עבודה זו נתמכת על ידי משרד המחקר והפיתוח, מחקר רפואי שירות, מחלקה לענייני יוצאי הצבא. מחקר זה מומן בפרס הצטיינות VA סקירה (לMCL) ואוניברסיטת טנסי קרן למדעי בריאות במרכז לחקר טרשת נפוצה על ידי.

1. תיוג נוגדן (איור 1) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך 0.1 3 חיץ NaHCO M: <ul type="circle" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 8.4 גרם NaHCO 3 </li><li style=";text-align:right;direction:rtl"> 29.2 גרם NaCl </li><li style=";text-align:right;direction:rtl"> 1 הליטר H 2 O </li></ul></li><li style=";text-align:right;direction:rtl"> להעלות את המאגר לרמת חומציות של 8.4 עם הפתרון הזה (10.6 גרם Na 2 3 CO, 29.2 גרם NaCl, 1 הליטר H 2 0). </li><li style=";text-align:right;direction:rtl"> הוסף 35 Atto550 NHS μl לאנטי hnRNPA1 ו500 3 חיץ μl μl 70 NaHCO ולסובב לשעה 1 בטמפרטורת חדר. אחרי הסיבוב באורך שעה, להזריק את התערובת לתוך dialyzer ודיאליזה ב2 ליטר של PBS בן לילה. </li><li style=";text-align:right;direction:rtl"> למחרת, לרכז את Atto550 NHS אנטי hnRNPA1-דיאליזה באמצעות עמודת ספין (Amicon Ultra 0.5). </li><li style=";text-align:right;direction:rtl"> לאחר Atto550 NHS כותרת הנוגדן אנטי hnRNPA1 כבר מרוכז, ננו טיפת מדגם כדי לקבוע את הריכוז. </li></ol> 2. Transfection נוגדן (איור 2) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זרעי 10 5 תאים / גם לתוך 500 μl של Dulbecco השינוי של הנשר הבינוני (+1% אנטיביוטיקת FBS DMEM/F12 +10%) בשקופית 24 שעות גם 8 לפני transfection. confluency תא צריך להיות לפחות 70%. </li><li style=";text-align:right;direction:rtl"> עשרים וארבע שעות לאחר זריעה, להוסיף 2 μl של ריאגנט Ab-סחורה בלתוך צינור אפנדורף. </li><li style=";text-align:right;direction:rtl"> בשלב בא, להוסיף 2 מיקרוגרם של Atto550 NHS כותרת הנוגדן אנטי hnRNPA1 (0.5 מיקרוגרם / μl) לאותו הצינור אפנדורף ודגירה 10-15 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> במהלך הדגירה, לשאוב תקשורת ולהוסיף 394 μl של תקשורת DMEM הטריה לתאים. </li></ol> שים לב: הוסף 500 DMEM μl לתא אחד של תאי תוליים לפעול כביקורת. <ol start="5" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר דגירה, להוסיף 100 μl של DMEM לתערובת הנוגדנים, מערבב על ידי pipetting למעלה ולמטה, ולהוסיף לתאים בDMEM להיקף כולל של 500 μl. </li><li style=";text-align:right;direction:rtl"> שינוי התקשורת לאחר 48 שעות של לידת נוגדן. </li></ol> הערה: הפרוטוקול חזר על עצמו עם FITC שכותרת ריג כביקורת חיובית. 3. הדמיה חייה וקבועה לקביעת יעילות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תמונת תאי החיים 48 שעות בשימוש במסנן Cy3 על מיקרוסקופ פלואורסצנטי לתמונה את Atto550 NHS transfected הנוגדנים שכותרתו. </li><li style=";text-align:right;direction:rtl"> לאחר הדמיה חייה הושלמה, לשאוב את התקשורת ותאים לתקן. </li><li style=";text-align:right;direction:rtl"> לאחר aspirating תקשורת, לטפל בתאים עם Paraformaldehyde 0.4% למשך 15 דקות בטמפרטורת חדר. שטוף תאים 4 × 5 דקות כל אחת עם PBS. ואז, לעגן את התאים עם מדיה הרכבה המכילה ריאגנט DAPI וcoverslip תיקון. למדוד יעילות transfection של התאים הקבועים עם AxioVision תוכנה. אם AxioVision תוכנה היא לא בהישג יד, פשוט לספור את האירועים מהתמונה שלך על ידי יד ותוצאות חישוב בתקע 3.7. לחלופין, תוכנת ImageJ יכולה לשמש לחישובים אלה. </li><li style=";text-align:right;direction:rtl"> ראשית, שימוש במדידה &quot;האירועים&quot; כדי לספור את המספר הכולל של תאים בתמונה על פי בחירתך ב20x הגדלה. </li><li style=";text-align:right;direction:rtl"> לאחר מכן השתמשתי במדידה &quot;האירועים&quot; לספור את התאים שלא היוtransfected בהצלחה עם נוגדנים באותה התמונה בהגדלה 20x. </li><li style=";text-align:right;direction:rtl"> כדי לגשת ל&quot; אירועים &quot;נמדדו בפורמט סגנון Excel, לחץ על&quot; מאפיינים &quot;, לחץ על הכרטיסייה&quot; המדידה &quot;, ולחץ לצפייה&quot; כשולחן. &quot; </li><li style=";text-align:right;direction:rtl"> לבסוף, השתמש בחישוב הבא כדי לקבוע את יעילות transfection שלך על ידי ההשוואה בין שני אירועים שנמדדו בעבר: ((תאים - תאי סך Untransfected) / תאים סה&quot;כ) = יעיל x 100 Transfection. </li></ol> 4. נציג תוצאות תמונות החיות של untransfected (האיור 3A) בהשוואה ל( איור 3B) נוירונים transfected מגלה כי התיוג של הנוגדנים (אדום או ירוק בכל דמות) הוא ציין באופן ברור בתוך תאים (האיור 3B, C, D). בעקבות שאיפה וכביסה עם תקשורת כדי להסיר נוגדנים או בין חסיד אל פני השטח של תאים, נוגדנים שכותרתו הם עדיין נמצאים בתאי עצב (האיור 3C, ד).<html> כדי לחשב את יעילות transfection, השתמש בתוכנת AxioVision לחשב תאים הכוללים בהשוואה לתאים שלא transfected בהצלחה. ערכים אלו נקשרו לנוסחת יעילות transfection לרכוש את יעילות transfection כאחוז מכלל תאים (איור 4). <img alt="איור 1" src="/files/ftp_upload/4154/4154fig1.jpg" /> איור 1. תרשים זרימה של הנהלים המשמשים לנוגדני תווית עם ATTO 550 NHS אסתר אחרי הצעדים הנדרשים כדי לרכז את הנוגדנים שהכותרת. <a href="http://www.jove.com/files/ftp_upload/4154/4154fig1large.jpg" target="_blank">לחץ כאן לצפייה בדמות גדולה.</a> <img alt="איור 2" src="/files/ftp_upload/4154/4154fig2.jpg" /> איור 2. תרשים זרימה של הנהלים המשמשים לtransfect נוגדנים שכותרתו לתוך תאי חיים בתרבות.arge.jpg &quot;target =&quot; _blank &quot;&gt; לחץ כאן לצפייה בדמות גדולה. <img alt="איור 3" src="/files/ftp_upload/4154/4154fig3.jpg" /> איור 3. חי תמונות של הנוירונים transfected 48 השעות transfection הבא. <img alt="איור 4" src="/files/ftp_upload/4154/4154fig4.jpg" /> איור 4. קבוע ושטף תמונות עם אירועים נספרו על מנת לקבוע יעילות transfection.

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אין ניגודי האינטרסים הכריזו.

על מנת לבדוק את ההשערות לגבי מולקולות ספציפיות וביטוייהם, גנים ווקטורים אחרים transfected נפוץ לתוך תאים. ההיבט הייחודי של הליך זה הוא היכולת בקלות transfect נוגדנים לתאי המטרה. בצענו בשיטה זו בחמישה ניסויים נפרדים, כל אחד בכפילויות. תוצאות יעילות transfection היו דומות בכל הניסויי?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td></tr><tr><td> מיקרוסקופ פלורסנט AxioObserver A1 </td><td> Zeiss </td><td></td></tr><tr><td> AxioVision גרסת 4.2 </td><td> Zeiss </td><td></td></tr><tr><td> אנטי rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> FITC מתויג אנטי ריג </td><td> העוז Biosciences </td><td> AI20100 </td></tr><tr><td> Atto550 NHS אסטר </td><td> סיגמא </td><td> 92835 </td></tr><tr><td> Ab-סחורה ב </td><td> העוז Biosciences </td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10% FBS אנטיביוטיקת 1% </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> פולי-D-ליזין שקופיות תרבות 8-כן </td><td> BD </td><td> 354632 </td></tr><tr><td> Rotator רב תכליתי </td><td> המדעי Industries Inc </td><td> דגם 151 </td></tr><tr><td> DAPI הרכבת מדיה </td><td> Millipore </td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion </td><td> 9626 </td></tr><tr><td> Dialyzer </td><td> תרם הפישר סיינטיפיק </td><td> 66380 </td></tr><tr><td> Amicon Ultra-0.5 </td><td> Millipore </td><td> UFC501096 </td></tr><tr><td> Paraformaldehyde (פתרון 4%) </td><td> מדעי מיקרוסקופ אלקטרונים </td><td></td></tr><tr><td> Nano-Drop </td><td> תרם הפישר סיינטיפיק </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

נוגדנים לספק את היכולת לקבל תובנה רומן לאירועים שונים המתרחשים במערכות חיים. היכולת לייצר נוגדנים ספציפיים מאוד לחלבוני היעד אפשרה לשאלות ביולוגיות מדויקות מאוד להתייחסות. חשוב לציין, הנוגדנים היו מעורבים בפתוגנזה של מספר המחלות אנושיות לרבות זאבת מערכתית (לופוס), דלקת מפרקים שגרוניים (RA), תסמונות paraneoplastic, טרשת נפוצה (MS) וטיפוס אנושי T-lymphotropic וירוס 1 (HTLV-1) קשור myelopathy / paraparesis ספסטית הטרופית (HAM / כפית) 1-9. איך לגרום למחלה היא נוגדני שטח של חקירה מתמשכת, והנתונים מצביעים על כך שאינטראקציות בין מולקולות נוגדנים ותוצאות תאיות שונות בדלקת, שינו סלולריים הודעות, ואפופטוזיס 10. הוכח כי חולים עם טרשת נפוצה וHAM / TSP לייצר נוגדנים עצמיים לחלבונים התאיים RNA מחייבים חלבונים הטרוגנית ribonuclear1 (hnRNP A1) 3, 5-7, 9, 11. הנתונים האחרונים מצביעים על כך נוגדנים לאנטיגנים משטח תוך עצבי ושניהם פתוגניים 3, 5-9, 11. לפיכך, הליך המאפשר לחקר נוגדנים תאיים: אינטראקציות חלבון הייתי משאיל תובנה גדולה להיווצרות מחלה. גני transfected נפוץ לתוך תאים ראשוניים וקווים סלולריים בתרבות, לעומת זאת transfection של נוגדנים לתאים התעכב בשל שינוי של מבנה נוגדנים או יעילות transfection עניה 12. שיטות אחרות של transfection כוללות transfection נוגדן המבוסס על יפוזומים קטיוני (מורכב DOTAP / סמים) וpolyethylenimines (PEI); שניהם גרמו לירידה של פי עשרה בtransfection נוגדן השוואה לקבוצת ביקורת 12. השיטה מבוצעת במחקר שלנו היא דומה לשיטות קטיוני שומנים בתיווך ומשתמשת במנגנון מבוסס שומנים ליצירת קומפלקסים שאינם קוולנטיים עם הנוגדנים דרך אלקטרוסטטי וhydrאינטראקציות ophobic 13. אנו מנוצלים מגיבים Ab-סחורה ב, שהוא ניסוח שומנים מסוגל ללכוד נוגדנים דרך אינטראקציות אלקטרוסטטיות והידרופובי שאינם קוולנטיים ואספקתם בתוך תאים. לפיכך כימי וגנטיים זיווגים אינם הכרחיים עבור משלוח של נוגדנים פונקציונליים לתוך תאי חיים. שיטה זו אפשרה לנו לבצע מעקב נוגדנים שונים וניסויי חלבון לוקליזציה, כמו גם את הניתוח של ההשלכות המולקולריות של נוגדנים תאיים: אינטראקציות חלבון 9. בפרוטוקול זה, אנו נראים כיצד transfect נוגדנים לנוירונים במהירות, reproducibly ועם רמה גבוהה של יעילות transfection. כדוגמה, ישתמשו נוגדני A1 ואנטי IgG נגד hnRNP. לכימות קל של יעילות transfection השתמשנו נוגדני A1 אנטי hnRNP כותרתו עם ATTO-550-NHS וFITC שכותרת IgG. Atto550 NHS הוא תווית חדשה עם ספיגה מולקולרית גבוהה והקוונטים ייישן. מקור עירור ומסנני ניאון לAtto550 דומה לCy3 (שמות 556 Em. 578). בנוסף, יש Atto550 photostability גבוה. FITC מתויג-IgG שמש כבקרה כדי להראות ששיטה זו היא תכליתית ולא לצבוע תלוי. גישה זו והנתונים שיופקו תסייענה בהבנה של התפקיד שנוגדנים לאנטיגנים תאיים היעד עשויים לשחק בפתוגנזה של מחלות בבני אדם.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

גישה מהירה כדי לחקור אינטראקציות והשפעות על מנגנונים מולקולריים הקשורים לנוכחות של נוגדנים בסביבה תאית מתוארת. השיטה כרוכה transfection של נוגדנים לתאים חיים באמצעות היווצרות מורכבת שאינן קוולנטיים המבוססת על ניסוח שומנים בדם. הטכניקה ניתנת להתאמה לשורות תאים הנציחו ותאים ראשוניים.

Transfection נוגדן לנוירונים ככלי ללימוד פתוגנזה מחלות

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

antibody-transfection-into-neurons-as-tool-to-study-disease

Research

JoVE Journal

Neuroscience

10.7K Views.  Veterans Administration Medical Center, Memphis, TN. גישה מהירה כדי לחקור אינטראקציות והשפעות על מנגנונים מולקולריים הקשורים לנוכחות של נוגדנים בסביבה תאית מתוארת. השיטה כרוכה transfection של נוגדנים לתאים חיים באמצעות היווצרות מורכבת שאינן קוולנטיים המבוססת על ניסוח שומנים בדם. הטכניקה ניתנת להתאמה לשורות תאים הנצי...

Video: Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

לייזר Microdissection לכידה של תסיסנית נוירונים היקפיים

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

Cut-טעינה: כלי שימושי לבחינת היקף Coupling צומת גאפ נותב בין הנוירונים רשתית

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

סידן פוספט Transfection של ראשוני נוירונים בהיפוקמפוס

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Analyzer הנייד מבוססת עכבה בזמן אמת ככלי להתוות מסלולים מולקולריים מעורבים ברעילות העצבית וneuroprotection בקו תאים עצבי

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

מערכת חוש הריח כמודל לחקר דפוסי axonal צמיחה ומורפולוגיה<em&gt; In vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

זריקות לשריר לאורך צלחות מוטוריות סיום: גישה פולשנית להסעות קליעים נותבים ישירות לתוך מנוע נוירונים

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Stripe Assay לחקר פעילות דוחה או מושך של מצע חלבון באמצעות ניתק נוירונים בהיפוקמפוס

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic תרבויות פרוסה בהיפוקמפוס כמודל מחקר Neuroprotection, Invasiveness של תאים סרטניים

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

הממשל אקסוגני של אלפא-synuclein Microsomes-הקשורים אגרגטים כדי נוירונים העיקרי כמודל תא עוצמה היווצרות הסיבים

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

מבוססת תא Assay ללמוד טאו בתיווך נוגדנים אישור מאת מיקרוגלייה

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...