CIBC fellowship (JA) and NSERC grant (RZ). We are grateful to Roch M. Comeau (Brainsight) for his help regarding the infrared camera, the MR compatible trackers and other hardware support. We are also grateful to Brian Hynes (Hybex Innovations Inc.) who designed the multi-jointed arm for coil holder and provided some of the figures displayed in the video. And a special thanks to all the MR technicians and M. Ferreira from the McConnell Brain Imaging Centre of the Montreal Neurological Institute who helped us optimizing the design of the experiment.

The protocol is divided in a two-day session (not necessarily consecutive). The first day consists of an fMRI localizer composed with an anatomical and a functional MR scans to define for each participant the areas to be targeted with TMS. The second day consists in the fMRI sessions pre- and post-TMS where TMS is applied inside the scanner using a special MR compatible TMS coil (Magstim Ltd., Wales, UK) and a frameless stereotactic system (Brainsight). The latter is used to position in real-time the TMS coil on cortical areas relative to each participant's anatomical and functional data.
1. Localizer Session
<ul>
 <li>Start with acquiring a high-resolution anatomical image of your participant.</li>
 <li>Then, acquire functional images using a gradient echo EPI pulse and a sparse sampling paradigm in order to minimize any BOLD effect or auditory masking due to MRI scanning noise 14,15. In our case, fMRI is carried out during a melody task in which participants have to determine if two consecutive 5-note melodies are the same or different 2,16. A nondiscrimination auditory control task is also included, in which subjects hear two equal-length patterns of five notes, all at the same pitch of C5 and are instructed to click the left button following the second stimulus. Periods of silence are also inserted randomly among the task trials in each run. In total, 72 trials are presented in a randomized order: 24 trials of melody discrimination, 24 auditory control trials and 24 periods of silence, for a total duration of 12 min 16 sec. </li>
 <li>Define the stimulation site using anatomical and/or functional landmarks. One has to be aware that TMS is limited regarding the depth of the stimulation site because of the attenuation of the electric field strength in depth, and cannot expect to reach areas deeper than 3 cm6,17 . A crucial step is to use similar landmarks for each participant, which could be difficult because of the differences in anatomy and function between participants. Here, we target Heschl's gyrus in each participant, located using both anatomical and functional landmarks. We use masks of Heschl's gyrus provided by the Harvard-Oxford structural atlases (<a href="http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html" target="_blank">http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html</a>) and the TMS target is defined individually by the peak of activation within the Heschl's gyrus 2. In addition, we also define the vertex position, which will be used as a control site to control for non-specific effects of TMS such as acoustic and somatosensory artifacts. The vertex is defined anatomically as a point midway between the inion and the bridge of the nose, and equidistant from the right and left intertragal notches. The order of site of stimulation (i.e. Heschl's gyrus or vertex) is counterbalanced across individuals.</li>
</ul>
2. Pre- and Post-TMS fMRI Experiment
Pre-TMS fMRI session
<ul>
 <li> Prepare the participant to go directly in the scanner. This includes removal of metal and filling the TMS and MR screening form.</li>
 <li>Start the MR acquisition with an anatomical and a functional scans (identical to the one carried out in the localizer session, see section 1).</li>
</ul>
Frameless stereotaxy and TMS in the MRI environment
The frameless stereotaxy system is composed of an infrared camera (Polaris Spectra), some tools and trackers (Brainsight) used for the registration procedure and a computer. The computer is located outside of the scanner room, but positioned at the entrance of the scanner room and the scanner door is kept opened during the TMS application. The tools and trackers are MR compatible, as well as the tripod (home-made) supporting the infrared camera and are therefore used inside the scanner room. The infrared camera is not MR-compatible, and therefore is positioned inside the scanner room, near the scanner door at approximately two meters from the scanner bed (see discussion for safety procedure). The TMS stimulator system is located in a room adjacent to the MRI scanner room. We use an MRI compatible TMS coil located inside the scanner room and connected to the TMS system via a 7-m cable through an RF filter tube.
<ul>
 <li> Load your participant's anatomical and functional images and the stimulation targets into the stereotactic software package (e.g. Brainsight). Here, we will be targeting the right Heschl's gyrus.</li>
 <li> After the pre-TMS fMRI acquisition, remove the upper MR head coil part of the 32-channel head coil (if using the Siemens 3T scanner and the 32-channel head coil configuration).</li>
 <li> Next, slide down the participant on the scanner bed.</li>
 <li> Fix the headband and tracker set on the participant's head.</li>
 <li> Mount the multi-jointed arm to the scanner bed and fix the MR compatible TMS coil onto the arm. </li>
 <li> Verify that all the trackers and the coil are in the field of view of the camera. Here, the camera is slightly moved to the right side of the participant to enable an easier tracking of the coil displacements when targeting the right hemisphere. </li>
 <li> Calibrate your subject's head with the stereotaxy tools (i.e. pointer tool). This is done by coregistering several landmarks on the participant's head (e.g. in our case the tip of the nose, the nasion and the tragus of both ears) with the same landmarks on the anatomical data. In this procedure, two experimenters are needed, one close to the participant's head to position the pointer tool on the participant's head, and the other experimenter at the entrance of the scanner room to perform the registration on the computer.</li>
 <li> Position the MR compatible TMS coil tangential to the scalp, and the coil trackers directed towards the infrared camera. The coil is oriented with the coil handle pointing backward and parallel to the midline 2. Fix the coil position using the screws on the multi-jointed arm. </li>
 <li> In the room adjacent to the MRI scanner, turn on the TMS system and begin stimulation. TMS is applied following a patterned protocol, i.e., continuous theta burst stimulation (cTBS) consisting in 3 pulses at 50Hz, repeated at 5Hz for 40s. We use a fixed stimulation intensity (41%) defined by the stimulator output 18,19. We chose this protocol as it has been shown to modulate cortical plasticity for a duration up to 30 min after stimulation cessation in healthy populations 20, (see discussion section for safety procedure). </li>
</ul>
Post-TMS fMRI session 
<ul>
 <li>Once stimulation is complete, it is important to get the subject back into the scanner as soon as possible. Remove the TMS coil from the scanner room, and remove the multi-jointed arm. Slide back the participant's head into the MR head coil. Ensure your scanner is prepared and ready to go. Our advice is to keep the body platform raised during the whole TMS session, and reduce the number and duration of localizer scans to a minimum.</li>
 <li>Because the effects of rTMS are transitory, the final scanning session should begin with the functional scan. Again, we conducted fMRI during a 12-min run of the melody task.</li>
 <li>After the final scan is complete, finish with an anatomical scan.</li>
</ul>
3. Representative Results
Analyses of fMRI data are conducted separately for both the pre- and post-TMS fMRI session. For each fMRI session (i.e., pre and post-TMS), the contrast between the melodies and the auditory control task shows task-related activity in the left and right Heschl's gyri, superior temporal gyri, inferior frontal gyri and precentral gyri (Figure 1 A, B). To evaluate differences between pre- and post-TMS fMRI sessions, we perform a random-effect analysis using Student's paired t-test. Significance is determined using clusters identified by a z &gt; 2 threshold and a corrected cluster threshold of p = 0.05. Figure 1 C represents the contrast post- minus pre-cTBS for a single participant. The data suggest that cTBS targeting the right Heschl's gyrus (black circle) induces an increase in fMRI response in the contralateral (left) auditory cortex, including the left Heschl's gyrus. Changes in fMRI response are also found in the left postcentral gyrus, left insula, and in the lateral occipital cortex bilaterally. However, no significant change in fMRI response is seen under the coil. In addition, similar combined TMS-fMRI protocol is repeated to stimulate the vertex (control site). Comparison of pre- and post-fMRI sessions with cTBS applied over the vertex did not show any significant effect (data not shown).
<img src="/files/ftp_upload/3985/3985fig1.jpg" alt="Figure 1"/> 
Figure 1. Analysis of individual pre-TMS fMRI data (A), post-TMS fMRI data (B) and post- minus pre-TMS fMRI data (C). A. Results of the contrast melody discrimination minus auditory control trials for a single participant in the pre-TMS fMRI session (A) and in the post-TMS fMRI session (B). From left to right: axial, coronal and sagittal views. In both (A) and (B), the TMS coil is targeting the right Heschl's gyrus (black circle) located at x=54, y=-13, z=1 (MNI152 standard space). For both pre- and post-TMS fMRI sessions, coordinates are displayed at x=-54, y=-13, z=1 (MNI152 standard space) to show changes in the left hemisphere at the site of stimulation (i.e. right Heschl's gyrus). C. Results of the contrast post- minus pre-TMS fMRI sessions using Student's paired t-test.

No conflicts of interest declared.

We describe a protocol combining offline TMS and fMRI to investigate the functional organization of the auditory cortex. In the next sections, we will discuss the methodological factors to consider when conducting such approach. 
Acquisition and timing for post-TMS fMRI session
Order of scans acquisition and counterbalancing of pre- and post- TMS fMRI sessions
It is crucial to acquire an MR anatomical scan before and after TMS in o...

<table border="1">
 <tbody>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>Transcranial magnetic stimulation</td>
 <td>Magstim super Rapid2 stimulator, Rapid-2 Plus One Module </td>
 <td>Magstim Ltd., Wales, UK </td>
 </tr>
 <tr>
 <td>Coil for magnetic stimulation</td>
 <td>MRI-compatible 70 mm figure-of-eight-coil </td>
 <td>Magstim Ltd., Wales, UK </td>
 </tr>
 <tr>
 <td>Magnetic resonance imaging</td>
 <td>3-T Siemens Trio scanner, 32-channel Head Coil </td>
 <td>Siemens, Inc., Germany</td>
 </tr>
 <tr>
 <td>Frameless Stereotaxy</td>
 <td>Brainsight </td>
 <td>Rogue Research Inc., Montreal, Canada </td>
 </tr>
 <tr>
 <td>Optical measurement system</td>
 <td>Polaris Spectra </td>
 <td>Northern Digital Inc, Ontario, Canada </td>
 </tr>
 <tr>
 <td>Multi-jointed arm for coil holder</td>
 <td>Standard</td>
 <td>Hybex Innovations Inc., Anjou, Canada</td>
 </tr>
 <tr>
 <td>MRI-Compatible Insert Earphones </td>
 <td>Sensimetrics, Model S14 </td>
 <td>Sensimetrics Corporation, MA, USA </td>
 </tr>
 </tbody>
</table>

mapping the after-effects of theta burst stimulation on the human auditory cortex with functional imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Watch this Scientific Journal Video about Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging at JoVE.com

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

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13.8K Views.  McGill University. עיבוד שמיעתי הוא בסיס לדיבור ועיבוד הקשורים למוסיקה. גרייה מגנטית (TMS) שמשה בהצלחה ללמוד מערכות קוגניטיביים, חושיות ומוטוריות, אך רק לעתים נדירות יושמה לאודישן. כאן אנו חקרנו TMS בשילוב עם סורק תהודה מגנטי תפקודי כדי להבין את הארגון התפקודי של קליפת המוח השמיעתי.

Video: Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...