This work was supported by Australian Research Council Linkage Infrastructure Equipment and Facilities Grant LEO668421 and Australian Research Council Linkage Project Grant LP0669471. The authors gratefully acknowledge the collaboration of Kanazawa Institute of Technology and Yokogawa Electric Corporation in establishing the KIT-Macquarie Brain Research Laboratory.

These guidelines describe equipment and procedures for measuring cognitive brain function in preschoolers using magnetoencephalography (MEG). First, we discuss general guidelines and issues that must be considered when carrying experimental tests of cognitive function in children. Second, we describe a protocol developed to align the requirements of MEG data collection with the motivational and behavioral capacities of four-year-old children.
1. General considerations for studying young children with MEG
MEG researchers face a number of unique challenges when working with awake healthy children. First, a stark and functional laboratory environment is likely to intimidate or frighten young children. Second, the basic requirements of an experimental MEG study include restriction of movement and actively attending to a task for an extended period, conditions that many adults find tedious and uncomfortable. When studying children it becomes imperative to adapt the experimental procedures to the capacities and limitations of the children. In this section, we describe the initial steps involved in preparing the subject to participate in a data acquisition session.
1.1 Familiarization/Training Session: For pre-school children a familiarization/training session is scheduled prior to the actual data acquisition session. This session allows the child to become familiar with the environment and the researchers at their own pace. We introduce the child and the parent to the researchers and familiarize them with the lab surroundings and routines. By the end of the introductory session the parent and child understand all the steps involved in participating and understand what they should expect during data recording. If during the first visit, a child appears shy or anxious, we have a period of playtime until they feel more comfortable. Then, we proceed with the introduction of all the steps.
1.2 Explaining the environment: To explain the MEG environment to the child, we employ a theme that provides a child-friendly justification for the surroundings. For instance, the MEG system is a spaceship waiting to take the children on a space adventure where some unfamiliar events may occur. The laboratory has also been decorated with wall stickers and toys (Fig. 1), and it has a dedicated playroom. When talking to the child, we use language that children can easily understand such as 'freezing' instead of 'minimizing movement', and 'astronaut helmet' instead of 'marker-coil cap'.
1.3 Explaining the procedures to the child and to the parents: We show the child and the parent the steps that are taken before the child goes inside the magnetically shielded room (MSR). One of the first steps is to fit a five marker-coil cap on the subject's head; these coils indicate the position of the subject's head inside the MEG helmet. With the child's help, we digitize the head shape of a puppet, and then we invite the child and the parent to accompany the puppet to the MSR/spaceship. Once inside the MSR, we place the puppet's head inside the helmet to watch a movie. We then ask the child if they want to watch the movie with the puppet. If the child is comfortable and relaxed and their parent is satisfied with our protocols, we invite them to play a game inside the spaceship the next time they visit our lab.
1.4 Workbooks: We have workbooks based on the space adventure theme that shows the steps children will take during the testing session. Children are given these worksbooks to take home and work through them with their parents in preparation for the next visit. The aim of the workbooks is to emphasize the importance of the three major requirements for testing: a) the avoidance of any ferrous material inside the MSR, b) the placement of marker coil cap to ensure accurate data co-registration and c) and the importance of remaining still during digitization and data acquisition.
2. A protocol for measuring MEG signals from pre-school aged children
2.1&nbsp; Equipment used during data acquisition and experimental task
Marker-coil cap: in order to align MEG data with the subject's structural information (e.g. MRI or digitized head shape), a set of five coils are placed in a swimming cap that the child wears inside the MEG helmet. These coils act as reference points and their positions are compared before and after the task to measure how much the subject's head has moved during acquisition.
Digitizer: Prior to data acquisition, the position of five marker coils and the head shape of the subject is digitized using a Polhemus Fastrak&reg; digitizer (Colchester, VT). The digitizer consists of a pen digitizer, three receivers, a transmitter and a motion tracking unit. This process measures the location of the coils. Later these locations are matched against the location information obtained by MEG to align the acquired magnetic data and head shape.
Child MEG system: Data acquisition is done using a whole-head child MEG system (KIT, Kanazawa, Japan) [2]. Data is acquired continuously at a sample rate of 1000Hz, with an online band pass filter between 0.03 Hz and 200Hz. Marker coil positions are obtained before and after recordings to measure head movement. Data are rejected if head movement is greater than 5mm.
MEG system peripherals: Experimental stimuli are controlled with a PC using Presentation&reg; (Neurobehavioral Systems, Albany, CA). Visual inputs are delivered by a projector that sits outside the MSR and is reflected onto the MSR screen. Auditory stimuli are delivered by Etymotic Research Model ER-30 insert earphones fitted with baby-sized foam earpieces (Etymotic Research Inc., Elk Grove Village, IL). The child is constantly monitored by closed circuit camera and the experimenter communicates with the child through a microphone.
2.2 Data acquisition: Data recording sessions involve the following stages:
Stage I: Prior to child's arrival
It is important to have all computers, software and equipment ready to start the experiment as soon as the child arrives. As a general recommendation, two experienced researchers should be present while the child and parent are in the laboratory. Clear communications between researcher, parent and child are crucial for the success of the experiment.
Stage II: After the child arrives
<ol>
 <li>Consent forms are signed and the experimenters check if the parents have questions. </li>
 <li>The experimenter checks if the child remembers all the steps introduced during the first meeting or in the booklets and reintroduces any that are not remembered. The importance of freezing or minimizing movement during digitization and data acquisition is emphasized. This can be demonstrated using a puppet.</li>
 <li>Then, the experimenter checks if the child is carrying any ferrous material (clothing with glitter patches, jewelry, toys forgotten inside pockets, hair pins, etc.). If they carry a piece of clothing that contains ferrous material, we provide them with MEG safe clothing. If the parent wishes to accompany the child during the experiment, we ensure that they also remove their ferrous material.</li>
</ol>
Stage III: Getting the child ready to perform the task inside the MSR
<ol>
 <li>The experimenter fits the cap that holds the 5 marker coils.</li>
 <li>The experimenter digitizes the location of the marker coils and the child's head shape. In order to maintain heads in a fixed position, we use a neck brace which has been fitted with three receivers. It is important to reinforce the idea of minimizing movement or 'freezing' regularly to obtain a good head shape. The child may be rewarded with a sticker for doing a good job at 'freezing'. </li>
 <li>The child is invited to enter the MSR. Earphones are inserted and the child is positioned in the MEG dewar helmet in a supine position. </li>
 <li>Once the child is lying down comfortably with their heads inside the MEG helmet, the marker coils are connected to their power unit and the MSR door is closed. One experimenter remains (and also a parent, if desired) in the room during the entire session. </li>
 <li>For entertainment during setup and between data collection blocks, a movie or cartoon can be projected onto the MSR back-projection screen.</li>
</ol>
Stage IV: Running the experiment
<ol>
 <li>Once the MSR door is closed, SQUIDs are engaged and data acquisition starts.</li>
 <li>As the child watches a movie, a coregistration pre-test is conducted to make sure that MEG signals can be coregistered to the digitized head shape. This pretest consists of matching the position of the marker coils obtained during digitization with the position shown by the MEG system.</li>
 <li>Once the first test shows that the child's head is correctly positioned inside the helmet, the researcher can start the experiment: (a) the movie is muted; (b) the researcher asks the child if they are ready to "play the game"; (c) once the game starts, the experimenter regularly provides the child with feedback. For example, a child may be asked to listen to a series of sentences, and to repeat a target word such as 'cheese', as soon as they hear it. These target words are embedded in a subset of catch trials that probe continued attentiveness to the task but are not included in the final analyses. </li>
 <li>Once the task is completed, another marker coil measurement is performed to monitor head position and movement. </li>
 <li>Once the SQUIDs are disengaged, the MSR door is opened and the child is invited to a playroom where they can choose a prize for their good work.</li>
</ol>
3. Representative results
A representative study in our laboratory used this protocol to study the development of logic in child language. Twenty-six children aged 3-4 years (mean age 4 years and 5 months) participated in at least one acquisition session. Seventeen of those children returned for a second testing session and five came for a third testing session. Average head movement was less than 3.7 mm (s.d. 3.1 mm), well within the 5mm threshold. Movements greater than 5mm occurred in 4.5% of the sessions, with variation ranging from 5.5 mm to 15 mm (Mean= 8.6mm, s.d.= 2.5 mm).
<img src="/files/ftp_upload/1693/1693fig1.jpg" alt="Figure 1" /> Figure 1. The MSR and MEG helmet have been decorated with stickers and soft toys.
<img src="/files/ftp_upload/1693/1693fig2.jpg" alt="Figure 2" /> Figure 2. Four year-old girl in adult-sized MEG (left). Note the padding at the top of her head to minimize movement and the distance between head and helmet. Right picture shows same girl in the child MEG.

<ol>
	<li>H&#228;m&#228;l&#228;inen, M., Hari, R., Ilmoniemi, R. J., Knuutila, J., Lounasmaa, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalography-+theory,+instrumentation,+and+applications+to+noninvasive+studies+of+the+working+human+brain.">Magnetoencephalography- theory, instrumentation, and applications to noninvasive studies of the working human brain.</a> Rev. Mod. Physics. 65 (2), 413-497 (1993).</li><li>Johnson, B. W., Crain, S., Thornton, R., Tesan, G., Reid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+brain+function+in+pre-school+children+using+a+custom+sized+whole-head+MEG+sensor+array.">Measurement of brain function in pre-school children using a custom sized whole-head MEG sensor array.</a> Clin. Neurophysiol. , (2009).</li><li>Kado, H., Higuchi, M., Shimogawara, M., Haruta, Y., Adachi, Y., Kawai, J., Ogata, H., Uehara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalogram+system+developed+at+KIT.">Magnetoencephalogram system developed at KIT.</a> IEEE Trans. Appl. Supercond. 9, 4057-4062 (1999).</li><li>Uehara, G., Adachi, Y., Kawai, J., Shimogawara, M., Higuchi, M., Haruta, Y., Ogata, H., Kado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Channel+SQUID+Systems+for+Biomagnetic+Measurement.">Multi-Channel SQUID Systems for Biomagnetic Measurement.</a> IEICE Trans. Electr. E86-C, 43-54 (2003).</li></ol>

This protocol is designed to facilitate measurements of cognitive brain function in healthy and awake 4 year-old children. We use a custom-sized MEG system designed to fit the heads of pre-school aged children. With this system, a child-friendly laboratory environment, and a protocol adapted to the capabilities and motivations of children, we are able to measure cognitive brain function in an age range for which there is presently very little data.
The KIT-Macquarie system is the first whole-head system custom sized for use with pre-school aged children. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children s heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children (see Fig. 2). These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. These features significantly facilitate setup, positioning, and measurement of MEG signals from children.
The advent of custom sized child MEG system is an important advance for cognitive neuroscience. Alternative functional neuroimaging techniques that employ radiopharmaceuticals or strong magnetic fields may not be suitable for routine use with young children. Since MEG is an entirely passive measurement technique there is no conceivable risk to developing tissues. There is currently very little MEG data from healthy preschool aged children, because conventional adult MEG systems are not well-suited to used with children. Child MEG systems will allow us to study brain function at at ages where a dramatic structuring of brain activity is occurring, providing new insights into normal and pathological development of cognitive function.

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Whole head child MEG</td>
<td>&nbsp;</td>
<td>Kanazawa Institute of Technology (Kanazawa) and Yokogawa Electric Corporation (Tokyo)</td>
<td>Model PQ1064R-N2m</td>
<td>&nbsp;</td>
<tr>
<td>Magnetically shielded room</td>
<td>&nbsp;</td>
<td>Fujihara Co. Ltd., (Tokyo)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Digitiser</td>
<td>&nbsp;</td>
<td>Polhemus (Colchester, VT)</td>
<td>Fastrack</td>
<td>&nbsp;</td>
<tr>
<td>Experimental control software</td>
<td>&nbsp;</td>
<td>Neurobehavioral Systems (Albany, CA)</td>
<td>Presentation</td>
<td>&nbsp;</td>
<tr>
<td>Earphones</td>
<td>&nbsp;</td>
<td>Etymotic Research Inc. (Elk Grove Village, IL)</td>
<td>ER-30</td>
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measurement of neuromagnetic brain function in pre-school children with custom sized meg

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

Watch this Scientific Journal Video about Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG at JoVE.com

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

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16.8K vues.  Macquarie University. L'avènement de systèmes MEG taille pour les jeunes enfants ouvre de nouvelles possibilités importantes pour étudier le développement du cerveau. Le nouveau système, avec un protocole qui aligne les exigences expérimentales avec les capacités des enfants, peuvent être utilisées pour étudier les processus cognitifs et linguistiques dans la santé, les enfants éveillés trois à six ans.

Vidéo: Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...