This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

כל הניסויים המתוארים במסמך זה בוצעו בהתאם לבעלי חיים בריטניה (הליכים מדעיים) Act of 1986. 1. הכנה של מכשירים, תרבות מדיה, ומנות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן שני מספריים מעבדה הנירוסטה לנתח ושני מלקחיים מעבדה נירוסטה. החיטוי כל המכשירים והמקום בO מכסה המנוע התרבות / N תחת אור אור (UV) אולטרה סגול לעיקור. </li><li style=";text-align:right;direction:rtl"> הפוך 500 מיליליטר של תקשורת חיונית בינוני מינימום (ממ) תרבות (10% עובריים שור [FBS] סרום, 20 מ&quot;מ KCl, 25 מ&quot;מ NaHCO 3, 30 מ&quot;מ D-גלוקוז, L-גלוטמין 2 מ&quot;מ, 100 U / פניצילין מיליליטר, 100 מיקרוגרם / מיליליטר סטרפטומיצין ו -6 מיקרוגרם / מיליליטר אמפיצילין) ולסנן לעקר. חנות ב 4 מעלות צלזיוס במשך חודשים. </li><li style=";text-align:right;direction:rtl"> הכן 24 צלחות תרבות היטב המכילות 14 מ&quot;מ, עובי coverslips מספר 1. החיטוי coverslips ומעיל ב/ L 100 מ&quot;ג פולי-ד-ליזין (PDL) כפי שתואר קודם לכן 3. אור UV לעקר O / N. </li></ol> 2. הכנת פתרונות CGC תרבות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן &#39;פתרון&#39; (100 מיליליטר) בautoclaved אור UV, טופל, סטרילי DH 2 O המכיל 250 מ&quot;ג של גלוקוז, 300 מ&quot;ג של סרום שור אלבומין [BSA], 955 מ&quot;ג של בופר פוספט [PBS] (Ca 2 + ו Mg + חינם), ו 1 מיליליטר של 3.8% MgSO 4 · 7H 2 O. </li><li style=";text-align:right;direction:rtl"> הכן &#39;פתרון ב&#39; (10 מיליליטר) מכיל 1 מיליליטר של 5 מ&quot;ג / מיליליטר טריפסין המדולל ב9 מיליליטר של א &#39;הפתרון </li><li style=";text-align:right;direction:rtl"> הכן &#39;C פתרון &quot;(10 מיליליטר) מכיל DNase 1,000 יחידות, 0.5 מ&quot;ג של מעכבי טריפסין הסויה, של 3.8% MgSO 4 · 7H 2 O 100 μl, בדילול מלא עד 10 מיליליטר עם א&#39; הפתרון </li><li style=";text-align:right;direction:rtl"> הכן &#39;D פתרון &quot;(20 מיליליטר) מכיל 3.2 מיליליטר של C פתרון, בדילול מלא עד 20 מיליליטר עם א&#39; הפתרון </li><li style=";text-align:right;direction:rtl"> הכן הפתרון מאוזן של BSA / ארל תמיסת מלח (EBSS) המכיל EBSS, 25 מ&quot;מ NaHCO 3, 3 מ&quot;מ MgSO 4 · 7H 2 O, ו4 BSA%, pH 7.4, כפי שתואר לעיל 3. </li><li style=";text-align:right;direction:rtl"> הכן LME ידי הוספת LME הטהור לתוך 5 מיליליטר של מדיום תרבות ממ לתת ריכוז סופי של 150 מ&quot;מ LME, להחזיר את הפתרון לpH 7.4 באמצעות מד pH benchtop, ומסנן לעקר באמצעות sulfone 28 מ&quot;מ polyether (PES) 0.2 מיקרומטר מזרק מסנן. </li></ol> 3. Culturing CGCs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף cerebella מגורי חולדה ישנים 4-7 יום כפי שתואר בעבר 3. הנח מייד בצלחת פטרי המכילה 5 מיליליטר פתרון על קרח. </li><li style=";text-align:right;direction:rtl"> יוצקים את הפתרון עודף מרקמת המוח קטנה ומקום במכסה צלחת פטרי. </li><li style=";text-align:right;direction:rtl"> קוצצים דק רקמה עם סכין גילוח בערה היטב בלפחות שלושה כיוונים שונים. </li><li style=";text-align:right;direction:rtl"> להוסיף רקמה קצוצה לפתרון B (לשטוף צלחת פטרי עם פתרון כדי להבטיח רקמה מוגבלת אבד) ומקום באמבט מים ב 37 מעלות צלזיוס למשך 5 דקות. ללחוץ בעדינות כל כמה דקות. </li><li style=";text-align:right;direction:rtl"> להוסיף D פתרון (20 מיליליטר) לצינור כדי לנטרל את טריפסין, לרעוד ואחוזיםrifuge ב 65 XG במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> יוצקים את supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend ב 4 מיליליטר של תמיסת ג Triturate היטב (כ 10 פעמים כל אחד) עם שלוש טפטפות זכוכית הלוהבת של הפחתת קוטר, עד ההשעיה היא הומוגנית ככל האפשר. </li><li style=";text-align:right;direction:rtl"> לאט ובעדינות להוסיף כמה טיפות של homogenate זה על גבי BSA / EBSS. אם זה מתחיל לשקוע דרך BSA / EBSS, triturate יותר ו / או להוסיף פתרון יותר קטן ג homogenate צריך לשבת בשכבה על גבי BSA / EBSS. לא לרעוד. ספין ב 100 XG במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> הסר supernatant וגלול גלולים (רך) ב 1 מיליליטר של מדיום ממ. </li><li style=";text-align:right;direction:rtl"> ספירת תאים באמצעות haemocytometer וצלחת ב800,000 תאים / coverslip ב500 μl של מדיום ממ. מקום בחממה על 37 מעלות צלזיוס עם 6% CO 2. </li><li style=";text-align:right;direction:rtl"> לשנות את המדיום למחרת (24-36 שעות לאחר ההכנה). הפוך פתרון של מדיום ממ המכיל 10 מיקרומטר של cytidine מחזור התא arabinofuranosyl המעכב (ARAÇ). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לכל אחדcoverslip, לשמור על 250 μl של המדיום הישן בצלחת גם טרי 24, לשאוב את השאר, להוסיף 250 μl של מדיום ממ הנורמלי ולשאוב זה לשטוף את התאים, ולאחר מכן להוסיף 250 μl של מדיום ישן חזרה לתאים והעליונים עד עם 250 μl של מדיום המכיל ARAÇ. הערה: ניתן להשתמש בתאים מ6 ימים במבחנה (DIV). </li></ol></li></ol> 4. סלקטיבי וכלת Microglia (הנערכת בשעה 6 DIV) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף LME הטהור עד 5 מיליליטר של aliquot נפרד של מדיום ממ לתת ריכוז סופי של 150 מ&quot;מ LME. </li><li style=";text-align:right;direction:rtl"> להחזיר את הפתרון לpH 7.4 ומסנן לעקר באמצעות sulfone 28 מ&quot;מ polyether (PES) מסנן 0.2 מיקרומטר מזרק. </li><li style=";text-align:right;direction:rtl"> לדלל פתרון לריכוז של 50 מ&quot;מ (2 x LME) במדיום ממ ומקום באמבט מים ב 37 מעלות צלזיוס במשך 10 דקות יחד עם מדיום ממ רגיל לתרבויות שליטה. </li><li style=";text-align:right;direction:rtl"> הסר מחצית (250 μl) בינוני ממ מתרבויות CGC ולשמור על 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> תאי פינוקעם מדיום ממ המכיל 2 x LME (250 μl), כלומר דילול 1: 1 נותן ריכוז טוב של 25 מ&quot;מ, או עם מדיום מראש חימם ממ ללא LME (250 μl) לתרבויות שליטה. </li><li style=";text-align:right;direction:rtl"> דגירה תאים ב 37 מעלות צלזיוס באווירת humidified עם 6% CO 2 עבור שעה 1. </li><li style=";text-align:right;direction:rtl"> לשטוף את תאי פעמיים במדיום ממ טרי מראש חימם להסיר תקשורת LME המכיל, ולאחר מכן להחליף את מדיום התרבות נשמרת ונפח שווה של מדיום ממ הטרי מראש חימם לבארות המקבילות. </li><li style=";text-align:right;direction:rtl"> חזור תרבויות לחממה (humidified עם 6% CO 2 על 37 מעלות צלזיוס) ולהשאיר למנוחה למשך 24 שעות לפני כל טיפול נוסף. </li></ol>

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The authors declare that they have no competing financial interests.

הצעדים החשובים ביותר כדי להבטיח את הסילוק בררני המוצלח של מיקרוגלים מCGC ו / או תרבויות מעורבות הן: 1) שמירה על תרבות CGC סטרילי ובריאה; 2) מסנן חיטוי הבינוני LME המכיל וחוזר הפתרון לpH 7.4; 3) שמירה על תקשורת CGC נשמרת וLME המכיל תקשורת ב 37 ° C כדי למנוע הלם חום; ו 4) עובד במהיר?...

המוח האנושי מורכב 85 מליארד נוירונים משוערים ותאים שאינם עצביים 85 מיליארדים נוספים כוללים גליה 1. לחלק הגדול יותר של 100 השנים האחרונות מדעני המוח התמקדו בעיקר באוכלוסיית התאים העצבית, תאי גליה להאמין להיות קצת יותר מאשר תאים פסיביים תמיכה שספקו תמיכה מבנית לנוירונים - ומכאן האטימולוגיה היוונית של &quot;גליה&quot; תורגמה לאנגלית כ &quot;דבק&quot;. לאחרונה, עם זאת, זה הפך להיות ברור יותר ויותר כי אינטראקציות עצביות-גליה עשויות להיות הרבה יותר בסיסיות להיבטים בסיסיים של נוירוביולוגיה, נוירופיזיולוגיה, והראשית וההתקדמות של מחלות ניווניות רבות. תאי המוח הקטן גרגיר (CGCs), האוכלוסייה הומוגנית העצבית הנפוצה ביותר במוח האנושי, שולטים במוח הקטן ומהווים יותר מ -90% מהבוחרים הסלולריים שלה. כתוצאה מכך, תאים אלה היו בשימוש נרחב במבחנה כמערכת מודל לtהוא חקר התפתחות עצבית, פונקציה, ופתולוגיה 2-6. עם זאת, תרבויות CGC עדיין מכילות מיקרוגלים וגליה אחרות בפרופורציות לטעון משמעותיות. כתוצאה מכך, נתוני CGC putatively מוצגות תגובות עצביות ישירות לטיפולי תאים שונים עשויים למעשה מתעוררים - בחלקו או בסך הכל - מהתגובה המשנית העקיפה של השכן גליה בתרבות. כדי להעריך את זה, אנחנו סלקטיבי בוטלו microglial מתרבויות עצביות CGC עם הסיוע של אסתר L-לאוצין תיל (LME). LME הוא סוכן lysomotropic שימש במקור כדי להרוס באופן סלקטיבי מקרופאגים 7, ומאז משמש לרוקן גם באופן סלקטיבי מיקרוגלים מעצביים, astrocyte, ותרבויות גליה מעורבות 8,9,10. LME מופנם על ידי מקרופאגים ומיקרוגליה, שבו היא גורמת להפרעת lysosomal ואפופטוזיס לאחר מכן 13,14. המקרופאגים ומיקרוגליה הם אופייני עשירים בlysosomes, גורמים להם להיות particularly פגיע בחשיפה לטיפול LME. פרוטוקול זה מספק דרך רבת עוצמה, אך פשוטה וקלה לברר את התרומה של מיקרוגליה בניסויי ניצול CGC ומערכות אחרות עצביות / גליה תרבות.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

היכולת של טכניקה זו כדי לחסל באופן סלקטיבי מיקרוגלים מCGC ו / או תרבויות מעורבות מסתמכת על היכולת העתידית של החוקר לזהות בצורה מדויקת ולהבדיל מיקרוגלים מהתאים המקיפים אותם. זה יכול להיות מושגים באמצעות יצרנית תא microglial הספציפי, כגון isolectin-B4, כפי שמודגם באיור 1. ?...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

דלדול סלקטיבית של Microglia מתא תרבויות המוח הקטן גרגיר שימוש L-לאוצין התיל אסתר

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Research

JoVE Journal

Neuroscience

8.6K Views.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

Video: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

תרבויות cerebellar Organotypic: אתגרים אפופטוטיים וזיהוי

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

ניתוח כמותי של רענון שלפוחיות Synaptic בריכת ב Cultured נוירונים גרגיר cerebellar באמצעות צבעים FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

תרבויות Explant עצביות מן<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

מניפולציה גנטית של Cerebellar גרגיר נוירונים<em&gt; במבחנה</em&gt; ו<em&gt; בVivo</em&gt; ללמוד עצבי מורפולוגיה וגירה

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo הדמיה של נדידת תאי המוח הקטן אחרי הלידה גרגיר שימוש Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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 ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

הערכת הכדאיות העצבית באמצעות פלואורסצין Diacetate-Propidium יודיד מכתים פעמיים Cerebellar גרגר Neuron תרבות

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

מידול מוות עצביים והתנוונות בנוירונים גרגר אסטרוציטומה העיקרי העכבר

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

לכימות מיקרוגלייה מורפולוגיה של Photomicrographs של אימונוהיסטוכימיה מוכן רקמות באמצעות ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Immunostaining של Myelinating Organotypic פרוסה אסטרוציטומה תרבויות והכנה

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...