We would like to thank Brain Products GmbH for providing their equipment, expertise and help. This study was funded by the Federal Ministry of Economics and Technology via a grant from the German Space Agency (DLR) 50WB0819.

1. Experimental Procedure
<ol>
 <li>On ground pre-flight preparation - The subject preparation is done in a separate room at the airport. (1-2 hours before the flight)
 <ol>
 <li>Mounting the EEG/NIRS cap
 <ol>
 <li>Electrodes and NIR sensors are attached to the scalp using an EEG cap. This method ensures the correct position of the sensors.</li>
 <li>The size of the cap is determined by the size of the subject's head</li>
 <li>The operator makes sure of the correct position of the cap. The Cz electrode is on the vertex (the point midway between the nasion and onion), the PO9-PO10 and the Fp1-Fp2 electrodes are horizontal, the cap is symmetric.</li>
 <li>The heart rate electrode is placed on the chest</li>
 </ol>
 </li>
 <li>Minimization of Impedance
 <ol>
 <li>The Brain Products actiCAP electrodes are connected to the control box. </li>
 <li>Every electrode contains LEDs, which turn red, when the impedance measurement is started.</li>
 <li>The hair is moved away from the tip of the electrode with a blunt-tipped needle.</li>
 <li>Gel is injected between the tip of the electrode and the surface of the skin.</li>
 <li>The color of the LEDs change, as the impedance decreases. The initial red color becomes yellow, the yellow becomes green, if the target impedance value is achieved.</li>
 <li>The target impedance is 25 kOhm, since the active electrodes provide good signal-to-noise ration below this value. Therefore the cap preparation is quick and convenient.</li>
 <li>The operator starts to work on the reference and ground electrodes, and repeats for all other electrodes.</li>
 </ol>
 </li>
 </ol>
 </li>
 <li>On board pre-flight preparation
 <ol>
 <li>Pre-measurements
 <ol>
 <li>Subjects are placed into the experimental setup, the seat belts are fastened loosely</li>
 <li>Cables are connected, the batteries are loaded. </li>
 <li>The operator starts the EEG and NIRS module, controls the connectivity and the quality of the EEG/NIRS signal.</li>
 <li>Recording resting state EEG/NIRS. The subjects do not have any task.</li>
 <li>The recording is stopped.</li>
 <li>The subjects perform the cognitive task on the ground. The cognitive task is an attention/calculation task (<a href="http://itunes.apple.com/us/app/chalkboard-challenge/id317961833?mt=8">http://itunes.apple.com/us/app/chalkboard-challenge/id317961833?mt=8</a>), where subjects have to identify that side of an equation which is larger than the other in relation to speed and accuracy.</li>
 </ol>
 </li>
 <li>Storing the equipment
 <ol>
 <li>The operator stores the camera and the IPhones for take off.</li>
 </ol>
 </li>
 </ol>
 </li>
 <li>In flight measurement
 <ol>
 <li>Preparation
 <ol>
 <li>Operator mounts the video-camera to handrail and starts recording.</li>
 <li>The iPhones are placed on the upper leg of the subjects.</li>
 <li>The operator starts the EEG and NIRS module, controls the quality of the EEG/NIRS signal, and starts the recording.</li>
 </ol>
 </li>
 <li>Measurement
 <ol>
 <li>The subjects perform the cognitive task during two blocks of five parabolas between parabola 11-15 and 16-20. Task will be performed in a random order in weightlessness or normal gravity. Only resting state EEG/NIRS is recorded during the first 10 parabolas. The last parabolas will be used in case of missing previous measurements (see Figure 1).</li>
 <li>The operator controls the recording, and instructs the subjects. The operator will write down all results of the cognitive tests and times.</li>
 </ol>
 </li>
 </ol>
 </li>
 <li>On ground post-flight measurement
 <ol style="list-style-type:none;">
<li>
 <ol>
 <li>Resting state EEG/NIRS measurement is carried out.</li>
 </ol>
</li>
 </ol>
 </li>
</ol>
We expect to find increased cerebral activation during weightlessness as shown before (Schneider et. al 2008+ 2009). We further expect so see increased oxygenated tissue in the frontal brain in weightlessness and lower oxygenated tissue in hypergravity. The attention task is supposed to be impaired during the whole flight compared to pre and post flight and maybe even more in weightlessness due to higher central activation and arousal in weightlessness.
2. Representative Results
Mapping the transition from the hypergravity phase to weightlessness we were able to observe increased brain cortical activity in the frontal cortex and decreased activity in the temporal and occipital cortex 2000 - 2350 ms after the onset of weightlessness (Figure 2a,b). sLORETA allowed localizing this increased frontal activation in Brodmann area 9 of the dorsolateral prefrontal cortex, which is known to be involved in executive functions with the integration of sensory and mnemonic information in the course of motor planning, organization and regulation, (Figure 3a, b). In addition, subject 2 showed an increase in Brodmann area 6, the premotor cortex, which plays a role in sensory guidance in the course of body stabilization (see Figure 3b).
Averaged over the first 10 parabolas, NIRS analysis revealed decreased oxygenated hemoglobin (HHb) concentration of both subjects in hypergravity as well as increased oxygenated hemoglobin (O2Hb) in weightlessness. For HHb hemoglobin in subject 1 we found a trend of an increase in the hypergravity phase prior to weightlessness as well as a decrease during weightlessness and the hypergravity phase after weightlessness. In this subject O2Hb was back to baseline 10 to 15 sec following the parabola. In contrast subject 2 showed a slight increase along with the decrease of O2Hb in the hypergravity phase prior to weightlessness, an increase during weightlessness and a decrease during hypergravity after weightlessness. For this subject O2Hb remained to be decreased for about 30 sec following parabola (Figure 4a,b)
The cognitive task resulted in decreased performance scores for both participants in normal gravity during the flight compared to a preflight session. Only subject 2 showed a decreased score in weightlessnes (Figure 5).
<img src="/files/ftp_upload/2670/2670fig1.jpg" alt="Figure 1" /> 
Figure 1. Parabolic flight sequence. Order of tasks and measurements during the flight; number of parabolas are indicated in grey, numbers with apostrophe indicate the length of longer breaks between parabolas.
<img src="/files/ftp_upload/2670/2670fig2a.jpg" alt="Figure 2a" /> <img src="/files/ftp_upload/2670/2670fig2b.jpg" alt="Figure 2b" /> 
Figure 2 Mapping view of two subjects over the timeframe of 500 ms before weightlessness (in hypergravity) until 2500 ms in weightlessness. View is from above the head; little circles indicate electrode positions, blue color decreases and yellow to red color increases of electrocortical activity in micro Volt.
<img src="/files/ftp_upload/2670/2670fig3a.jpg" alt="Figure 3a" /> 
<img src="/files/ftp_upload/2670/2670fig3b.jpg" alt="Figure 3b" /> 
Figure 3. Three LORETA views (top: from above, bottom left: from the left side, bottom right: from the back) of two subjects over the timeframe of 2000 ms until 2350 ms after onset of weightlessness. Red color indicates increased brain activity.
<img src="/files/ftp_upload/2670/2670fig4a.jpg" alt="Figure 4a" /> 
<img src="/files/ftp_upload/2670/2670fig4b.jpg" alt="Figure 4b" /> 
Figure 4. NIRS traces (red: oxygenated hemoglobin, blue: deoxygenated hemoglobin, black: gravity level) over the period of one parabola from 40 sec before the parabola in normal gravity (1G: yellow area), over the first hypergravity phase (1.8G: blue area), weightlessness (0G: red area) and the second hypergravity phase (1.8G: blue area) until 40 sec after the parabola. Gravity level is displayed inverse (decrease of trace means increase of gravity starting from 0 equal to normal gravity (1G). Data shown is an average over 10 parabolas.
<img src="/files/ftp_upload/2670/2670fig5.jpg" alt="Figure 5" /> 
Figure 5. Performance score of cognitive task of participant 1 (blue trace) and 2 (red trace) for the measurements training before flight and inflight in weightlessness (0G) and normal gravity (1G).

<ol>
	<li>Santo, N. G. D. e., Cirillo, M., Kirsch, K. A., Correale, G., Drummer, C., Frassl, W., Perna, A. F., Stazio, E. D. i., Bellini, L., Gunga, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anemia+and+erythropoietin+in+space+flights.">Anemia and erythropoietin in space flights.</a> Semin Nephrol. 25, 379-387 (2005).</li><li>Ellis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collisions+in+space.">Collisions in space.</a> , 4-9 (2000).</li><li>Genik, R. J. 2. n. d., Green, C. C., Graydon, F. X., Armstrong, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+avionics+and+watching+spaceflight+crews+think:+generation-after-next+research+tools+in+functional+neuroimaging.+Aviat+Space+Environ+Med+76.">Cognitive avionics and watching spaceflight crews think: generation-after-next research tools in functional neuroimaging. Aviat Space Environ Med 76.</a> , 208-212 (2005).</li><li>Schneider, S., Brummer, V., Carnahan, H., Dubrowski, A., Askew, C. D., Struder, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+happens+to+the+brain+in+weightlessness?+A+first+approach+by+EEG+tomography.">What happens to the brain in weightlessness? A first approach by EEG tomography.</a> Neuroimage. 42, 1316-1323 (2008).</li><li>Schneider, S., Brummer, V., Mierau, A., Carnahan, H., Dubrowski, A., Strueder, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+brain+cortical+activity+during+parabolic+flights+has+no+influence+on+a+motor+tracking+task.">Increased brain cortical activity during parabolic flights has no influence on a motor tracking task.</a> Exp Brain Res. 185, 571-579 (2008).</li><li>Schneider, S., Brummer, V., Gobel, S., Carnahan, H., Dubrowski, A., Struder, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabolic+flight+experience+is+related+to+increased+release+of+stress+hormones.">Parabolic flight experience is related to increased release of stress hormones.</a> Eur J Appl Physiol. 100, 301-308 (2007).</li></ol>

The production of this article was sponsored by Brain Products, GmbH. Roland Csuhaj is an employee of Brain Products, GmbH, which manufactures an instrument used in this article.

Due to missing brain-imaging methods under extreme conditions so far the underlying neurophysiological processes for impairment in cognitive 
performance and mental state have not been evaluated. In this paper we were able to display changes in brain cortical activity and oxygenation level in 
the course of a parabolic flight and to localize these changes within the brain using EEG combined with LORETA and NIRS. As expected, we found an 
increase in electrocortical activity during weightlessness, which was localized in frontal brain regions (Brodmann areas 9+6). Results indicate 
that approximately 2000 ms after the transition brain cortical activity is altered mainly in frontal brain regions. It might be assumed that this 
increased activity in Brodmann area 6 and 9 reflects the mechanisms of the brain detecting and processing changed gravity conditions in order to keep 
body stability as well as motor ability in altered gravity conditions.
Regarding hemodynamic changes, NIRS revealed that the O2Hb of the frontal brain dramatically decreases in the first hypergravity phase and increases in weightlessness, whereas, HHb showed only moderate changes. Accordingly this effect cannot be attributed to a shift of blood volume solely. More likely this seems to reflect a kind of cerebral autoregulation, especially as the increase of O2Hb occurs long before the transition from 1.8 G to 0G (especially in figure 4). In contrast O2Hb and HHB both decrease in the second hypergravity phase.
The results of the cognitive task indicate no clear impairment during normal gravity or weightlessness inflight compared to a preflight session. Based on the results of two subjects no clear statement is possible whether parabolic flights or weightlessness together with its increase in brain activity and oxygenation level have an influence on cognitive performance. Earlier studies give reason to believe that in this context stress might also play a role (Schneider et al. 2007), nevertheless no changes in cortisol concentration could be obtained for both subjects. Further data is needed to validate these findings and to allow correlation of changes in brain cortical activity, hemodynamic changes as well as cognitive performance. 
This paper intended to show that monitoring of local changes in brain cortical activity and oxygenation level throughout different phases of changed gravity is possible by using EEG in combination with NIRS and LORETA. These results are a success for space research and will enable displaying complex and local changes of brain cortical activity in hypergravity or weightlessness and correlating mental or motor test with objective changes in the brain. The next step is to apply this method during long-term space missions.

coherence between brain cortical function and neurocognitive performance during changed gravity conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

Watch this Scientific Journal Video about Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions at JoVE.com

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

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19.4K Views.  German Sport University Cologne. The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Video: Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Subtype-selective Electroporation of Cortical Interneurons

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

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

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

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

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 ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...