The work was supported by the NIH/National Center for Complementary and Alternative Medicine (R21AT00978) (1-P01-002048-01) (K01-AT-002166-01), the National Center for Research Resources (P41RR14075), the Mental Illness and Neuroscience Discovery Institute (MIND) and the Brain Project Grant (NS 34189).

Part 1: Informed consent and subject preparation
Healthy adult volunteers are recruited for the study according to Institutional Review Board approved procedures. Informed consent by the subject is signed before beginning the study.
Part 2: Setting up physiological monitoring equipment
The AD Instruments PowerLab system allows for physiological data to be synchronized with the fMRI scan by using the fMRI trigger signal recorded on an input channel. This is a laptop based system with inputs for ECG, respiration, skin conductance response, pulse oximetry, and any other physiological signal that would need to be recorded.
<ol>
	<li>If a respiratory belt system is being used, the transducer must be plugged into a power outlet in the room adjacent to the scanner, with the tube leading to the belt fed via inlet into the scan room and a BNC connector leading to the PowerLab in the scan control room.</li>
	<li>If a trigger is being used to synchronize the physiological data to the fMRI data, then a BNC cable must also connect the MRI scanner trigger signal to the PowerLab in the scan control room.</li>
	<li>Gather monitoring devices. Four ECG electrodes will be needed, as well as an ECG cable connected to an MR-compatible In Vivo system rollcart.</li>
	<li>If skin conductance is being measured, plug the skin conductance connectors into the breakout box in the scan room, and have skin conductance gel ready in the scan room.</li>
</ol>
Part 3: Preparing subject for MR scanning
<ol>
	<li>It is important to rule out undue anxiety and fatigue on the day of the scan because basic state can affect the response of the brain to acupuncture stimulation. Suggest the subject eat a light meal prior to the procedure so that weakness and untoward reactions can be avoided.</li>
	<li>Instruct the subject to lie supine in the machine wearing ear plugs to suppress scanner noise and with the head immobilized by cushioned supports. Since the signal/noise ratio of acupuncture stimulation scans is low compared with common motor or visual tasks, the data tends to be more easily corrupted by head motion. A support is placed under the knees to keep the heels form the surface of the stretcher to reduce body motion. The head coil is fastened and cushions are adjusted for comfort. For an average scanning session of 2 hours, comfort is vital for uninterrupted scanning.</li>
	<li>If physiological monitoring is desired, subject should be prepared according to the previous section before being put into the scanner. It is desirable to monitor the wakefulness of the subject with eye-tracking during the scans if available.</li>
	<li>Attach 4 ECG electrodes to the subject&#39;s left upper chest (for female subjects, take privacy precautions such as lowering the blinds on the window to the bay in the scan control room and shutting the scanner door). Attach cable to the electrodes and to the In Vivo device. For safety this cable and all other cables leading to the subject should run directly along the scan table, without forming any loops. Turn on the In Vivo device and check for ECG signal quality. Adjust electrode positioning if necessary.</li>
	<li>Fill the wells of the SCR electrodes with conductive gel. This special SCR gel should be isotonic to the skin. Perform open circuit zero in Chart software. Attach SCR electrodes to 2 adjacent distal finger pads of the subject&#39;s hand by wrapping the velcro around as tightly as is comfortable.</li>
</ol>
Part 4: Functional MR imaging sequences tailored for imaging acupuncture stimulation.
<ol>
	<li>We use a 1.5 or 3.0 Tesla Siemens Trio MRI system (Siemens, Erlangen, Germany) equipped for echo planar imaging with a standard quadratic head coil.</li>
	<li>Our studies over the years consistently demonstrated that acupuncture had extensive action over functional systems at multiple levels of the brain. The structures located at the base of the brain such as the amygdala, hippocampus, ventral medial prefrontal cortex and subgenual areas are particularly susceptible to susceptibility artifacts. It is important to check each EPI functional scan to rule out signal loss in these regions as we found differences in loss of signals between two models of a Siemens scanner with similar field strength and with a similar MRI sequence for data collection.</li>
	<li>Thinner brain slices are preferable for reducing the susceptibility artifacts. Slices 3 mm thick with 0.6 mm or 0.75 mm gap in the sagittal or axial orientation are used to cover the entire brain including the brainstem and cerebellum depending on the memory capacity of the imaging system. Other orientations and slice thickness can be used for partial brain imaging. The special attention we pay to these issues contribute to the more consistent findings of acupuncture effects on the structures at the base of the brain.</li>
	<li>The functional scans are acquired with a T2*-weighted gradient echo sequence (TR/TE 4000/30 ms, FOV 200 mm, matrix 64x64, flip angle 90&#176;, slice thickness 3.0 mm, gap 0.6 mm). We acquired a total of 150 time points in 10 min. Image collection is preceded by 4 dummy scans to allow for equilibration of the fMRI signal. The shorter TE reduces susceptibility artifacts. The relatively long TR permits whole brain coverage with high spatial resolution.</li>
</ol>
Part 5: The initial localizer and anatomic scans
<ol>
	<li>Before functional scans with acupuncture are run, a localizer scan, a high resolution anatomical scan, and the T1 weighted 3D MP-RAGE are acquired to assure correct positioning and anatomical soundness. General observation at this point can assure that unnecessary scans are not run. The localizer, a very short scan, assures that the whole brain is included in the scan and allows you to line it up with the AC-PC line. If there are any suspect anomalies, researchers will send images to a trained radiologist but should not make any claims about what is on the scan to subjects.</li>
	<li>The initial scans take about 20 minutes total. Brief viewing can tell the investigator if it will be useful to continue scanning. This is a good time to speak with the subject and make sure that they are still comfortable and aren&#39;t experiencing any adverse effects from being in the Magnet.</li>
</ol>
Part 6: Acupuncture with fMRI monitoring
Of the two forms of treatment (manual needle manipulation and electric stimulation), this video will be limited to manual acupuncture, which has a much longer history of clinical use. Acupuncture stimulation is administered in duplicate runs to classical acupoints LI4 on the hand, LV3 on the foot or ST36 on the leg, employing sterile, one-time use only acupuncture needles.
<ol>
	<li>Instruct subjects to relax, keep their eyes closed and refrain from motions during the scanning. To avoid noxious stimulation, we specifically instruct the subject to raise one finger if any of the acupuncture sensations approaches a score of 8, and raise two fingers in case of sharp pain of any degree.</li>
	<li>After inserting the needle, pretest the subject&#39;s sensitivity and tolerance to needle manipulation to help choose the depth of insertion and the force of manipulation for data collection.</li>
	<li>During the 10 min functional imaging, the needle is rotated for 2 min during M1 and M2, and left in place during the rest periods R1 (2min) ), R2 (3min), R3 (1 min). The acupuncturist makes sure not to approach or leave the subject directly before or after the stimulation period, as even that motion could affect the results. (Paradigm, Fig 1) 
	<img alt="Figure 1" src="/files/ftp_upload/1190/1190fig1.n.jpg" /> 
	Figure 1. Acupuncture fMRI Paradigm.</li>
	<li>Upon the completion of acupuncture stimulation, the acupuncturist records the force with which the needle resists manipulation. At the end of each scan, another staff investigator asks the subject to grade the sensations experienced on a scale of 1-10. The list, aching, soreness, pressure, fullness, heaviness, numbness, tingling, dull pain, sharp pain, is based on the experience of the acupuncturist community and previous experimental data rather than pain questionnaires in the literature. The psychophysical response is categorized as a typical deqi group and another group of deqi mixed with sharp pain based on these records.</li>
	<li>Speak to the subjects between scans to make sure they feel comfortable and they stay awake. If any part of their body begins to cramp or feel uncomfortable, the research staff on site should take appropriate measures to relieve the discomfort. Movements generated by the subject would cause data corruption. Occasional subjects may fall asleep during scanning, which would affect the hemodynamic response.</li>
</ol>
Part 7: Data Processing
<ol>
	<li>Multiple levels of analysis can be run. A general linear model uses rest periods as a baseline to determine the % BOLD MR signal change at every voxel of the brain during the task. It also determines the statistical significance of these changes and removes BOLD data that do not reach 3 contiguous voxels at a specific p-value.</li>
	<li>Perform seed-based cross correlation analysis to show the functional connectivity between one neurological region and the rest of the brain. This is done by selecting a reference regions and comparing its time-course with that of every other voxel in the brain. Other analyses such as independent component analysis, fuzzy cluster analysis, structural equation modeling, Granger Causality can be applied.</li>
	<li>Physiological data can be analyzed by averaging response within windows related to acupuncture needle stimulation. For instance, ECG data can be annotated for QRS complex timing followed by analysis of heart rate and heart rate variability.</li>
</ol>
Part 8: Results
<img alt="Figure 2" src="/files/ftp_upload/1190/1190fig2.n.jpg" /> 
Figure 2. Hemodynamic response in acupuncture deqi vs. tactile stimulation at right LI4, ST36 and LV3 (52 runs/37 subjects). The clusters of deactivated structures at the MPFC (medial prefrontal cortex) (1), MPC (medial parietal cortex) (2), MTL (medial temporal lobe) (3), were more prominent in acupuncture (Fig A) than in tactile stimulation (Fig B). Extensive deactivation of the right BA22/21 (4) in acupuncture was not observed in tactile stimulation. The sensorimotor BA43 (5) and association cortex (6) showed more activation in tactile stimulation than in acupuncture (not shown). Interestingly, the right anterior insula,(7) was activated in acupuncture and not in tactile stimulation. (p&#60;0.0001).
<img alt="Figure 3" src="/files/ftp_upload/1190/1190fig3.n.jpg" /> 
Figure 3. Hemodynamic response in deqi (52 runs/37 subjects) versus deqi mixed with sharp pain (52 runs/29 subjects) during acupuncture stimulation at right LI4, ST36 and LV3, The prominent deactivation of the MPFC (1), MPC (2) and MTL (3) seen with deqi absent pain (Fig A) was attenuated in the presence of pain (Fig B). With pain, activation of the sensorimotor and association cortices (4) became more prominent, and a subset of the limbic and paralimbic regions such as the midC/SMA (5), postC_BA23 (6), Amy (7), and cerebellar vermis (8) became activated. The right anterior insula (9) was markedly activated in deqi while smaller areas of anterior and posterior insula (10) were activated bilaterally in pain. P&#60;0.0001.
<img alt="Figure 4" src="/files/ftp_upload/1190/1190fig4.n.jpg" /> 
Figure 4. Overlap of limbic-paralimbic-neocortical network (LPNN) in acupuncture with the default mode network (DMN); The BOLD response of the LPNN by GLM analysis (Fig.A) and by model-free Fuzzy Cluster analysis (Fig B) during acupuncture stimulation at LI4, ST36 and LV3 show virtually identical patterns with the DMN (Fig.C, adapted from Shulman et al, 1997). Clusters of regions with coherent signal decreases appear in the medial prefrontal cortex (1), postero-medial cortex (2) and temporal lobe (3). p&#60;0.0001.

<ol>
	<li>Hui, K. K. S., Liu, J., Marina, O., Napadow, V., Haselgrove, C., Kwong, K. K., Kennedy, D. N., Makris, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrated+response+of+the+human+cerebro-cerebellar+and+limbic+systems+to+acupuncture+stimulation+at+ST+36+as+evidenced+by+fMRI.">The integrated response of the human cerebro-cerebellar and limbic systems to acupuncture stimulation at ST 36 as evidenced by fMRI.</a> Neuroimage. 27 (3), 479-496 (2005).</li><li>Hui, K. K. S., Marina, O., Claunch, J. D., Nixon, E. E., Fang, J., Liu, J., Li, M., Napadow, V., Vangel, M., Makris, N., Chan, S. T., Kwong, K. K., Rosen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acupuncture+mobilizes+the+brain's+default+mode+and+its+anti-correlated+network+in+healthy+subjects.">Acupuncture mobilizes the brain's default mode and its anti-correlated network in healthy subjects.</a> Brain Res. 1287, 84-103 (2009).</li><li>Napadow, V., Dhond, R. P., Purdon, P., Kettner, N., Makris, N., Kwong, K. K., Hui, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+acupuncture+fMRI+in+the+human+brainstem+with+heart+rate+variability.">Correlating acupuncture fMRI in the human brainstem with heart rate variability.</a> Conf Proc IEEE Eng Med Biol Soc. 5, 4496-4499 (2005).</li><li>Shulman, G. L., Corbetta, M., Buckner, R. L., Raichle, M. E., Fiez, J. A., Miezin, F. M., Petersen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+modulation+of+early+sensory+cortex.">Top-down modulation of early sensory cortex.</a> Cereb. Cortex. 7 (3), 193-206 (1997).</li></ol>

The employment of proper techniques, the experience of the acupuncturist and a large sample size to permit separation from confounding effects of inadvertent noxious stimulation are important in the acquisition and interpretation of acupuncture fMRI data. Our studies of several classical acupoints indicate that acupuncture modulates the limbic network, an important intrinsic regulatory system of the human brain. The network shows significant overlap with the default mode of the resting brain. These associated autonomic n...

monitoring acupuncture effects on human brain by fmri

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

Watch this Scientific Journal Video about Monitoring Acupuncture Effects on Human Brain by fMRI at JoVE.com

Monitoring Acupuncture Effects on Human Brain by fMRI

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

monitoring-acupuncture-effects-on-human-brain-by-fmri

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15.3K Views.  Massachusetts General Hospital and Harvard Medical School. FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Video: Monitoring Acupuncture Effects on Human Brain by fMRI

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

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.

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 ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

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 ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...