 10 to 15 trials. Each trial involves a period of relaxation, to be used as a baseline, followed by language activation. Blood flow velocity during the period of activation is compared against the baseline. We have found that it is possible to use the period while the child passively watches a 12 second video clip as a baseline. This helps avoid boredom. The baseline is followed by a 10 second response period in which the child is asked to describe what happened. This is followed by a 16 second rest period.</li>
 <li> The timing of the procedure is important in order to detect changes in blood flow velocity due to metabolic demands, which may take up to 7 seconds. We use computerised tasks, written in Presentation or Matlab, which control the timing of each trial. Critically, these programs also send pulses to the hardware which records a marker in the Doppler data file to indicate onset of the activation period. These are used in the data analysis to determine periods of baseline and activation blood flow.</li>
 <li> We use a Multi-dop system which enables ultrasound recording from left and right sides simultaneously. It is best to have the Multi-dop system set up and ready to go before the participant arrives. Make sure the probes are connected to the Multi-dop system before switching on. Check that the stimulus computer is connected, via parallel port, to the Multi-dop computer and pulses from one are seen on the other. Ensure the highpass filter is set to 300 Hz for both sides and check the depth of insonation (45-55 mm) and the power of the signal. The system has limits set on the different parameters to ensure only safe levels can be used.</li>
 <li> A participant code should be recorded in the Multi-dop data files in order for condition, group, or demographic data matching.</li>
 <li> The system should then be paused while preparing the probes. Each ultra-sound probe is covered with gel which provides a contact between the probe and the skin. Use a generous amount of gel to maintain the best contact possible.</li>
 <li> Now it is time to start the session. Check whether the child needs to go to the toilet before starting! We demonstrate the headset using toy animals, and make sure that the child understands about what is going to happen and what they will be asked to do. It is particularly important that they understand the need to keep quiet after the activation period, so that the signal can return to baseline. Children should be seated comfortably as a full task may last between 20 and 30 minutes and a lot of gross body movements can interfere with the Doppler signal.</li>
 <li> The hardest part of the procedure is the positioning of the gel-covered probes. New users should be aware that it takes practice to become expert at this part of the process. There is a useful computer simulation by Rune Aaslid that can help with training (<a href="http://www.transcranial.com/">http://www.transcranial.com/</a>), but the best way to learn is by practising on friends and colleagues. Once you have assessed about 10 people, you will start to find it much easier. One good thing about working with children is that it is usually easier to find a signal from a child than an adult, because they have thinner skulls.</li>
 <li> Each probe is placed at the temporal window of the skull on each side of the head. This is the thinnest section of bone in the head and provides the best area through which a signal can be obtained from the middle cerebral arteries. These windows can be found just forward of each ear. When working with children, it can help reduce the set-up time if you have two experimenters, one adjusting each probe. Most children are happy to watch a DVD during this part of the procedure, but it is important to monitor the child's behavior and ensure they are comfortable and relaxed. Terminate the session if the child requests this or if there is any sign of distress.</li>
 <li> The position of the probes is then adjusted in order to insonate the middle cerebral artery. It may take a few minutes before a satisfactory signal is found. This signal has a characteristic sound as well as visual pattern which should be monitored from the Multi-dop computer.</li>
 <li> The characteristic sound is a low whooshing noise of the rate of the participant's heart beat. The characteristic visual pattern is a sharp rise followed by an exponential decay fall off which also repeats at the rate of the heart beat. You may pick up activity from other blood vessels, depending on the depth of insonation and direction of the probe. A depth of approximately 45 to 50 mm usually works well in children. Once a strong signal has been found, reduce the power as much as possible while maintaining a good signal. The gain can also be increased to amplify a weak signal.</li>
 <li> Once the signals for each middle cerebral artery are satisfactory, the auditory output of the Multi-dop signal should be turned down, and the procedure can begin. The experimenter explains to the child that they are going to watch a video clip quietly. When they see the question mark come up on the computer they need to tell the experimenter as much as they can about what happened in the video. When they see the picture of the boy going 'shhh' they have to stop talking immediately and sit quietly to wait for the next video to start (see Figure 2 and .avi examples).</li>
 <li> Recording devices are then started, i.e., a voice recorder for auditory responses; along with the Multi-dop system recording; and the computer controlling stimulus presentation.</li>
 <li> The experimenter's job is then to monitor the child and Doppler signal to a) make sure the child is comfortable and attending to the task and b) ensure that a suitable signal is maintained. The Doppler signal is far less sensitive to movement than an fMRI signal, but it can still be disrupted by gross body movements, or if the probe moves. If the signal becomes significantly disrupted, the display computer should be paused and the probes reset appropriately. At times it may be necessary to apply more gel in order to gain re-establish a satisfactory signal. We find that testing of children works best if one experimenter is responsible for keeping the child on task and recording what they say, and the other monitors the Doppler signal.</li>
 <li> When the experimental procedure is finished, the Multi-dop recording can be stopped and the probes removed. It is useful to have some tissues available to remove any excess gel.</li>
 <li> The raw data files are processed in order to compare the left and right Doppler signals. We have developed our own Matlab software based on the methods developed by Deppe7. This processing involves down-sampling the data and correcting for fluctuations due to heart beat. Single trials are excluded from the analysis if the left or right activation is outside a specified range, commonly 70 to 130% of baseline blood flow velocity. The activation in these trials is likely to be affected by problematic probe contact. Figure 3 shows activation relative to probe signals for a good signal, whereas Figure 4, shows a case where the signal is periodically lost.</li>
 <li> Relative to baseline blood flow velocity, activation in the right channel is subtracted from the left in order to calculate the difference activation (see Figure 5). The lateralization index is calculated as the average of the difference activation for one second either side of the maximum difference within a pre-defined period of interest. This period of interest is 7 to 17 seconds following the start of the 'talk' command, indicated by the marker sent from the display computer to the Multi-dop data file. As the difference activation is the left minus right channels, positive lateralization indices reflect left lateralization and negative reflect right.</li>
</ol>

2. Representative Results
<img src="/files/ftp_upload/2161/2161fig1.jpg" alt="Figure 1" /> 
Figure 1: Outer surface of cerebral hemisphere, showing areas supplied by cerebral arteries. Pink is region supplied by middle cerebral artery. (from: <a href="http://en.wikipedia.org/wiki/File:Gray517.png">http://en.wikipedia.org/wiki/File:Gray517.png</a>)
<img src="/files/ftp_upload/2161/2161fig2.jpg" alt="Figure 2" /> 
Figure 2: Schematic outline of experimental trial with 12 second baseline video, 10 second response interval, and 16 second rest phase. Period of interest for laterality calculation is also depicted.
<img src="/files/ftp_upload/2161/2161fig3.jpg" alt="Figure 3" /> 
Figure 3. Clean data recording of raw Doppler velocity (cm/sec) for the Left (blue) and Right(red) Doppler channels from 130 to 150 seconds during a single experimental session. Each visible pulse corresponds to one heart beat. Event marker indicating stimulus onset displayed in green.
<img src="/files/ftp_upload/2161/2161fig4.jpg" alt="Figure 4" /> 
Figure 4. Disrupted data recording of raw Doppler velocity (cm/sec) for the Left (blue) and Right (red) Doppler channels from 400 to 450 seconds during a single experimental session. Event marker indicating stimulus onset displayed in green. Significant evidence of dropout is visible for both channels.
<img src="/files/ftp_upload/2161/2161fig5.jpg" alt="Figure 5" /> 
Figure 5. Left (blue) and right (red) channel (Panel A) and left minus right difference (Panel B) Doppler Velocity (cm/sec) averaged across all acceptable trials for a group of participants. Participants were cued to "Talk" at 12 seconds and the period of interest for the laterality calculation is also depicted in green. The grey error bars surrounding the difference velocity in Panel B represent the standard error of the mean.
Avi examples: the video sequences to tell the "Freezefoot" story can be downloaded from: <a href="http://psyweb.psy.ox.ac.uk/oscci/Miscellaneous.htm">http://psyweb.psy.ox.ac.uk/oscci/Miscellaneous.htm</a>

<ol>
	<li>Bishop, D. V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handedness and developmental disorder. , (1990).</li><li>Pelletier, I., Sauerwein, H., Lepore, F., Saint-Amour, D., Lassonde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+alternatives+to+the+Wada+test+in+the+presurgical+evaluation+of+language+and+memory+functions+in+epilepsy+patients.">Non-invasive alternatives to the Wada test in the presurgical evaluation of language and memory functions in epilepsy patients.</a> Epileptic Disorders. 9, 111-126 (2007).</li><li>Deppe, M., Ringelstein, E. B., Knecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+investigation+of+functional+brain+lateralization+by+transcranial+Doppler+sonography.">The investigation of functional brain lateralization by transcranial Doppler sonography.</a> NeuroImage. 21, 1124-1146 (2004).</li><li>Knecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+determination+of+language+lateralization+by+functional+transcranial+Doppler+sonography+:+A+comparison+with+the+Wada+test.">Noninvasive determination of language lateralization by functional transcranial Doppler sonography : A comparison with the Wada test.</a> Stroke. 29, 82-86 (1998).</li><li>Lohmann, H., Drager, B., Muller-Ehrenberg, S., Deppe, M., Knecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Language+lateralization+in+young+children+assessed+by+functional+transcranial+Doppler+sonography.">Language lateralization in young children assessed by functional transcranial Doppler sonography.</a> Neuroimage. 24, 780-790 (2005).</li><li>Bishop, D. V. M., Watt, H., Papadatou-Pastou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+reliable+method+for+measuring+cerebral+lateralization+during+speech+with+functional+transcranial+Doppler+ultrasound.">An efficient and reliable method for measuring cerebral lateralization during speech with functional transcranial Doppler ultrasound.</a> Neuropsychologia. 47, 587-590 (2009).</li><li>Deppe, M., Knecht, S., Henningsen, H., Ringelstein, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AVERAGE:+a+Windows+program+for+automated+analysis+of+event+related+cerebral+blood+flow.">AVERAGE: a Windows program for automated analysis of event related cerebral blood flow.</a> Journal of Neuroscience Methods. 75, 147-154 (1997).</li><li>Whitehouse, A. J. O., Bishop, D. V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+dominance+for+language+function+in+adults+with+specific+language+impairment+or+autism.">Cerebral dominance for language function in adults with specific language impairment or autism.</a> Brain. 131, 3193-3200 (2008).</li><li>Illingworth, S., Bishop, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+cerebral+lateralisation+in+adults+with+compensated+developmental+dyslexia+demonstrated+using+functional+transcranial+Doppler+ultrasound.">Atypical cerebral lateralisation in adults with compensated developmental dyslexia demonstrated using functional transcranial Doppler ultrasound.</a> Brain and Language. 111, 61-65 (2009).</li><li>Whitehouse, A. J. O., Bishop, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemispheric+division+of+function+is+the+result+of+independent+probabilistic+biases.">Hemispheric division of function is the result of independent probabilistic biases.</a> Neuropsychologia. 47, 1938-1943 (2009).</li><li>Whitehouse, A. J. O., Badcock, N., Groen, M. A., Bishop, D. V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+a+novel+paradigm+for+determining+hemispheric+lateralization+of+visuospatial+function.">Reliability of a novel paradigm for determining hemispheric lateralization of visuospatial function.</a> Journal of the International Neuropsychological Society. 15, 1028-1032 (2009).</li></ol>

No conflicts of interest declared.

As well as studying normal development, we have been using fTCD to study language lateralization in adults and children with developmental disorders of language and literacy 8, 9. We are also interested in developing methods of using fTCD for assessment of lateralization of visuo-spatial abilities10, 11.

A correction was made to <a href="http://www.jove.com/video/2161">Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)</a>. There was a misstated time interval for the point of interest.

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

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<td>Multi-Dop T with Upgrade to 2 Channel MultiFlow Monitoring</td>
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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.

Watch this Scientific Journal Video about Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD at JoVE.com

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.

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 ...

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20.1K Views.  University of Oxford. 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. 

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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 ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

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, ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

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 ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...