אנו מודים לאנג&#39;לה Kralemann-קוהלר לתמיכה טכנית מצוינת. עבודה ניסויית שתוארה במסמך זה בוצעה במעבדה שלנו ונתמך על ידי מענקים של מועצת המחקר הגרמנית (DFG) לCK וBK ומענק של המשרד הגרמני למחקר וחינוך (BMBF) לBK.

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החוקרים מצהירים כי אין להם ניגוד אינטרסים.

neurogenesis למבוגרים, ואת האפשרות של המניפולציה שלה באמצעות עיכוב של NF-κB בתאי עצב, ומאוחר יותר מחדש שלה באמצעות דוקסיציקלין, מציע מערכת מרתקת לחקירות לנוירונים יילוד במוח הבוגר, כמו גם לדה ודור מחדש עצבי . היופי של השיטה הזו הוא שעיכוב מסלול איתות NF-κB בנוירונים תוצאות לא ר...

אונטולוגית, ההיפוקמפוס הוא אחד המבנים העתיקים ביותר במוח אנטומיים הידועים. הוא אחראי על משימות מורכבות מגוונות, כגון פונקציות מרכזיות בוויסות של זיכרון לטווח ארוך, התמצאות במרחב, ועל היווצרות וגיבוש של הזיכרון, בהתאמה. מבחינה אנטומית, ההיפוקמפוס מורכב משכבות פירמידה תא (pyramidale שכבה) כוללים cornu Ammonis (CA1, CA2, CA3, וCA4) האזורים וgyrus משוננת (dentatus gyrus), המכיל תאי גרגיר וכמה אבות עצביים באזור subgranular . תאי גרגיר להקרין לכיוון אזור CA3 באמצעות הסיבים שנקרא אזובי (האקסונים של תאי גרגיר). עד סוף המאה שעברה, היה האמין המוח של היונקים הבוגרים להיות איבר סטטי חסר פלסטיות ונוירוגנזה סלולריות. עם זאת, בשני העשורים האחרונים, כמות הולכת וגדלה של עדויות מראה בבירור neurogenesis למבוגרים המתרחשים בלפחותשני אזורים במוח, אזור subventricular (SVZ) ואזור subgranular של ההיפוקמפוס. המחקרים הקודמים שלנו, ואלה של קבוצות אחרות, הראו כי גורם שעתוק NF-κB הוא אחד הרגולטורים המולקולריים החיוניים של neurogenesis למבוגרים, ושתוצאות דה הרגולציה שלה בפגמים חמורים מבניים בהיפוקמפוס וליקויים קוגניטיביים 1-6. NF-κB הוא השם הגנרי של גורם שעתוק מושרה מורכב משילובים שונים dimeric של חמש יחידות משנה-DNA מחייבת: p50, P52, c-Rel, RelB, וp65 (רלה), יש להם את שלוש האחרונים שבם תחומים transactivation. בתוך המוח, הצורה נפוצה ביותר נמצאת בציטופלסמה היא heterodimer של p50 וp65, אשר נשמר בצורה פעילה על ידי מעכב של kappa B (IκB), חלבונים. ללמוד ולתפעל ישירות נוירוגנזה מונעת-NF-κB, אנו משתמשים במודלי עכבר מהונדסים כדי לאפשר עיכוב פשוט של כל subun NF-κBשלה, במיוחד במוח הקדמי 7 (ראה איור 1). לצורך כך, אנו חוצים גידלו-השורות הבאות מהונדסים עכבר, IκB / - ו-/tTA. IκB / המהונדס - הקו נוצר באמצעות מוטציה שלילית טרנס דומיננטי של NF-κB-המעכב IκBa (סופר מדכא IκBa-Aa1) 8. בניגוד לIκBα wild-type, יש IκBα--1AA שתי שאריות סרין מוטציה לalanines (V32 וV36), אשר מעכב את זירחון והשפלה proteasomal הבא של המעכב. לביטוי נוירון הספציפי מוח קדמי של IκBa--1AA-transgene, IκB / - עכברים הכליאו עם עכברי מחסה קינאז בסידן calmodulin תלוי IIα (CAMKIIα), אמרגן שיכול להיות מונע על ידי טרנס activator טטרציקלין (TTA) 9. יש עכברים עקום החוצה p65 פנוטיפ הקטלני עוברי, בשל אפופטוזיס כבד המסיבי 10, ולכן הגישה המוצגת כאן מספקת שיטה אלגנטיתלחוקר את התפקיד של NF-κB בלידה וneurogenesis למבוגרים. מבחן ההתנהגות הקלאסי ללמוד למידה וזיכרון המרחביים שתואר ב1980s על ידי ריצ&#39;רד מוריס, מבחן המכונה המים מבוך מוריס (MWM) 11. במי מבוך פתוח בתחום זה, בעלי חיים ילמדו לברוח ממים אטומים על גבי פלטפורמה מוסתרת מבוססת על רמזי התמצאות ובמיוחד מבוך. גרסה יבשה של MWM היא המבוך שנקרא ברנס (BM) 12. בדיקה זו עושה שימוש בצלחת עגולה עם 20 חורים עגולים מסודרים בגבול של צלחת, עם חור אחד מוגדר כתיבת בריחה, ורמזים נוסף מבוך חזותיים לאורינטציה. שני פרדיגמות הניסוי מסתמכות על הטיסה הנגרמת על ידי מכרסמים `s סלידה למים, או שטחים פתוחים, מוארים ההתנהגות. בשני המבחנים יאפשר חקירה של התמצאות במרחב, וביצועי זיכרון הקשורים. למרות ההיפוקמפוס ממלא תפקיד בכלל וחיוני להיווצרות הזיכרון המרחבי, r בהיפוקמפוסegions המעורב להשתנות בהתאם למבחן המוחל. הזיכרון נבדק בBM נובע מפעילות עצבית בין קליפת מוח enthorinal (EC) ונוירונים פירמידליים ממוקמים באזור CA1-של ההיפוקמפוס בלי תרומה של 13-16 DG. בפרט, BM הקלאסי נשען בעיקר על ניווט באמצעות מסלול monosynaptic temporo-ammonic מEC III לCA1 לEC V. חשוב לציין, DG הוא מעורב באופן מכריע בהכרה שנקרא מרחבית דפוס 17, מה שמרמז לא רק העיבוד של מידע חזותי ומרחבית, אלא גם את הפיכתו של ייצוג או זכרונות דומים לייצוגים שונים, nonoverlapping. משימה זו דורשת מעגל פונקציונלי תלת סינפטי מEC השני לDG לCA3 לCA1 ו-EC השישי, שלא ניתן לבדוק ב15 BM. כדי לטפל באתגרים אלה, יש לנו המציאו SPS-BM כמבחן התנהגותי כדי לבדוק את הביצועים קוגניטיביים משוננת gyrus תלוי בשיתוף במיוחדבעלי החיים ntrol, ובמודל סופר המדכא IκB / TTA הבא עיכוב NF-κB. חשוב לציין, בניגוד לMWM או BM, SPS-BM יכול לחשוף גירעונות התנהגותיים עדינים הנובעים מירידת הערך של נוירוגנזה. מאז מרחבית דפוס ההפרדה היא תלויה אך ורק על מעגל פונקציונלי בין EC השני וDG וCA3 וCA1 ו-EC VI, המבחן הזה הוא רגיש מאוד לשינויים אפשריים בנוירוגנזה, שינויים של מסלול אזובי סיבים או שינויים של הומאוסטזיס רקמה בתוך DG. מבחינה טכנית, ההגדרה של הבדיקה שלנו מבוססת על המחקר של Clelland et al., שבו דפוס ההפרדה המרחבית נבדק באמצעות מבוך 8 זרוע עץ זרוע הרדיאלית (RAM) 19. בהגדרה שונה שלנו, הוחלפו שמונה זרועות על ידי שבעה בתי אוכל צהובים זהים. לסיכום, השיטות שמוצגים כאן, ובכלל זה ניתוח (DCX +)-להביע תאי doublecortin בתוך ההיפוקמפוס, תחזיות אזובי סיבים, דה תאים עצביתATH ובמיוחד SPS-BM שהוצג כאן, יכול להיות מיושם על חקירות של דגמי עכבר אחרים המשלבים transgenes שיש להם השפעה על neurogenesis למבוגרים. יישומים נוספים עשויים לכלול את המחקר של סוכנים תרופתיים ומדידת ההשפעה שלהם על הפרדת דפוס DG ומרחבים.

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Moria MC17 Perforated Spoon&#160;</td>
			<td style="width:127px;">FST</td>
			<td style="width:132px;">10370-18</td>
			<td style="width:167px;">removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi SV8</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14084-08</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical scissors&#160;</td>
			<td style="width:127px;">FST</td>
			<td>14381-43</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td style="width:127px;">FST</td>
			<td>11254-20</td>
			<td>removal of the brains</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SuperFrost Slides</td>
			<td>Carl Roth&#160;</td>
			<td align="right">1879</td>
			<td>slides for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>fixative</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">TissueTek OCT compound</td>
			<td>Sakura Finetek</td>
			<td style="width:132px;">1004200018</td>
			<td>embedding of the brains</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Goat Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">005-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">Normal Rabbit Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">011-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Normal Donkey Serum</td>
			<td style="width:127px;">Jackson Immunolabs</td>
			<td style="width:132px;">017-000-001</td>
			<td>blocking in IHC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Neurofilament-M antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>2H3</td>
			<td>IHC, Dilution 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-doublecortin&#160;antibody</td>
			<td>sc-8066</td>
			<td>Santa Cruz</td>
			<td>IHC, Dilution 1:800</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-GFP antibody</td>
			<td>ab290</td>
			<td>abcam</td>
			<td>IHC, Dilution 1:2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">anti-BrdU antibody</td>
			<td>OBT0030G</td>
			<td>Accurate Chemicals</td>
			<td>IHC, Dilution 1:2000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Fluoro Jade-C</td>
			<td>FJ-C</td>
			<td>HistoChem</td>
			<td>Determination of neuronal cell death</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Betadine</td>
			<td style="width:127px;">&#160;MUNDIPHARMA</td>
			<td>D08AG02&#160;</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cryomicrotome</td>
			<td>Leica</td>
			<td>CM1900</td>
			<td>preparation of brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Heparin sodium salt</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">H3393</td>
			<td>perfusion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">circular plate made from hard-plastic (diameter 120 cm)</td>
			<td style="width:127px;">lab made</td>
			<td style="width:132px;">none</td>
			<td>plate for SPS-BM, diameter 120cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buraton rapid disinfectant&#160;</td>
			<td style="width:127px;">Sch&#252;lke &#38; Mayr</td>
			<td style="width:132px;">113 911</td>
			<td>disinfectant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">video-tracking system TSE VideoMot 2 with Software Package VideoMot2</td>
			<td>tse systems</td>
			<td style="width:132px;">302050-SW-KIT</td>
			<td>tracking and analysis of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>permeabilisation/IHC</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:447px;">cryotome</td>
			<td style="width:127px;">Reichert&#160; Jung/Leica</td>
			<td style="width:132px;">Frigomobil 1206</td>
			<td>preparation of 40&#181;m brain slices</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Mowiol 4-88</td>
			<td style="width:127px;">Carl Roth&#160;</td>
			<td>Art.-Nr. 0713</td>
			<td>embedding of the slides</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">SYTOX green</td>
			<td style="width:127px;">Invitrogen</td>
			<td>S7020</td>
			<td>Nuclear staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Food pellets (Kellog`s Froot Loops)</td>
			<td>Kellog`s</td>
			<td style="width:132px;"></td>
			<td>SPS-BM</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:447px;">Prism, Version 3.0</td>
			<td style="width:127px;">Graph Pad Software, San Diego, USA&#160;</td>
			<td style="width:132px;"></td>
			<td>Statistical evaluation of SPS-BM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">Zen 2008 or Zen 2011 Software</td>
			<td style="width:127px;">Carl Zeiss</td>
			<td style="width:132px;"></td>
			<td>Software (Confocal microscope)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:447px;">D.P.X</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:132px;">317616</td>
			<td>mounting medium for Fluoro Jade C staining</td>
		</tr>
	</tbody>
</table>

methods for the modulation and analysis of nf-&#954;b-dependent adult neurogenesis

ההיפוקמפוס ממלא תפקיד מרכזי בהיווצרות והגיבוש של זכרונות אפיזודי, ובהתמצאות במרחב. מבחינה היסטורית, ההיפוקמפוס המבוגר נצפה כאזור אנטומי מאוד סטטי של המוח היונקים. עם זאת, הממצאים אחרונים הראו כי הרכס משוננת של ההיפוקמפוס הוא אזור של פלסטיות עצומה במבוגרים, הכולל לא רק שינויים של מעגלים עצביים קיימים, אלא גם neurogenesis למבוגרים. פלסטיות זה מוסדרת על ידי רשתות תעתיק מורכבות, שבו גורם שעתוק NF-κB משחק תפקיד בולט. ללמוד ולטפל neurogenesis למבוגרים, מודל עכבר מהונדס לעיכוב עצבי מוח קדמי ספציפי של פעילות NF-κB ניתן להשתמש. במחקר זה, שיטות מתוארות לניתוח נוירוגנזה NF-κB תלויה, לרבות היבטיה המבניים, אפופטוזיס העצבי ושגשוגם מולידם, ומשמעות קוגניטיבית, אשרשעות היו דווקא העריכה באמצעות gyrus משוננת (DG) תלוי מבחן התנהגות, מבוך ההפרדה בארנס דפוס המרחבי (SPS-BM). פרוטוקול SPS-BM יכול להיות פשוט מותאם לשימוש עם מודלים של בעלי חיים מהונדסים אחרים שנועדו להעריך את השפעתם של גנים מסוימים בneurogenesis בהיפוקמפוס המבוגר. יתר על כן, SPS-BM יכול לשמש בהגדרות ניסיוניות אחרות שמטרתם לחקור ומניפולצית למידת DG תלויה, למשל, באמצעות סוכנים תרופתיים.

הכלאות של IκB / - קווי העכבר מהונדסים וTTA מובילה לעיכוב מותנה של פעילות NF-κB בהיפוקמפוס. כדי לחקור את הביטוי של IκBα--1AA-transgene בעכבר (איור 1 א) המהונדס הכפול, המוח היו מבודד, cryosectioned ומוכתם באמצעות נוגדן כנגד GFP (חלבון פל?...

Watch this Scientific Journal Video about Methods for the Modulation and Analysis of NF-ÎºB-dependent Adult Neurogenesis at JoVE.com

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

שיטות למניפולציה והניתוח של neurogenesis בהיפוקמפוס המבוגר NF-κB תלוי מתוארות. פרוטוקול מפורט מוצג לבדיקה משוננת gyrus תלוי התנהגותי (שמכונה במבוך ההפרדה בארנס דפוס המרחבי) לחקירת תוצאה קוגניטיבית בעכברים. טכניקה זו צריכה גם לעזור לאפשר חקירות בהגדרות ניסיוניות אחרות.

שיטות לאפנון וניתוח של Neurogenesis מבוגרים NF-κB תלוי

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

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Neuroscience

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

14.2K Views.  University of Bielefeld.  שיטות למניפולציה והניתוח של neurogenesis בהיפוקמפוס המבוגר NF-κB תלוי מתוארות. פרוטוקול מפורט מוצג לבדיקה משוננת gyrus תלוי התנהגותי (שמכונה במבוך ההפרדה בארנס דפוס המרחבי) לחקירת תוצאה קוגניטיבית בעכברים. טכניקה זו צריכה גם לעזור לאפשר חקירות בהג?...

Video: Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

יציקה של כלי דם במוח העכבר למבוגרים הדמיה 3D ו ניתוח מורפולוגי

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

חקירה תפקודית של מבוגרים ההיפותלמוס Neurogenesis עם הפוקוס ברדיולוגית עיכוב

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

פציעה דקירת פצע של דג הזברה telencephalon מבוגרים: שיטה לחקר המוח של בעלי החוליות Neurogenesis והתחדשות

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

אימונוהיסטוכימיה ותיוג מרובה עם נוגדנים ממיני Host אותו ללימוד למבוגרים בהיפוקמפוס נוירוגנזה

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

הערכת Neurogenesis יסודי<em&gt; Xenopus</em&gt; עוברים באמצעות Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

ניתוח Synaptic אפנון של<em&gt; תסיסנית</em&gt; קולטני אור לאחר חשיפה לאור ממושך

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

העכבר Hindbrain כמודל ללימוד נוירוג'נסיס עובריים

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

ניתוח מטבולית של melanogaster דרוזופילה Larval ושכל למבוגרים

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

בידוד של חוט השדרה למבוגרים גרעינים עבור רצפי RNA חד-גרעין בנפט מקבילים

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...