עבודה זו נתמכה על ידי מענקים ממשרד ההגנה (פרס פיתוח רעיון, W81XWH-10-1-0041, א.ב.), CT תאי גזע המענק (א.ב.), והמכון לאומי לבריאות NRSA 10668225 (DMF). החומר זה מבוסס על העבודה הנתמכת בחלקו על ידי מדינת קונטיקט במסגרת התכנית לקונטיקט לתאי הגזע למחקר המענקים. התוכן שלה הוא באחריות בלעדית של הכותבים ואינם מייצג בהכרח את עמדתה הרשמית של מדינת קונטיקט, המחלקה לבריאות ציבור של מדינת קונטיקט או CT חידושים, Inc הממנים לא היו כל תפקיד בתכנון מחקר, איסוף נתונים וניתוח, החלטה לפרסם, או הכנה של כתב היד.

הליך זה בהתאם לדרישות ייל IACUC. מדענים צריכים לוודא שהנחיות IACUC מאושרות ואחריו על פי הדרישות המוסדיות שלהם. 1. סעיף 1. הכנת דנ&quot;א, פתרונות, וזכוכית פיפטות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור טוהר גבוה (OD 260/280&gt; 1.80) ריכוז גבוה (מיקרוגרם / μl&gt; 2.5) אנדוטוקסין נטול ה-DNA. </li><li style=";text-align:right;direction:rtl"> הכן תמיסת מלח 0.9% ומסנן סטרילי דרך מסנן מיקרומטר 22. </li><li style=";text-align:right;direction:rtl"> 10 צינורות Place סנטימטר אש מלוטשים ורוסיליקט זכוכית נימים (OD: 1.5 מ&quot;מ, מזהה: 1.10 מ&quot;מ) למודל PP-830-10 Narishige מחשב זכוכית פיפטה חולץ עם חוט kanthal. הגדר חולץ למשיכת צעד אחד משוקללת ב70.5 מעלות צלזיוס לשבור טיפים עם מלקחיים מעגליים ולבדוק תחת מיקרוסקופ לנתח לקצוות משוננים. טיפי שיפוע ומקום תחת מנורת UV במשך 15 דקות. pipettes משך צריך להיות מסומן ב 2 מ&quot;מ מהקצה של הקצה. </li><li style=";text-align:right;direction:rtl"> הכן 0.1% משקל / נפח המהיר הירוק solutiעל ידי ערבוב פתרון סטרילי מסונן 0.9% עם מלח ירוק מהיר יבשה. </li></ol> 2. סעיף 2. הכנת בעלי חיים, זכוכית פיפטה טוענת והזרקת פלסמיד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר יום לאחר לידה בנפרד 0-1 גורים מכלובים, מקום על צלחת פטרי זכוכית מראש צוננת עד 4 מעלות צלזיוס, ולאחר מכן מקום על קרח רטוב. </li><li style=";text-align:right;direction:rtl"> לאחר כ 5 דקות, לקבוע את המצב של הרדמה על ידי ניצול את תגובת דחק הרגל. אם אין תנועה מתרחשת, גורי בכפוף להזרקה תוך חדרים. </li><li style=";text-align:right;direction:rtl"> חשוב לציין כי שילוב של ירוק מהיר, ה-DNA, ותמיסת מלח יכול לגרום לאידוי מהיר, התגבשות, ומצור על pipettes זכוכית. לכן, לייצר פתרון מניות ו1-2 μl aliquot על parafilm מייד לפני זריקות. </li><li style=";text-align:right;direction:rtl"> לשאוב נפח aliquoted כל עם הכוס טפטפה משך. </li><li style=";text-align:right;direction:rtl"> החזק את הראש של הגור בין אגודל ואצבע המורה של היד הדומיננטית פחות שלך. </li><li style=";text-align:right;direction:rtl"> הפעל מנורהד להחזיק את הראש של גור, ממש מחוץ לאלומת אור. במידת צורך, לשים מלח על ראש כדי להפחית את כמות האור מוחזר על ידי גור. חדרי הלב עכשיו צריכים להיות מוארים. </li><li style=";text-align:right;direction:rtl"> עם היד הדומיננטית שלך, הכנס את המחט לתוך חלל רוחב הקרוב אליך (האיור 1A ו-1B). במקום הזרקה צריך להיות כ מרחק שווה מהתפר והעין ו2 מ&quot;מ לרוחב כדי תפר sagittal lambdoid. הכנס משכתי פיפטה לסימן המ&quot;מ 2 עבור P0 גור, שאמור להבטיח חדירה לתוך חלל הרוחב. בצירוף מקרים, אתה עשוי לראות חזרה של מילוי CSF לתוך פיפטה משחרור לחץ תוך גולגולתי. כדי להפחית את לחץ תוך גולגולתי, המסייע בהזרקה של פלסמיד, לשחרר את האחיזה שלך על ראש pup.Step 2.8. להזריק כ -0.5 μl של פלסמיד לתוך חדר לב לרוחב באמצעות הזרקת אוויר בלחץ (Picospritzer, פארקר). </li></ol> 3. סעיף 3. Electroporation ושחזור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Seלא המתח של הגנרטור electroporation במשך 5 פולסים מרובעים, 50 msec / דופק של 100 וולט, עם 950 מרווחי msec. הסגולי המרחבי של electroporation פלסמיד מוכתב על ידי הכיוון של העברת תשלום. תשומת לב צריכה להינתן למיקום נתון ההטרוגניות של אוכלוסיות תאי גזע לאורך SVZ 8. הבדלים בשיעורי התפשטות וגורל תא זוהו למקומות גחון גב וזנב מקוריים-9,10. </li><li style=";text-align:right;direction:rtl"> ברגע פלסמיד מוזרקת, לטבול אלקטרודות פינצטה לתוך PBS. צעד זה הוא למקסם את העברת תשלום וכדי למנוע כוויות שנגרמו על ידי התנגדות. </li><li style=";text-align:right;direction:rtl"> הנח את האלקטרודה החיובית על הקיר לרוחב הגב של הגור ליד אוזן ipsilateral לאתר של הזרקת פלסמיד (האיור 1C ו1D). הנח את האלקטרודה השלילית על רוחב גחון ההמיספרה נגדית לחוטמו של הגור. </li><li style=";text-align:right;direction:rtl"> ליזום העברה נוכחית על ידי לחיצה על רגלי הדופקמכשפת דוושה ולסחוף את האלקטרודות מהגבה לרוחב באמצעות ~ 25 ° מרווחי זווית. </li><li style=";text-align:right;direction:rtl"> מקום electroporated גורים על כרית חימום למשך 5 דקות. בעדינות לסובב גורים בכל 30 שניות עם גירוי קל. </li></ol>

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המחברים אין גילויים שיבצעו.

כאן אנחנו פירוט הטכניקה של electroporation SVZ יילודים, טכניקה במהירות ובאופן עמיד לתייג ולטפל בתאי גזע SVZ וצאצאיהם. ישנם מספר יתרונות שיש electroporation בהשוואה לשיטות אחרות. ראשית, מאחר תיוג המוקד של תאים, אחד הוא מסוגל להבחין סלולריות השפעות אוטונומיות אוטונומיות ולא תא. מניפולצ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> אבובי ורוסיליקט מלוטשים כבדים </td><td> מכשיר סאטר </td><td> BF150-110-10 </td><td></td></tr><tr><td> Dual-שלב הזכוכית micropipette פולר </td><td> Narishige </td><td> PC-10h </td><td></td></tr><tr><td> גרין המהיר </td><td> הפישר סיינטיפיק </td><td> 0521192205 </td><td></td></tr><tr><td> ECM 830 Wave הכיכר פולסה גנרטור </td><td> הרווארד Apparatus </td><td> 45-0052 </td><td></td></tr><tr><td> Tweezertrodes </td><td> הרווארד Apparatus </td><td> 45-0488 </td><td></td></tr><tr><td> מקור אור בסיבים אופטי </td><td> הפישר סיינטיפיק </td><td> 12-562-36 </td><td></td></tr><tr><td> הלוגן טונגסטןמנורה </td><td> USHIO אמריקה, Inc </td><td> 1002247 </td><td></td></tr><tr><td> Picospritzer השני </td><td> פארקר מכשירים </td><td> 052-0312-900 </td><td></td></tr></tbody></table>

neonatal subventricular zone electroporation

תאי גזע עצביים (NSCs) קו רוחב חדרי לב לאחר הלידה ולהצמיח סוגי תאים רבים הכוללים נוירונים, האסטרוציטים, ותאי ependymal 1. הבנת המסלולים המולקולריים האחראים למל&quot;ל התחדשות עצמית, מחויב, וההתמיינות היא קריטית לרתימת הפוטנציאל הייחודי שלהם כדי לתקן את המוח ולהבין טובים יותר את הפרעות במערכת עצבים מרכזיות. קודם שיטות למניפולציה של מערכות יונקים נדרשו זמן מאמץ רב ויקר של הנדסה גנטית ברמת החיים השלמה 2. לכן, הרוב המכריע של מחקרים בחן את הפונקציות של מולקולות המל&quot;ל במבחנה או בחסרי חוליות. הנה, אנחנו מדגימים את הטכניקה הפשוטה ומהירה כדי לתפעל NPCs יילודים המכונה האזור electroporation subventricular יילודים (SVZ). טכניקות דומות פותחו לפני עשור כדי ללמוד NSCs העוברי וסייעו מחקרים על cortical פיתוח 3,4. לאחרונה זה יושם ללמוד המוח הקדמי המכרסם לאחר לידת 5-7. טכניקה זו מביאה לתיוג חזק של SVZ NSCs וצאצאיהם. לפיכך, לאחר לידת SVZ electroporation מספק עלות וזמן חלופי יעילה להנדסה גנטית של יונקי המל&quot;ל.

תוצאות ילודות subventricular אזור electroporation בתיוג של כמעט כל גליה הרציפה עם האזור הגבה, לרוחב גב, ורוחב subventricular בעקבות התנועה &quot;הגורפת&quot; של האלקטרודה פינצטה (האיור 1E) רדיאלי. עם זאת, electroporation ניתן להתאים לדרישה של הניסוי המתאים אך לא מטאטא את האלקטרודה ושימוש במיקו...

Watch this Scientific Journal Video about Neonatal Subventricular Zone Electroporation at JoVE.com

Neonatal Subventricular Zone Electroporation

אנחנו מדגימים את טכניקה פולשנית המכונית electroporation האזור subventricular ילוד. הטכניקה מורכבת של הזרקת DNA פלסמיד לתוך חדרי לב לרוחב של גורים ילודים ויישום זרם חשמלי כדי לספק ולתפעל גנטיים תאי גזע עצביים

Electroporation האזור Subventricular ילודים

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

neonatal-subventricular-zone-electroporation

Research

JoVE Journal

Neuroscience

12.8K Views.  Yale University School of Medicine. אנחנו מדגימים את טכניקה פולשנית המכונית electroporation האזור subventricular ילוד. הטכניקה מורכבת של הזרקת DNA פלסמיד לתוך חדרי לב לרוחב של גורים ילודים ויישום זרם חשמלי כדי לספק ולתפעל גנטיים תאי גזע עצביים

Video: Neonatal Subventricular Zone Electroporation

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

אזור Subventricular En פנים: מכתים Wholemount ושפל Ependymal

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

ב electroporation vivo של הרשתית עכבר פיתוח

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

עכבר בתוך הרחם Electroporation: ג&#39;ין מבוקרת transfection Spatiotemporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

הכנת Slices האזור Subventricular החריף להדמית סיד

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Electroporation שטח pial ילודים

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Electroporation תת סוג סלקטיבי של בקליפת המוח interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

בידוד והתרבות של תאי גזע בוגרים עצביים מאזור העכבר Subcallosal

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

הקלטה EEG בחופשיות העברת חולדות neonatal בשיטת רומן

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

נוירונים חוט השדרה בידוד ותרבות מ עכברים ניאונטליים

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...