עבודה זו נתמכה על ידי מענקים מהמוסד הקנדי לבריאות מחקר (CIHR), את פיטר Lougheed למחקר רפואי הקרן, תוכנית מקגיל ב Neuroengineering, Fonds דה משוכלל ונדיר en סנטה קוויבק (FRSQ), וכן קרן קנדה עבור חדשנות (CFI ). סבסטיאן ד Langlois נתמך על ידי פרס הדרכה מוסמך של Fonds de la משוכלל ונדיר en סנטה קוויבק (FRSQ) ועל ידי פרדריק בנטינג וצ&#39;רלס בסט פרס קנדה מלגות לתואר שני מן המכון הקנדי לבריאות מחקר (CIHR). אנחנו אסירי תודה על ג&#39;סיקה MT פאם לסיוע עם דמויות.

<ol>
	<li>Charron, F., Stein, E., Jeong, J., McMahon, A. P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. , 113-1111 (2003).</li><li>Fabre, P., Shimogori, T., Charron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+ipsilateral+retinal+ganglion+cell+axons+at+the+optic+chiasm+requires+the+Shh+Receptor+Boc.">Segregation of ipsilateral retinal ganglion cell axons at the optic chiasm requires the Shh Receptor Boc.</a> Journal of Neuroscience. 30, 266-275 (2010).</li><li>Helms, A. W., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitors+of+dorsal+commissural+interneurons+are+defined+by+MATH1+expression.">Progenitors of dorsal commissural interneurons are defined by MATH1 expression.</a> Development. 125, 919-928 (1998).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1, 2406-2415 (2006).</li><li>Okada, A., Charron, F., Morin, S., Shin, D. S., Wong, K., Fabre, P. J., Tessier-Lavigne, M., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boc+is+a+receptor+for+sonic+hedgehog+in+the+guidance+of+commissural+axons.">Boc is a receptor for sonic hedgehog in the guidance of commissural axons.</a> Nature. 444, 369-373 (2006).</li><li>Yam, P. T., Langlois, S. D., Morin, S., Charron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+guides+axons+through+a+noncanonical,+Src-family-kinase-dependent+signaling+pathway.">Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway.</a> Neuron. 62, 349-362 (2009).</li></ol>

תיארנו שיטה לנתח ותרבות נוירונים commissural מחוט השדרה עכברוש עובריים. הליך זה נעשה שימוש שגרתי במעבדה שלנו באופן מהימן להכין נוירונים כדי ללמוד את המנגנונים תאית ומולקולרית של הדרכה האקסון. עבור ביולוגיה של התא וניסויים immunochemistry, דיסקציה של פסולת מהן מניבה מספיק נוירונים. כאשר תאים יותר יש צורך, כמו בניסויים רבים ביוכימיה, דיסקציה של שתי המלטות ייתכן שתידרש. עבור אדם מאומן, דיסקציה ו דיסוציאציה של ~ 20 עוברי יכול להתבצע תוך פחות מ 4 שעות. לוחות הזמנים ארוכים תגרום לנתיחה קשה יותר של מיתרי השדרה עקב שינויים שלמות רקמות, והוא יכול גם להתפשר התאוששות יעיל של נוירונים קיימא. לאחר ביצוע הליך בפעם הראשונה, תאים על צלחת PLL מצופים משטחים שונים (חומצה שטף coverslips זכוכית, תרבות מנות רקמות) לצורך השוואה. בדרך כלל, התאים צריכים לצרף וחסונה כדי PLL מצופה פלסטיק רקמות מנות התרבות או רב גם הצלחות. זה יכול לשמש כבדיקה כללית כדאיות התא ותחזוקה אם יש בעיות עם ההדבקה של תאים על צמיחה coverslips זכוכית. הגורם העיקרי האחראי על הידבקות התא עניים זכוכית הוא איכות ניקוי זכוכית coverslip (חומצה כביסה עיקור). זה ישפיע על הידבקות התא בחלקו על ידי הקטנת היעילות PLL ציפוי. גורמים משותפים נוספים כוללים ציפוי PLL רע (זמן קצר מדי או ציפוי פתרון PLL מבוגר מדי), זיהום חיידקי או פטרייתי, או את השימוש בתקשורת או תוספי מזון שהם ישנים או פג. השתמשנו תרבויות נוירון commissural מוכן על פי נוהל זה סוג של מספר ניסויים, כולל immunochemistry, ביוכימיה, מבחני מפנה (ים et al. 2009). למרבה הפלא, נוירונים commissural מצופה זכוכית על PLL מצופה לשמור על היכולת להגיב לאותות הנחיה, כלומר, הם יוכלו לשנות את כיוון הצמיחה שלהם בתגובה גורם chemotropic מיושם, כמו סוניק הקיפוד, הראו בעבר להיות אות הנחיות האקסון ( Charron et al, 2003; אוקאדה, et al, 2006; ים et al 2009; פאבר et al, 2010).. לכן, זו היא מערכת רבת עוצמה כדי לחקור את ההשפעה של רמזים האקסון הדרכה במבחנה מאפשרת ניסויים שאינן אפשריות in vivo.

Dissection של חוט השדרה הגבי עוברי עכברים (ראה גם טבלה I) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> E13 להריון מבוים עכברוש </li><li style=";text-align:right;direction:rtl"> אתנול 70% </li><li style=";text-align:right;direction:rtl"> מספריים כירורגיות, כלי המדע פיין </li><li style=";text-align:right;direction:rtl"> מלקחיים, דומון # 5, כלים המדע פיין </li><li style=";text-align:right;direction:rtl"> צלחות פטרי </li><li style=";text-align:right;direction:rtl"> L-15 בינוני </li><li style=";text-align:right;direction:rtl"> Dissection מיקרוסקופ, Leica </li><li style=";text-align:right;direction:rtl"> העברת pipettes פלסטיק </li><li style=";text-align:right;direction:rtl"> Microscissors, כלי המדע פיין </li><li style=";text-align:right;direction:rtl"> טונגסטן מחטים בעלי פינים (אחד וו בצורת מחט, אחת L-צורות מחט, אחד מחט ישר), כלי המדע פיין </li><li style=";text-align:right;direction:rtl"> חום מומת סוס סרום (HiHS) </li><li style=";text-align:right;direction:rtl"> 15 צינורות פלסטיק מ&quot;ל </li></ul> Commissural נוירון תרבות (ראה גם טבלה I) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות רקמות החממות (37 מעלות צלזיוס, 5% CO 2, לחות מבוקרים) </li><li style=";text-align:right;direction:rtl"> הגרמני Desag coverslips זכוכית ו / או פלסטיק תרבות מנות רקמה </li><li style=";text-align:right;direction:rtl"> פולי-L-ליזין, סיגמא </li><li style=";text-align:right;direction:rtl"> סטרילי milliQ H 2 O </li><li style=";text-align:right;direction:rtl"> Neurobasal, Invitrogen </li><li style=";text-align:right;direction:rtl"> חום מומת בסרום שור עוברית (HiFBS) </li><li style=";text-align:right;direction:rtl"> L-גלוטמין </li><li style=";text-align:right;direction:rtl"> B27, Invitrogen </li><li style=";text-align:right;direction:rtl"> פניצילין / סטרפטומיצין אנטיביוטיקה </li><li style=";text-align:right;direction:rtl"> 37 ° C waterbath </li><li style=";text-align:right;direction:rtl"> סטרילי פסטר pipettes </li><li style=";text-align:right;direction:rtl"> בונזן מבער גז </li><li style=";text-align:right;direction:rtl"> HBSS, Ca 2 + / Mg 2 + חינם, Invitrogen </li><li style=";text-align:right;direction:rtl"> DNAse, וורטינגטון </li><li style=";text-align:right;direction:rtl"> MgSO 4 </li><li style=";text-align:right;direction:rtl"> סרכזת </li><li style=";text-align:right;direction:rtl"> Hemacytometer </li><li style=";text-align:right;direction:rtl"> Trypan פתרון כחול </li></ul>

dissection and culture of commissural neurons from embryonic spinal cord

נוירונים Commissural היה בשימוש נרחב כדי לחקור את המנגנונים הדרכה האקסון במהלך ההתפתחות העוברית חוט השדרה. גופי התא של הנוירונים האלה נמצאים בעמוד השדרה הגבי ו האקסונים שלהם לעקוב אחר מסלולי סטריאוטיפית במהלך ההתפתחות העוברית. אקסונים Commissural בתחילה הפרויקט ventrally כלפי floorplate. לאחר חציית קו האמצע, אקסונים אלה להפוך anteriorly הפרויקט לכיוון המוח. כל אחד משלבים אלה מוסדר על ידי פעולה של אותות הדרכה מספר. תרבויות מועשר בנוירונים commissural מתאימות בצורה אידיאלית עבור ניסויים רבים בהתמודדות עם מנגנוני pathfinding האקסון, כולל מבחני מפנה, immunochemistry וביוכימיה. כאן אנו מתארים שיטה לנתח ותרבות נוירונים commissural מחוט השדרה הגבי E13 חולדה. ראשית, חוט השדרה מופרד רצועות הגב הם גזור החוצה. הרקמה הגב אז הוא ניתק את ההשעיה על ידי תא trypsinization והפרעה מכאנית. נוירונים הם מצופה על פולי-L-ליזין coverslips מצופים זכוכית או רקמות תרבות הכלים. לאחר 30 שעות<em&gt; במבחנה</em&gt; רוב הנוירונים הרחיבו האקסון. טוהר התרבות (ים<em&gt; Et al.</em&gt; 2009), בדרך כלל מעל 90%, ניתן להעריך על ידי immunolabeling עם DCC נוירון סמנים commissural, LH2 ו TAG1 (הלמס וג&#39;ונסון, 1998). זו הכנה העצבית הוא כלי שימושי עבור<em&gt; במבחנה</em&gt; מחקרים של המנגנונים תאית ומולקולרית של צמיחה האקסון הדרכה commissural ובמהלך התפתחות חוט השדרה.

Watch this Scientific Journal Video about Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord at JoVE.com

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

סרט הווידאו הזה מדגים שיטה לנתח ותרבות נוירונים commissural מחוט השדרה הגבי E13 חולדה. נוירונים commissural ניתק מועילים כדי ללמוד את המנגנונים תאית ומולקולרית של צמיחה האקסון והדרכה.

Dissection והתרבות של נוירונים Commissural מחוט השדרה עובריים

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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16.7K Views.  Institut de Recherches Cliniques de Montréal. סרט הווידאו הזה מדגים שיטה לנתח ותרבות נוירונים commissural מחוט השדרה הגבי E13 חולדה. נוירונים commissural ניתק מועילים כדי ללמוד את המנגנונים תאית ומולקולרית של צמיחה האקסון והדרכה.

Video: Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection ותרבות ראשונית של נוירונים בהיפוקמפוס עכבר עובריים קליפתיים

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Dissection והתרבות של גנגליון צ&#39;יק Statoacoustic Explants ואת חוט השדרה ג&#39;ל קולגן עבור מבחני תוצאה Neurite

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DiI-תיוג של נוירונים DRG לחקר axonal מסעף בהכנת הר שלם של חוט השדרה עובריים בעכבר

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, תרבות וניתוח של<em&gt; Xenopus laevis</em&gt; רקמת רשתית עוברית

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

פרוטוקול עוף עובר חוט שדרת Slice תרבות

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

נתיחה והרכבה לרוחב של עוברי דג הזברה: ניתוח התפתחות חוט השדרה

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq ניתוח של ביטוי הגנים בדיפרנציאל חוט השדרה Electroporated אפרוח עוברי

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

בידוד, תרבות והטווח ארוך תחזוקה של יסודיים mesencephalic דופאמין נוירונים ממוח עוברי מכרסם

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

זברה<em&gt; באתרו</em&gt; הכנת בחוט השדרה עבור הקלטות אלקטרו מתוך השדרה חושית הנוירונים המוטוריים

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...