המחברים מבקשים להודות Università degli Studi di Milano לתמיכה לאליאנה Di Cairano (מלגת פוסט-דוקטורט) וסטפניה Moretti (מלגת דוקטורט). עבודה זו נתמכה על ידי המחקר באוניברסיטת תכנית PUR לCP ברצוננו להודות לפרופ &#39;ג&#39;רמי מ&#39; הנלי, בית הספר לביוכימיה באוניברסיטת בריסטול, בריטניה, לpHluorin לבנות וד&quot;ר דוטי פרנצ&#39;סקו לסיוע בניתוח נתונים, וסילביה Marsicano לקבלת סיוע טכני.

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The authors declare that they have no competing financial interests.

<html> מאמר זה מציג פרוטוקול לתמונה ולנתח את דינמיקת שלפוחית ​​בתאי מפרישים, באמצעות וקטורים בקידוד cDNA ניאון וTIRFM. מרכיבים עיקריים של הדמיה מוצלחת על ידי TIRFM הם הבחירה של המודל הסלולרי וtransfection תא עם אינדיקטורים אופטיים גנטי מקודדים של שחרור שלפוחית ​​ומחזור. <p class="jov...

שידור סינפטי כימי בין תאי העצב הוא מנגנון עיקרי של תקשורת במערכת העצבים. היא מסתמכת על שחרורו של נוירוטרנסמיטורים דרך מחזור דינמי של איחוי שלפוחית ​​ואחזור באתר presynaptic. רבים מהחלבונים מעורבים בדינמיקת שלפוחית ​​זוהו; עם זאת, התרומה הספציפית שלהם לתופעה עדיין לא הבהירה 1. ההבנה שלנו מוגבלת וחלקו על ידי העובדה שהמבחנים הנפוצה ביותר לexo / אנדוציטוזה לא תמיד המתאימים ביותר. מספר מחקרים הקשורים לאיחוי שלפוחית ​​ודינמיקה מסתמכים על טכניקות אלקטרו. טכניקה זו מספקת פתרון זמני אופטימלי והוא מצוין לחקירת ההיתוך הראשוני של שלפוחית ​​לקרום הפלזמה אבל אינה מסוגל לזהות רב של האירועים המולקולריים שבבסיס שתומכים בפונקצית presynaptic. במיקרוסקופ אלקטרונים, בצד השני, מספק morphologica הטוב ביותרl תיאור של כל צעד יחיד, אבל בהיבט הדינמי של האירוע לא יכול לנפול בפח, כי דגימות חייבות להיות קבועה כדי להיות מנותחת. הכניסה של טכניקות חדשות הקלטה האופטית 2,3, בשילוב עם התקדמות בפיתוח בדיקות מולקולריות ניאון 4-6, מאפשרת הדמיה של תהליכי exocytic בתאים חיים, ובכך לספק רמות חדשות של מידע על המבנה והתפקוד הסינפטי. מחקרים ראשוניים צבעים ניצלו פעילות תלויה styryl (FM1-43 וצבעים אורגניים קשורים) 7,8. מדינה-של-the-art טכניקות הדמיה להעסיק גרסאות pH רגיש של החלבון פלואורסצנטי הירוק (GFP) (pHluorin) קשורים לחלבוני שלפוחית ​​luminal 9. בדיקות אלה הן בדרך כלל כבויים כאשר קיימים בשלפוחית ​​בגלל pH luminal הנמוך. לאחר התמזגות עם קרום הפלזמה, הפנים השלפוחית ​​חשופים למרחב תאי הניטרלי, pH<html> בפתאומיות מגביר, מקל על מרווה הפרוטון-תלוי של pHluorin ואות הניאון במהירות מופיעה. כשינוי בpHluorin הוא מהיר יותר מאשר אירוע ההיתוך, על ידי ניטור הקרינה עולה, איחוי שלפוחית ​​עם הקרום ניתן למדוד ולנתח. בגלל מולקולות מתויג pHluorin פני שטח endocytosed, אות הקרינה לאחר מכן חוזרת לרמה בסיסית, ולכן אותו המבנה עשוי לשמש גם כדי לפקח על שלפוחית ​​מחזור 9. בעוד pH-החיישן מתויג השלפוחית ​​מבטיח ההדמיה רק ​​של אלה שבאמת שלפוחית ​​מתאחה עם קרום הפלזמה, נדרשת הדמיה ברזולוציה מרחב ובזמן גבוהה לתאר בפרטים את השלבים כרוכים בתהליכי endocytic exo /. הטכניקה האופטית המספקת רזולוציה מרחב ובזמן הדרושה היא מיקרוסקופיה הכוללת ההשתקפות הפנימית הקרינה (TIRFM), יישום של מיקרוסקופ פלואורסצנטי 10. &quot;&gt; השתקפות פנימית מוחלטת מתרחשת בממשק שבין הכיסוי להחליק זכוכית והמדגם. כאשר נתיב האור מגיע לכיסוי להחליק זכוכית עם זווית אירוע גדולה יותר מהזווית הביקורתית, אור העירור אינו מועבר למדגם אך הוא משתקף לחלוטין בחזרה. בתנאים אלה, צורות גל אור חלוף בממשק ומתפשט במדיום עם פחות צפיפות אופטית (לדוגמא). כעוצמת שדה החלוף דועכת אקספוננציאלית עם מרחק מהממשק (עם עומק חדירה של כ -100 ננומטר) רק fluorophores בקרבה הקרובה ביותר לכיסוי החלקה יכולים להיות נרגשים ואילו אלה רחוקים יותר מהגבול אינם. בתאים transfected עם GFP-בונה, עומק זה מתאים לחלבונים הביעו בקרום הפלזמה או במבנים שלפוחי שמתקרב לזה. כפי שלא יכול להיות נרגשת fluorophores בפנים התא, הקרינה הרקע היא ממוזערת, ותמונה עם אות / עכברוש רקע גבוה מאודio נוצר 11. כמה מאפיינים להפוך TIRFM הטכניקה של בחירה לניטור דינמיקת שלפוחית. לעומת זאת המושלם ויחס אות לרעש הגבוה מאפשרים זיהוי של אותות נמוכים מאוד הנובעים משלפוחית ​​אחת. רכישת תמונה מבוססת שבב בכל מסגרת מספקת רזולוציה של הזמן הנחוץ כדי לזהות תהליכים דינמיים מאוד. לבסוף, החשיפה המינימלית של תאים לאור בכל מטוס אחר במדגם מאוד מפחיתה phototoxicity ומאפשרת הקלטת הזמן לשגות לאורך זמן 12. ניתוח הנתונים נשאר ההיבט המאתגר והחשוב ביותר של טכניקה זו. הדרך הפשוטה ביותר כדי לפקח על איחוי שלפוחית ​​היא למדוד את ההצטברות של חלבוני ניאון כתב על פני התא, לאורך זמן 13. כתוצאת מגדילה היתוך, עליות אות הקרינה נטו גם כן. עם זאת, שיטה זו עשויה להקל הראש בתהליך, במיוחד בתאים גדולים ובתנאי מנוחה,בגלל תהליכי אנדוציטוזה וphotobleaching לקזז את העלייה בעוצמת הקרינה בשל exocytosis שלפוחית. שיטה חלופית היא לעקוב כל אירוע היתוך אחד 14. שיטה זו אחרונה היא מאוד רגישה ויכולה לחשוף פרטים חשובים על מנגנוני ההיתוך. עם זאת, זה דורש בחירה הידנית של אירועים בודדים, משום שנהלים באופן אוטומטי לגמרי לעקוב שלפוחית ​​ולרשום את התנודות של אותות הניאון שלהם לא תמיד זמינים. תצפית של דינמיקת שלפוחית ​​דורשת תאי דגימה בתדירות גבוהה. זה מייצר כמות גדולה של נתונים שבקושי ניתן לנתח באופן ידני. הצעתו של מאמר זה היא לייעל את טכניקת ההדמיה TIRFM לניטור הבסיסית ושחרור הנוירוטרנסמיטר מגורה בשורת תאי נוירובלסטומה SH-5YSY, ולתאר, צעד-אחרת-צעד, שיטה שפותחה במעבדה לניתוח נתונים, שניהם ברמות כל תא וחד-השלפוחית.

<table border="0" cellpadding="0" cellspacing="0" width="1414">
	<tbody>
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			<td height="21" style="height:21px;width:259px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td style="width:371px;"></td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Axio Observer Z1</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">491912-9850-000</td>
			<td>inverted microscope 
			<a href="http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction">http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Multiline Argon Laser Lasos 77</td>
			<td style="width:199px;">Lasos</td>
			<td style="width:119px;">00000-1312-752</td>
			<td>multi-line (458/488/514 nm), 100mW argon-ion laser 
			<a href="http://www.lasos.com/products/argon-ion-laser">http://www.lasos.com/products/argon-ion-laser</a></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:259px;">Laser TIRF slider</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">423681-9901-000</td>
			<td><a href="http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html">http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">100x Objective</td>
			<td style="width:199px;">Zeiss</td>
			<td>421190-9900-000</td>
			<td>Oil, NA 1.45 Alpha-Plan 
			<a href="https://www.micro-shop.zeiss.com/?l=en&amp;p=us&amp;f=o&amp;a=v&amp;m=a&amp;id=421190-9900-000&amp;ss=1">https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=o&#38;a=v&#38;m=a&#38;id=421 
			190-9900-000&#38;ss=1</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">CCD Camera RetigaSRV Fast 1394&#160;</td>
			<td style="width:199px;">QImaging</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf">http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">LAMBDA 10-3 optical filter changer with SmartShutter</td>
			<td style="width:199px;">Sutter Instrument Company</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.sutter.com/IMAGING/lambda103.html">http://www.sutter.com/IMAGING/lambda103.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Software</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image ProPlus 6.3 Software</td>
			<td style="width:199px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>spot selection, ROI selection, fluorescence intensity determination 
			<a href="http://www.mediacy.com/index.aspx?page=IPP">http://www.mediacy.com/index.aspx?page=IPP</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Excel</td>
			<td style="width:199px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>photobleaching correction, whole-cell and single-vesicle analyses 
			<a href="http://office.microsoft.com/it-it/excel/">http://office.microsoft.com/it-it/excel/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 4.00&#160;</td>
			<td>GraphPad Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>statistical analysis 
			<a href="http://www.graphpad.com/scientific-software/prism/">http://www.graphpad.com/scientific-software/prism/</a></td>
		</tr>
	</tbody>
</table>

tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis

שלפוחית ​​סינפטית לשחרר נוירוטרנסמיטרים בסינפסות כימיות דרך מחזור דינמי של היתוך ואחזור. ניטור פעילות הסינפטית בזמן אמת ולנתח את השלבים השונים של Exo-אנדוציטוזה ברמה חד-השלפוחית ​​הם קריטיים להבנת פונקציות הסינפטי בבריאות ובחוליים. גנטי מקודד בדיקות pH רגיש ישירות ממוקדות לשלפוחית ​​סינפטית וסכתי פנימית השתקפות מיקרוסקופ פלואורסצנטי (TIRFM) מספקים רזולוציה המרחב ובזמן דרושה כדי לעקוב אחרי דינמיקת שלפוחית. שדה החלוף שנוצר על ידי השתקפות פנימית מוחלטת יכול רק לעורר fluorophores ממוקם בשכבה דקה (&lt;150 ננומטר) מעל מכסה הזכוכית שבו תאים לדבוק, בדיוק איפה התהליכים של Exo-אנדוציטוזה יתקיימו. תמונות בחדות גבוהה וכתוצאה מכך מתאימות בצורה אידיאלית לשלפוחית ​​מעקב וניתוח כמותי של אירועי היתוך. בפרוטוקול זה, n אדם SH-SY5Yתאי euroblastoma מוצעים כמודל בעל ערך ללימוד שחרור הנוירוטרנסמיטר ברמה חד-השלפוחית ​​על ידי TIRFM, בגלל המשטח השטוח שלהם ואת הנוכחות של שלפוחית ​​התפזרה. השיטות לגידול SH-SY5Y כתאים חסיד ולtransfecting עם synapto-pHluorin מסופקות, כמו גם את הטכניקה לבצע TIRFM והדמיה. לבסוף, אסטרטגיה שמטרתה לבחור, לספור, ולנתח אירועי היתוך ברמות כל תא וחד-השלפוחית ​​מוצגת. כדי לאמת את גישת ניתוח הליך ההדמיה ונתונים, הדינמיקה של שלפוחית ​​מתויגת pHluorin מנותחים תחת מנוחה ומגורה (depolarizing ריכוזי אשלגן) תנאים. שלילת קוטביות הממברנה מגבירה את התדירות של אירועי היתוך וגורמת להעלאה מקבילה של אות הקרינה הנקי שנרשמה בכל תא. ניתוח חד-שלפוחית ​​מגלה שינויים של היתוך-אירוע התנהגות (גובה שיא מוגבר ורוחב). נתונים אלה מצביעים על הבשלילת קוטביות אשלגן לא רק גורם לשחרור הנוירוטרנסמיטר מאסיבי, אלא גם משנה את המנגנון של איחוי שלפוחית ​​ומחזור. עם הבדיקה הניאון המתאימה, טכניקה זו יכולה להיות מועסק במערכות סלולריות שונות לנתח את מנגנוני הפרשה מכוננת ומגורה.

נהלי ניתוח ההדמיה TIRF ונתונים שתוארו נועדו ללמוד דינמיקת שלפוחית ​​במערכות סלולריות. טכניקה זו יכולה לשמש כדי לקבוע את ההשפעות של מולקולות איתות וסמים על אירועי היתוך ודינמיקת שלפוחית ​​הנוירוטרנסמיטר 17. שימוש בחלבוני קרום פלזמה מתויג GFP, ניתוח TIRFM כבר מועסק ...

Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM וGFP-בדיקות pH רגיש להערכת נוירוטרנסמיטר שלפוחיות Dynamics בתאי SH-SY5Y נוירובלסטומה: הדמיה נייד וניתוח נתונים

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

tirfm-ph-sensitive-gfp-probes-to-evaluate-neurotransmitter-vesicle

Research

JoVE Journal

Neuroscience

10.7K Views.  Università degli Studi di Milano. This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Video: TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-תא הדמיה של תאים מבשר חושי האיברים אינטקט תסיסנית גלמים

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

השתלה של תאי ensheathing חוש ריח להערכת החלמה תפקודית לאחר פגיעה עצבית היקפית

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

ויזואליזציה של Endosome Dynamics בחי מסוף עצב עם דימות פלואורסצנטי ארבעה ממדים

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

תרבויות Organotypic Slice ללמוד oligodendrocyte Dynamics וmyelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

מיון תא של תאי גזע ועצביים ממבוגרי עכבר Subventricular האזור וחי-הדמיה של Dynamics תא המחזור שלהם

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

בידוד גרעיני הבסיס העזוב והתרבות העיקריים ללמוד שחרור נוירוטרנסמיטר

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

במבחנה הדמיה כמותית אסאי עבור פאגוציטוזיס של תאי נוירובלסטומה מתים על ידי iPSC-מקרופאגים

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from &#946; cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis.
This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.

מדידת תחלופת שומנים בממברנה עם ND6 אנלוגי פלואורסצנטי רגיש ל- pH

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

ניתוחים של אקטין דינמיקה, מצמד צימוד וכוח המתיחה לקידום קונוס צמיחה

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...