עבודה זו מומנה על ידי ממכון מקס פלנק לפסיכיאטריה מארי האיחוד האירופי רשת קירי ההכשרה הראשונית (חוזה NINA 238665).

<ol>
	<li>Jirtle, R. L., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+epigenomics+and+disease+susceptibility.">Environmental epigenomics and disease susceptibility.</a> Nat. Rev. Genet. 8, 253-262 (2007).</li><li>Murgatroyd, C., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+programming+of+the+HPA+axis:+Early+life+decides.">Epigenetic programming of the HPA axis: Early life decides.</a> Stress (Amsterdam, Netherlands). , (2011).</li><li>Gluckman, P. D., Hanson, M. A., Buklijas, T., Low, F. M., Beedle, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+mechanisms+that+underpin+metabolic+and+cardiovascular+diseases.">Epigenetic mechanisms that underpin metabolic and cardiovascular diseases.</a> Nat. Rev. Endocrinol. 5, 401-408 (2009).</li><li>Murgatroyd, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+DNA+methylation+programs+persistent+adverse+effects+of+early-life+stress.">Dynamic DNA methylation programs persistent adverse effects of early-life stress.</a> Nat. Neurosci. 12, 1559-1566 (2009).</li><li>Murgatroyd, C., Wu, Y., Bockm&#252;hl, Y., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+learn+from+stress:+How+infantile+trauma+programs+us+for+depression.">Genes learn from stress: How infantile trauma programs us for depression.</a> Epigenetics. 5, (2010).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates, Compact: The Coronal Plates and Diagrams: Compact. The Coronal Plates and Diagrams. , (2008).</li><li>Bettscheider, M., Murgatroyd, C., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+DNA+and+RNA+isolation+from+brain+punches+for+epigenetics.">Simultaneous DNA and RNA isolation from brain punches for epigenetics.</a> BMC research notes. 4, 314 (2011).</li><li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Reed, K., Poulin, M. L., Yan, L., Parissenti, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+bisulfite+sequencing+PCR+with+pyrosequencing+for+measuring+differences+in+DNA+methylation.">Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation.</a> Anal. Biochem. 397, 96-106 (2010).</li></ol>

אין ניגוד עניינים הצהיר.

תכנות epigenetic מוכרת יותר ויותר לבריאות מנגנון חשוב הבסיס לבין המחלה. כאן אנו מציגים שיטה מעודנת לבידוד בו זמנית של DNA ו-RNA מ micropunches ואת הצעדים הבאים כדי לקבל פרופילים מתילציה דנ&quot;א באמצעות רצף bisulfite. עבודה אופטימיזציה מאפשר ניתוח נוח של תכנות epigenetic במוח בתגובה לגירויים סביבתיים. יתר על כן, גישה זו מקלה על חקירת הקשר הפונקציונלי בין מתילציה דנ&quot;א ביטוי גנים כמו גם מנותחים מדגם זהה. לבסוף, מספר בעלי החיים הדרושים לניסוי ניתן לצמצם באופן משמעותי. אמצעי זהירות מסוימים והנחיות חייבות לבוא בעת ביצוע העבודה שהוצג. בהתחשב במורכבות יוצאת דופן ההטרוגניות של המוח ומאז דפוס מתילציה והביטוי עשויה להשתנות באופן תאים ורקמות ספציפי זה של מירבכי micropunching חשיבות מתבצע בצורה סטנדרטית. כמו במקרה של הנקודה, תכנות epigenetic של AVP מתגלה paravaventriuclar, אבל לא בגרעין supraoptic (איור 4 ב &#39;, ד), 2 AVP להביע אזורי ההיפותלמוס 4. מבחינה זו, הזמינות של DNA ו-RNA של אגרוף רקמות זהה יש יתרון ביטוי RNA של גנים מסוימים ברקמות ספציפיות סמן יכול לשמש במקרים מסוימים לאשר כי השטח הנכון היה אגרוף. למרות micropunching יכול להפחית ההטרוגניות הסלולר, הוא צריך לזכור כי גישה זו אינה מובילה לבידודה של סוגי תאים בודדים או אוכלוסיות. צעדים נוספים טיהור עשוי להיחשב לטפל בנושא זה. עבור bisulfite רצף עיצוב פריימר האופטימלי לטיפול PCR bisulfite הבאה הגברה חיוני. מוצרי ה-PCR העולה על 400 נ&quot;ב באורך עלולה להיות בעייתית בשל הידרדרות של ה-DNA על ידי בisulfite הטיפול. יש לציין גם כי ה-PCR מקונן יכול לתת תוצאות מוטות אם רק כמה תבניות שלמות כמה לתרום לתהליך ההגברה. זוגות פריימר ספציפיים עבור amplicon שונה צריך להיות מתוכנן, רצפים שלהם לא יכילו dinucleotides CPG על מנת למנוע מתילציה מוטות הגברה. בנוסף, primers צריך להכיל לא CPG cytosines כדי למנוע הגברה של DNA ללא שינוי או המרה באופן חלקי. הגברה של מספר תבניות עשויות להפיק תועלת בניהול חם תחילת PCR. בכל מקרה, התאמת זוגות פריימר צריך להיות מאומת בניסויים הטייס כדי למנוע קלקול של דגימות ניסויים חשובים. סידור bisulfite מייצג שיטה סטנדרטית לניתוח באתר מתילציה ספציפי כפי שהוא מסוגל לזהות באופן אמין ומדויק מתילציה של שאריות CPG יחיד באופן אלל ספציפי 8. רצף ישיר של מוצר ה-PCR לא מומלץ, היות מתילציה o מעורבתשאריות fa CPG יופיעו השיא C / T כפול electropherogram, אשר יכול למנוע כימות של מתילציה באתר מודאג. מסיבה זו הצעד שיבוט ביניים מבוצע שיבוטים כמה הם רצף (~ 20-30) כדי לקבוע את מידת מתילציה. Pyrosequencing מייצג שיטה חלופית לכמת מתילציה בדנ&quot;א. הוא גם מנצל ההמרה bisulfite ו bisulfite PCR אבל במקום השיבוט רצף amplicon נתונה התגובה pyrosequencing ישיר, שבו מצב מתילציה של שאריות CPG נקרא פולימורפיזם כמו C / T נוקלאוטיד יחיד, שניתן לכמת בקלות. שתי השיטות לזהות רמות דומות של מתילציה 9 אבל מאז שיבוט שלב ניתן להשמיט pyrosequencing יותר זמן יעיל. עם זאת, בהתאם לתבנית רק 40-100 נוקלאוטידים יכול להיות רצף בבת אחת, כך מספר סבבי pyrosequencing עם פריימר סידור שונה נחוצים. זוהי מגבלה קריטית, בהתחשב thaסכומים לא גבוהים יותר של ה-DNA (כ 1 מיקרוגרם לכל תגובה) נדרשים, דבר הנראה לעין יעלה על הסכומים הזמינים מן המכות רקמה זעירים במוח. כך סריקה ראשונית של אזורים בטווח של כמה kilobases ידי pyrosequencing נראה פחות מתאים כמו קריאה שיבוט אחד. אף על פי כן, לאחר זיהוי על אזור קטן של pyrosequencing עניין יש לשקול. באופן קולקטיבי, את הפרוטוקול המתואר לעיל מספק גישה מדויק, יעיל יעיל לניתוח מהיר וחסכוני של micropunches המוח לשינויים epigenetic. דגימות מרובות ניתן לעבד בטווח אחת השיטות מתאימות בזמן הרצת דגימות ביניים בגודל ניסויים.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> דם DNeasy &amp; ערכת רקמות </td><td> Qiagen </td><td> 69506 </td></tr><tr><td> Nucleospin RNA II </td><td> Macherey-נגל </td><td> 740,955.50 </td></tr><tr><td> Epitect ערכת bisulfite </td><td> Qiagen </td><td> 59104 </td></tr><tr><td> Nucleospin חלץ </td><td> Macherey-נגל </td><td> 740,609.50 </td></tr><tr><td> pGEM-T וקטור שיבוט Kit </td><td> Promega </td><td> A3600 </td></tr><tr><td> Nucleofast 96 </td><td> Macherey-נגל </td><td> 743,100.50 </td></tr><tr><td> מונטאז רצף 96 ניקוי </td><td> Millipore </td><td> LSK09624 </td></tr><tr><td> Hotstar פולימראז Taq </td><td> Qiagen </td><td> 203203 </td></tr><tr><td> Taq פולימראז </td><td> Fermentas </td><td> EP0401 </td></tr></tbody></table>

optimized analysis of dna methylation and gene expression from small, anatomically-defined areas of the brain

חשיפה דיאטה, תרופות מצוקות החיים בשלב מוקדם של Windows רגישים של 1,2 החיים יכולים להוביל לשינויים בר קיימא ביטוי גנים התורמים התצוגה של פנוטיפים פיזיולוגיים והתנהגותיים. תכנות סביבתי כזה עלול להגביר את הרגישות, מחלות מטבוליות לב וכלי דם הנפש 3,4. מתילציה דנ&quot;א שינויים היסטון נחשבים תהליכים מרכזיים בגישור של דיאלוג גנים הסביבה ומופיעות גם רובד תחתון תכנות הסביבה 5. אצל יונקים, מתילציה בדנ&quot;א בדרך כלל כוללת בנוסף קוולנטי של קבוצת מתיל במיקום-5 של ציטוזין בהקשר של dinucleotides CPG. מתילציה CPG מתרחשת באופן רקמות ותאים ספציפי מאוד מה שהופך אותו לאתגר ללמוד בדידים, אזורים קטנים של המוח שבו ההטרוגניות הסלולר הוא גבוה וכמות הרקמה מוגבלת. יתר על כן, בביטוי גנים מתילציה ecause ו מתקיים קשר הדוק אירועים, ערך מוגבר ניתן להשיג על ידי השוואת שני הפרמטרים במדגם זהה. כאן, פרוטוקול צעד אחר צעד (איור 1) לחקירה של תכנות epigenetic במוח מוצג באמצעות פרדיגמה &quot;הפרידה מן האם&quot; צרה חייו המוקדמים לשם המחשה. הפרוטוקול מתאר את הכנת micropunches של מוחות עכבר דיפרנציאלי בגיל שממנו DNA ו-RNA יכול להיות מבודד בו זמנית, ובכך מאפשר מתילציה בדנ&quot;א וביטוי גנים ניתוחים במדגם זהה.

Watch this Scientific Journal Video about Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain at JoVE.com

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

זרימת עבודה יעילה ללמוד מתילציה דנ&quot;א ביטוי שינויים גנטיים על הלחץ מוקדם בחיים מוצג. החל מ הפרידה מן האם של עכברים בני יומם ובידוד של רקמות מוח נפרדים, אנחנו מייצגים את הפרוטוקול בו זמנית לבודד DNA ו-RNA של אגרופים רקמות המוח רצף bisulfite עתידיים RT-PCR ניתוח.

ניתוח אופטימיזציה של מתילציה דנ&quot;א ביטוי גנים מ, אזורים קטנים אנטומית מוגדרים של המוח

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

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Neuroscience

18.7K Views.  Max Planck Institute of Psychiatry. זרימת עבודה יעילה ללמוד מתילציה דנ"א ביטוי שינויים גנטיים על הלחץ מוקדם בחיים מוצג. החל מ הפרידה מן האם של עכברים בני יומם ובידוד של רקמות מוח נפרדים, אנחנו מייצגים את הפרוטוקול בו זמנית לבודד DNA ו-RNA של אגרופים רקמות המוח רצף bisulfite עתידיים RT-PCR ניתוח.

Video: Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

חוט השדרה Electrophysiology II: ייצור תאי אלקטרודה יניקה

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

בידוד באף גזע תאים חוש הריח של מכרסמים או בני אדם

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

ברזולוציה גבוהה הדמיה חיה של התנהגות התא ב Neuroepithelium פיתוח

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

ניתוח Stereotaxic על הנגע Excitotoxic של אזורי מוח ספציפיים עכברוש למבוגרים בלבד

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Microdissection לכידת ליזר אוכלוסיות מועשרות של הנוירונים או נוירונים יחידים לניתוח ביטוי גנים לאחר פגיעה מוחית טראומטית

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

מיקרומניפולציה של ביטוי גנים במוח דג הזברה למבוגר באמצעות Microinjection Cerebroventricular של oligonucleotides morpholino

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

ניתוח שינויי ביטוי הגנים בהיפוקמפוס העכברוש לאחר Deep Brain Stimulation של Anterior התלמוס הגרעין

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

ביטוי גנים מסוג התא ספציפי באמצעות סלולארי קרינה המופעל מיון לבדיקת רקמות במוח החולדה

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

הכנת פרוסות המוח חריפה באמצעות אופטימיזציה N-מתיל-D-שיטת שחזור מגן glucamine

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

רקמות קרינה הופעלו מיון תאים (FACS) וניתוח ביטוי גנים של נוירונים Fos להביע מ טרי קפוא מוח החולדה

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...