הממומן על ידי NINDS, איגוד הלב האמריקני, איגוד אמריקאי לחקר הזדקנות, מצעד הפרוטות

1. הכנה לפני שמתחילים, להרכיב את מכשירי ניתוח הנדרש, דיסקציה התקשורת לתרבות, עכברושית 7 יום או גורים העכבר. הפרוטוקול זהה עבור העכבר ההכנות חולדה. (I) חומרים וציוד <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מספריים הפעלה (אורך ישר 6 &quot;, חתול # RS6818, Roboz, Gaithersburg, MD) </li><li style=";text-align:right;direction:rtl"> מיקרו לנתח מספריים, (אורך 4 &quot;, חתול # RS5910. Roboz, Gaithersburg, MD) </li><li style=";text-align:right;direction:rtl"> פחמן פלדה דומון מלקטת (חתול # RS-5045.Roboz, Gaithersburg, MD) </li><li style=";text-align:right;direction:rtl"> השתנה &quot;טרנספר&quot; זכוכית pipettes (בחלק התחתון של פסטר פיפטה להסיר והחליקה קצה) </li><li style=";text-align:right;direction:rtl"> הסרט Aclar פלסטיק לחתוך 2 &quot;x 2&quot; ריבועים (Honeywell, # 812071, Pottsville, PA) </li><li style=";text-align:right;direction:rtl"> כיסוי ספוגים 4 על ידי 3 ב (קנדל, חתול # 3157, מנספילד, MA) </li><li style=";text-align:right;direction:rtl"> 30 מ&quot;מ Millicell מוסיף קרום (0.4 מיקרומטר; Millipore, בדפורד, מסצ&#39;וסטס) </li><li style=";text-align:right;direction:rtl"> Mcllwain המסוק רקמות עם להב פיפיות גילוח (המסוק McILwain רקמות, החברה Vibratome, סנט לואיס, מיזורי) </li><li style=";text-align:right;direction:rtl"> מיקרוסקופ הפוך עם מצלמת CCD וניתוח תוכנה תמונה </li></ul> (Ii) בינוני Dissection (DM, 1 ליטר ב-pH 7.3) <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> מלח מאוזן של האנק </td><td> 1 בקבוקון (95.2 g/1L) (סיגמא: H2387) </td></tr><tr><td> NHCO 3 (4.2 מ&quot;מ) </td><td> 350 מ&quot;ג </td></tr><tr><td> HEPES (10 mM) </td><td> 2.83 גר &#39; </td></tr><tr><td> גלוקוז (33.3 mM) </td><td> 6.0 גרם </td></tr><tr><td> פניצילין / סטרפטומיצין (100x) </td><td> 10 מ&quot;ל </td></tr><tr><td> ארגון ה-BSA (0.3%) </td><td> 300 מ&quot;ג </td></tr><tr><td> MgSO 4-7H 2 O </td><td> 1.44 גר &#39; </td></tr></tbody></table> (Iii) תרבות בינוני (400 מ&quot;ל) <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> ממ (Invitrogen 11575-032) </td><td> 200 מ&quot;ל </td></tr><tr><td> האנק *** מאזן של מלח (Invitrogen 24020-117) </td><td> 100 מ&quot;ל </td></tr><tr><td> סוס בסרום (Invitrogen 26050-070) </td><td> 100 מ&quot;ל </td></tr><tr><td> HEPES חיץ (1 מ &#39;; Invitrogen 15630-080) </td><td> 5 מ&quot;ל </td></tr><tr><td> פניצילין / סטרפטומיצין (100x) </td><td> 4 מ&quot;ל </td></tr><tr><td colspan="2"> *** הוסף 12.8 גרם גלוקוז לסולט מאזן 500 מ&quot;ל של האנק לפני ביצוע בינוני תרבות </td></tr></tbody></table> (Iv) בעלי חיים חולדה הישנה 7 יום או גורים העכבר לאחר הלידה, בדרך כלל אחד בכל המלטה הניסוי. 2. ביום התרבות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כל הכלים לנתיחה הסרט Aclar חייב להיות טבול ETOH 70% במשך 30 דקות לפני תחילת. פני השטח של מכסה המנוע לנתיחה, להב, וכן המסוק רקמה יש לחטא עם אתנול. </li><li style=";text-align:right;direction:rtl"> הכן מספר (3-4) 6 גם תרבות צלחות רקמות; להוסיף 1 מ&quot;ל בינוני לכל תרבות היטב, להוסיף קרום חדיר Millipore היטב כל אחד. מקום 6 גם צלחות של 37 ° החממה עד מוכן להעביר את הפרוסות. </li><li style=";text-align:right;direction:rtl"> הכן מספר (4-6) 60 מ&quot;מ צלחות עם DM ומניחים על קרח. </li><li style=";text-align:right;direction:rtl"> במהירות לערוף עם מספריים לטבול 70% ETOH, במהירות לחשוף את המוח עם חתך saggital הגולגולת. מוציאים בזהירות את המוח ואת המקום מיד DM קר. מפריד אותה לשני חצאי ומקום לכל צד האונה המדיאלי על ספוג כיסוי 2-3 שניות. </li><li style=";text-align:right;direction:rtl"> הרם בעדינות והנח את הכדור אל הכיכר הסרט מקום Aclar על המסוק רקמות; לחתוך את המוח coronally ב סעיפים 350 מיקרומטר. בסיום, בעדינות לקחת את הכיכר Alcar עם האונה לצלול מחולק וב DM קר. </li><li style=";text-align:right;direction:rtl"> באמצעות מיקרוסקופ לנתח, להפריד את הסעיפים בהיפוקמפוס. עבור החולדה, צריך להיות 5 חלקים פורש מקורי להיפוקמפוס הזנב, ועל העכבר צריך להיות בסעיפים 3-4. </li><li style=";text-align:right;direction:rtl"> קח את 6 גם צלחות עם קרום מוסיף מן הרקמה 37 ° התרבות בחממה. העברת פרוסות בהיפוקמפוס בנפרד על קרום חדיר בעזרת פיפטה העברת זכוכית. אם יותר מדי DM הוא שוחרר על הממברנה, להסיר עודף DM עם פיפטה פסטר. מקום חמש פרוסות בהיפוקמפוס על כל קרום. אפשר לפחות 3 בארות לכל מצב, או 15 פרוסות. </li><li style=";text-align:right;direction:rtl"> דגירה צלחות תרבות ב 37 מעלות ב CO החממה 5% humidified במשך 12-14 ימים לפני תחילת ניסוי רעילות. בינוני התרבות ניתן לשנות פעמיים בשבוע. </li></ol> 3. גרימת וכימות פגיעה עצבית בהיפוקמפוס בתרבות Slice <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר 12-14 ימים בתרבות, להוסיף 5 מיקרוגרם / מ&quot;ל ​​יודיד propidium (PI) לתקשורת ותרבות. צעד זה יאפשר להדמיה וכימות של מוות התאים הבזליים. </li><li style=";text-align:right;direction:rtl"> למחרת, לכמת PIהקרינה subregion CA1 (ו / או CA3 משוננת או במקרה הצורך) של כל פרוסה בהיפוקמפוס להשיג מתכוון מדידות הקרינה לצרכן המייצג המוות של התאים הבזליים (המכונה זמן = 0 שעות, או T 0). לרכישת תמונה וכימות, אנו משתמשים בתוכנות OpenLab (אימפרוביזציה, בריטניה), אך רכישת תמונה אחרת ותוכנה ניתוח יכול לשמש כדי לכמת את עוצמת הקרינה. </li><li style=";text-align:right;direction:rtl"> מיד לאחר רכישת התמונה של T 0 PI הקרינה, לגרום לפגיעה עצבית לפי שיטת הבחירה שלך (השתמשנו 10 מיקרומטר NMDA, חמצן, גלוקוז קיפוח, או LPS במשך שעה אחת כדי לגרום לרעילות עצבית 2,5). לאחר הגירוי רעילים, להחליף בינוני עם בינוני טרי המכיל 5 מיקרוגרם / מ&quot;ל ​​לצרכן ולשמור פרוסות לילה. </li><li style=";text-align:right;direction:rtl"> ביום 3, לכמת אומר לצרכן הקרינה של תנאי הניסוי ב 24 שעות, או טי 24. </li><li style=";text-align:right;direction:rtl"> לאחר רכישה זו התמונה, מיד לשנות בינוני ומוסיפים 10 מיקרומטר NMDA ו - 5 מיקרוגרם / מ&quot;ל ​​לצרכן ופרוסות דגירה לילה להשיג מוות עצבי מקסימלי. </li><li style=";text-align:right;direction:rtl"> ביום 4, לכמת אומר הקרינה לצרכן של מוות עצבי מקסימלי, כהגדרתו T max. חישוב התא למוות אחוז כדלקמן: (ט 24 - T 0) / (T max-T 0). </li></ol> 4. נציג תוצאות <img src="/files/ftp_upload/2106/2106fig1.jpg" alt="איור 1" /> איור 1.) A (קו זמן הדור של פגיעה ניסיוני בהיפוקמפוס ובעקבות רכישת התמונה. (ב) נציג תמונות של פרוסות בהיפוקמפוס basally (ט 0), 24 שעות לאחר חשיפה 1 שעה 10 מיקרומטר NMDA (ט 24), ואחרי 24 שעות או 10 מיקרומטר נוסף NMDA כדי להשיג פגיעה מירבית העצבית (ט מקסימום).

<ol>
	<li>Stoppin, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-173 (1991).</li><li>McCullough, L., Wu, L., Haughey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Function+of+the+PGE2+EP2+Receptor+in+Cerebral+Ischemia.">Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.</a> J Neurosci. 24 (1), 257-257 (2004).</li><li>Czapiga, M., Colton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+microglia+in+organotypic+slice+cultures.">Function of microglia in organotypic slice cultures.</a> J Neurosci Res. 56, 644-644 (1999).</li><li>Newell, D. W., Barth, A., Papermaster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+non-glutamate+receptor+mediated+toxicity+caused+by+oxygen+and+glucose+deprivation+in+organotypic+hippocampal+cultures.">Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures.</a> J Neurosci. 15, 7702-7702 (1995).</li><li>Wu, L., Wang, Q., Liang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Effects+of+prostaglandin+recetor+signaling+on+neuronal+survival.">Divergent Effects of prostaglandin recetor signaling on neuronal survival.</a> Neuroscience letter. 421, 253-253 (2007).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, R., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+in+vitro:+Analysis+of+morphology+and+adhesion+molecule+expression+in+organotypic+hippocampal+slice+cultures.">Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.</a> Glia. 18, 319-319 (1996).</li></ol>

אין ניגודי אינטרסים הכריז.

ישנן שתי נקודות חשובות לעקוב כדי להבטיח תוצאות לשחזור ועקבית כאשר משכפלים ניסויים. ראשית, חשוב לתת את גיל פרוסות בהיפוקמפוס עבור 12-14 ימים לפני ניסויים. עם בידוד פרוסה, יש תגובה microglial משמעותית, microglia להישאר פעיל במשך זמן מה, עם חזרה למצב מנוחה ענפה מתרחשת ביום 10 במבחנ?...

the organotypic hippocampal slice culture model for examining neuronal injury

פרוסה Organotypic תרבות בהיפוקמפוס היא שיטה חוץ גופית כדי לבחון את המנגנונים של פגיעה עצבית שבו הארכיטקטורה הבסיסית והרכב של ההיפוקמפוס נשמר יחסית 1. מערכת תרבות organotypic מאפשר בדיקה של השפעות עצביות, astrocytic ו microglial, אבל כהכנה vivo לשעבר, אינו מתייחס להשפעות של זרימת הדם, או גיוס של תאים דלקתיים היקפי. לשם כך, שיטה זו התרבות משמש לעתים קרובות כדי לבחון פגיעה excitotoxic ו hypoxic לנוירונים פירמידה של ההיפוקמפוס, אך שימש גם כדי לבחון את התגובה הדלקתית. בזאת אנו מתארים את שיטות להפקת תרבויות פרוסה בהיפוקמפוס מהמוח מכרסם לאחר הלידה, מתן גירויים רעילים כדי לגרום פגיעה עצבית, ו assaying וכימות מוות נוירונים בהיפוקמפוס.

Watch this Scientific Journal Video about The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury at JoVE.com

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

תרבות organoptypic בהיפוקמפוס מודל פרוסה היא<em&gt; במבחנה</emמודל&gt; בעבר לבחון פגיעה עצבית במגוון של פרדיגמות. במאמר זה, אנו מתארים את שיטות להפקת תרבויות פרוסה וכימות הפגיעה העצבית.

תרבות Organotypic בהיפוקמפוס Slice דגם לבחינת פגיעה עצבית

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

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Neuroscience

19.0K Views.  Stanford University School of Medicine. תרבות organoptypic בהיפוקמפוס מודל פרוסה היא במבחנה בעבר לבחון פגיעה עצבית במגוון של פרדיגמות. במאמר זה, אנו מתארים את שיטות להפקת תרבויות פרוסה וכימות הפגיעה העצבית.

Video: The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

פורסים תרבות Organotypic של ה-GFP-לבטא עוברי עכברים עבור הדמיה בזמן אמת תולדה של עצבים היקפיים

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypic Slice תרבויות בהיפוקמפוס

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Assay Slice Organotypic עבור זמן לשגות הדמיה ברזולוציה גבוהה נדידת הנוירונים במוח אחרי לידה

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

פורסים תרבויות Organotypic של המוח התיכון הגחון עובריים: מערכת מחקר ופיתוח עצבי דופאמין במבחנה</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

הניתוח של מורפולוגיה דנדריטים סלולרי פורקינג&#39; בתרבויות Slice Organotypic

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

הקלטות תא כל לזווג בפרוסות בהיפוקמפוס Organotypic

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

תרבויות Organotypic Slice ניתוח של עצבים וכלי דם שיפוץ בEntorhino-היפוקמפוס

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

בהיפוקמפוס ראשי בצפיפות נמוכה תרבות Neuron

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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. ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic Entrainment התנודות תטא בהיפוקמפוס ב מתנהג עכברים

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...