במימון NIMH, NARSAD וקרן גטסבי.

ציוד ההתקנה הצעד הראשון ריצה ניסוי fMRI הוא הגדרת ובדיקת הציוד. צעדים אלה יכול להיעשות בכל סדר. מיזוג אותות - הסורק נמצא בחדר מוגן חשמלית מגנטית. כל האותות החשמליים הולך מתוך חדר הבקרה לחדר סורק לעבור דרך פאנל מסנן כדי להסיר כל התדרים שיכול ליצור חפץ בתמונה MR. Airpuff - המכשיר airpuff מספקת משב מבוקר של הלחץ משך קבוע. כאשר מכוון העין, זה מרתיעה אבל לא טראומטי. וסת לחץ (WPI פניאומטיים PicoPump) למקור של אוויר דחוס, אשר יכול להיות טנק באוויר אבל אנחנו משתמשים בבית האוויר. הלחץ מותאם 30 psi. העיתוי מותאם לספק משב 50 msec. שסתום סולנואיד הוא נשלט על ידי האות מחשב. הפלט של הרגולטור את הלחץ מועבר הנושא באמצעות צינור פלסטיק 1/6th אינץ&#39; (Tygon). חשוב כי הצינור לא ארוך מדי או לא יהיה עיכוב בין פתיחת סולנואיד ואת זמן האוויר הדחוס מגיע בסוף הצינור. מסיבה זו, הרגולטור הלחץ יושב בתוך החדר סורק ואת אותות הבקרה מוזנים דרך לוח מסנן כדי להבטיח כי אין ממצא הדמיה. מיץ - מנפק מיץ שולט על גודל ומספר טיפות מיץ נמסר הפה על ידי צינורית פלסטיק. מנפק מיץ מכילה מאגר, שסתום סולנואיד מבוקרת מחשב, ו צינור ארוך המספק מיץ לנושא. כל הרכיבים האלקטרוניים הם מחוץ לחדר סורק ולכן אין להכניס חפצים האות MR. מנפק מיץ (מכשירים Crist) היא שטפה פעמיים על ידי מילוי המאגר ופתיחת שסתום סולנואיד. ואז הוא מלא עם משקה מועדף של הנושא. המערכת נבדק לפני סריקה על ידי הפעלת תוכנית שגורמת את המחשב כדי לייצר זרם של פעימות מבחן שתוצאתו מספר טיפות של מיץ מועבר הנושא. הנושא מוודאת כי מיץ זורם. Bite בר - ראש התנועה יוצרת כמה חפצים בתמונה fMRI. בניסוי זה, הנבדקים לקבל airpuffs אשר יכול לגרום תנועות רפלקסיביות, ומיץ המחייב בליעה. כדי למזער את תנועת הראש, bitebar משמש כדי לייצב את הראש. Bitebar מחובר סליל ראש RF. כל נושא יש שופר מותאם אישית עשוי מחומר תרמופלסטי. דופק oximeter - FMRI אמצעים חמצון הדם (BOLD). חמצון הדם במוח עשויה להיות מושפעת הפעילות העצבית, אבל זה גם יכול להיות מושפע פעימות הלב. כדי למדוד שינויים חמצון הקשורים למחזור לב, אנו משתמשים כדי למדוד את הדופק oximeter חמצון הדם האצבע באמצעות חיישן אינפרא אדום. Oximeter הדופק הוא MR תואם אותות הפלט שלה מוזן דרך לוח מסנן לחדר הבקרה שבו הוא דיגיטציה המאוחסנים במחשב. לפקח גז נשימה - חמצון דם יכול להיות מושפע גם הנשימה. אנו מודדים את הנשימה עם צג גז הנשימה המודד רמות CO2 פג. RGM הוא MR תואם אותות הפלט שלה מוזן דרך לוח מסנן לחדר הבקרה שבו הוא דיגיטציה המאוחסנים במחשב. גירוי חזותי - פאנל אחורי הקרנת ממוקם בקצה אחד של הסורק נשא קרוב למיקום ראש הנושא. נושאים מבט למסך הקרנה לאחור דרך הראי רכוב על גבי סליל ראש. התמונות מוקרנת על מסך ההקרנה בחזרה על ידי מקרן LCD הממוקם מחוץ לחדר הסורק. אלומת האור מן המקרן מכוון בתוך החדר סורק דרך צינור מוגן RF. מעקב עיניים - חשוב לדעת איפה הנושא הוא מחפש סיבות. תנועות עיניים להשפיע על המיקום של גירויים ויזואליים על הרשתית, אשר משפיע על התגובות ויזואלי במוח. תנועות עיניים יכול לשמש גם כאמצעי שיש בנושא מצביעים על תגובה התנהגותית. אנו משתמשים בשיטה הנקראת &quot;oculography וידאו אינפרא אדום&quot; כדי לעקוב אחר העיניים. בשיטה זו, מצלמת אינפרא אדום משמשת כדי לעקוב אחר תלמיד. פולט מצלמת אינפרא אדום בנויים לתוך זוג משקפי במיוחד. אלה הם משקפי אותו המספקים גירוי ויזואלי. הנתונים מן משקפי הוא מעובד על ידי מחשב ייעודי אשר ממירה תמונות של העין לתוך אותות אנלוגיים לתפקיד עין אופקי ואנכי. נושא ההכנה תיעוד הסכמה &amp; ndash; לפני הסריקה, כל נושא צריך למלא טפסים שונים לגבי הבטיחות הסכמה. גיליון תוכנית ניסויית - גיליון התוכנית מציין את פרוטוקול הניסוי ואת רצפי הדופק ואת הסדר שבו הם מתנהלים. הכנה גופנית - כאשר הנושא הוא מוכן להיות סרוקים, אחד הדברים הראשונים שהם עושים זה לשים אטמי אוזניים כדי להגן על השמיעה שלהם מן רעש גבוהה סורק תדרים. הם גם לשים על MR-תואם אוזניות לתקשר גם עם נושאים לסורק כדי לספק הוראות משוב במהלך הסריקה. הנושא מכניס לפה שלהם, הכולל קווים אינטש 1 / 16 ה למסירה מיץ ניטור גז הנשימה. הנושא מעמיד על ולהתאים את משקפי MR תואם המשמש גירוי ויזואלי תנועה ניטור העין. הנושא נמצא על המיטה סורק את סליל RF ממוקם מעל הראש שלהם. שופר (bitebar) מחובר סליל ה-RF כדי למנוע תנועת הראש. צינורות airpuff (1 / 6 tygon אינץ&#39;) ממוקם ליד עין אחת באמצעות צינור גמיש (loc-line). קליפ הדופק oximeter אצבע מחוברת אצבע אחת ומאובטחת עם הקלטת. סריקה נוהל דופק רצפים: מבנה: אנו משתמשים T1-משוקלל תמונות (תמונות מבנית), כי לתת הגדרה ברורה של מורפולוגיה של המוח של הנבדקים. הנושא נמצא פאסיבי ונשאר בשקט ככל האפשר בשלב זה, שנמשך כ -10 דקות. פונקציונלית: אנו משתמשים T2 משוקלל רצפים EPI (תמונות תפקודית) כדי להראות שינויים חמצון הדם מתואמים עם פעילות עצבית. זה בשלב זה כי הנושא נחשף מיץ, airpuff, גירויים שמיעתיים. קישוריות: השתמשנו דיפוזיה משוקלל תמונות (DWI) כדי לקבוע קישוריות מבנית בין אזורים במוח. הנושא פשוט שוכב ללא תנועה בשלב זה.

functional imaging with reinforcement, eyetracking, and physiological monitoring

אנו משתמשים הדמיה מוחית תפקודית (fMRI) כדי לחקור המעגלים העצביים העומדים בבסיס קבלת ההחלטות. כדי להבין כיצד להשפיע על תהליכים ותוצאות ההחלטה, משימות תפיסתיות פשוטות בשילוב עם תיאבון מתוגבר ו מרתיעה. עם זאת, השימוש של החיזוקים כמו מיץ airpuffs יכול ליצור אתגרים fMRI. משלוח חיזוק יכולות לגרום ראש התנועה, אשר יוצרת חפצים האות fMRI. חיזוק יכול גם להוביל שינויים בקצב הלב והנשימה, כי הם בתיווך מסלולים האוטונומית. שינויים בקצב הלב והנשימה יכולים להשפיע ישירות על האות fMRI (BOLD) במוח יכול להיות מבולבל עם שינויים האות כי הן בשל הפעילות העצבית. במצגת זו, אנו מדגימים שיטות לניהול החיזוקים באופן מבוקר, לייצוב הראש, למדידת הדופק והנשימה.

Watch this Scientific Journal Video about Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring at JoVE.com

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

מצגת זו ממחישה את השימוש של ה-fMRI לבחון המעגלים העצביים העומדים בבסיס קבלת ההחלטות. משימות תפיסתיות פשוטות בשילוב עם תגבורת תיאבון ו מרתיעה כדי לחקור כיצד להשפיע על תהליכים ותוצאות ההחלטה.

הדמיה תפקודית עם חיזוק, Eyetracking, ניטור פיזיולוגית

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

functional-imaging-with-reinforcement-eyetracking-physiological

Research

JoVE Journal

Biology

11.3K Views.  Columbia University. מצגת זו ממחישה את השימוש של ה-fMRI לבחון המעגלים העצביים העומדים בבסיס קבלת ההחלטות. משימות תפיסתיות פשוטות בשילוב עם תגבורת תיאבון ו מרתיעה כדי לחקור כיצד להשפיע על תהליכים ותוצאות ההחלטה.

Video: Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

ניטור אקטין פירוק עם מיקרוסקופיה זמן לשגות

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.

תיוג hESCs ו hMSCs עם ברזל אוקסיד חלקיקים עבור פולשני של מעקב vivo עם 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 ...

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.

תיוג בתאי גזע עם צבעי ניאון על איתור פולשני עם דימות אופטי

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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

זיהוי של קומפלקסים חלבונים עם proteomics כמותיים cerevisiae ס

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissection של gyrus משוננת בהיפוקמפוס מתוך עכבר למבוגרים

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

ניטור הלב לתפקד הזחל תסיסנית melanogaster ללימודים פיזיולוגיים

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

לטווח ארוך רעילות מבחן קטלני עם לסרטן הארטמיה franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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.

מיפוי לאחר הלוואי של תטא Burst גירוי בקורטקס השמיעתי האנושי בהדמיה תפקודית

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

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.

הדמיה תפקודית של שומן חום בעכברים עם 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 ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

בידוד ופיזיולוגי ניתוח של שריר לב העכבר

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...