המחברים מודים המקור לרעיון לבצע התרבות פרוסה על עוברי עכברים 5. ברצוננו להודות יואכים קירש תמיכה מדעית נדיב אנה Degen עבור מתנהג כמו גופר שלנו במהלך הצילומים. עבודה זו מומנה על ידי קרן המחקר הגרמנית (דויטשה Forschungsgemeinschaft: Sonderforschungsbereich 488, Teilprojekt B7/B9) של אוניברסיטת היידלברג (Cluster אקסלנס רשתות סלולריות).

חלק 1: הכנת לחתוך culturing. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 10 ס&quot;מ תרבות צלחות רקמה עם חיתוך בינוני (DMEM, 25% HBSS 1x, 25% בסרום שור עוברית, גלוקוז 0.5%, גלוטמין 1 מ&quot;מ, 2.5 HEPES מ&quot;מ, pH 7.3) ו - 3 ס&quot;מ Millicell-CM-0.4 מיקרומטר תרבות מוסיף קרום ושומרים החממה ב 37 ° C ו 5% CO 2. </li><li style=";text-align:right;direction:rtl"> מחממים 4% נמוך נקודת התכה agarose ב PBS במיקרוגל ולשמור אותו על צלחת חימום כך שהוא נשאר על כ 37 ° C. </li><li style=";text-align:right;direction:rtl"> מלאו 10 ס&quot;מ צלחות פטרי עם בקטריולוגית PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4, 1.8mM KH 2 PO 4) ומניחים על קרח. </li><li style=";text-align:right;direction:rtl"> הגדרת התקן קירור microtome או להבטיח מגש חיץ וקירור אלמנטים מאוחסנים במקפיא מראש לקרר אותו. </li></ol> חלק 2: הטבעה העובר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתח עוברים מן הרחם ולבחון אותם עם מיקרוסקופ פלואורסצנטי הפוכה כדי לבדוק ביטוי של GFP. </li><li style=";text-align:right;direction:rtl"> עוברים מקום על הפוך 10 ס&quot;מ צלחת פטרי ו להתמצא אותם באמצעות רצועות נייר Whatman להסיר PBS מוגזמת. </li><li style=";text-align:right;direction:rtl"> החל agarose על העובר כדי לתקן את זה במצב הזה. בואו agarose לחזק. </li><li style=";text-align:right;direction:rtl"> הגבל את האזור המקיף את העובר על ידי חיתוך בסכין גילוח. </li><li style=";text-align:right;direction:rtl"> סובב את העובר מוטבעים על הצד השני שלה. </li><li style=";text-align:right;direction:rtl"> החל agarose נוסף על הרקמה העוברית כדי להבטיח את העובר מוטבע לחלוטין. </li><li style=";text-align:right;direction:rtl"> הכן לחסום agarose עם קצוות נקי להתקינה על vibratome צ&#39;אק באמצעות Loctite 406, דבק מיוחד דומה &quot;דבק בדבק מגע&quot;. </li></ol> חלק 3: בחותכו ההליך. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדרת מגש חיץ precooled ואת אלמנט הקירור. </li><li style=";text-align:right;direction:rtl"> הכנס את צ&#39;אק עם הרקמה דבוק ולהוסיף 1x HBSS (Ca 2 +-Mg 2 + ללא HBSS, 10 mM HEPES חיץ pH 7.3, 500 U / ml פניצילין / סטרפטומיצין) מכוסה עד. </li><li style=";text-align:right;direction:rtl"> הכנס ולתקן להב precleaned (70% אתנול) microtome. </li><li style=";text-align:right;direction:rtl"> הכן 350-450 מיקרומטר פרוסות ולהעביר אותם באמצעות קיצור זכוכית pipettes פסטר לתוך צלחות תרבית הרקמה כל הזמן על הקרח. </li><li style=";text-align:right;direction:rtl"> בעזרת זוג מלקחיים, להסיר בזהירות agarose מתוך כל פרוסה ולהעביר Millicell ממברנות תרבות. כ 4 פרוסות יכול להיות מתורבת על קרום אחד צלחת 10 ס&quot;מ רקמה תרבות מלא מ&quot;ל 6 של המדיום לתרבות. </li><li style=";text-align:right;direction:rtl"> דגירה פרוסות בשעה 37 ° C ו 5% CO 2 (בינוני תרבות: DMEM, 25% HBSS 1x, 25% בסרום שור עוברית, גלוקוז 0.5%, 1mm גלוטמין, HEPES 2.5 מ&quot;מ, pH 7.3). אם אחד מבצע זמן לשגות סדרה הדמיה אחד צריך לשמור על נפח קבוע בינוני. במקרה של סדרה הדמיה מתוזמנים עבור פרק זמן קצר זה מספיק כדי להשאיר את המדיום כפי שהוא. במשך תקופות ארוכות יותר שינויים בתרבות לאחר 12-20 שעות מומלץ. </li></ol> חלק 4: תוצאה הדמיה עצב עמוד השדרה על המיקרוסקופ. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תמונה העצבים של עמוד השדרה הצבת 10 ס&quot;מ תרבות צלחת רקמות, המכיל ממברנות Millicell תרבות עם פרוסות, תחת מיקרוסקופ פלואורסצנטי זקוף. </li><li style=";text-align:right;direction:rtl"> תווית הכיוון של ממברנות Millicell התרבות על הבמה מיקרוסקופ לעמדה נכונה במהלך הנקודה הדמיה בפעם הבאה. </li><li style=";text-align:right;direction:rtl"> עצב השדרה תמונה תולדה באמצעות 4x (מספרי הצמצם [NA] 0.1), 10x (NA 0.3), או 20x (NA 0.5) מטרות. </li></ol> איור 1 מציג סדרת הדמיה המתארת ​​את תולדה עצב עמוד השדרה במהלך 20 שעות של תרבות עם מטרות 10x ו 4x. <img src="/files/ftp_upload/2309/2309fig1.jpg" alt="איור 1" /> איור 1 Imaging סדרת תולדה עצב השדרה פרוסת רוחבי של העובר tauGPF הומוזיגוטים. * = הנוירונים המנוע של חוט השדרה הגחון, חץ = DRG. הגבי (D)-הגחוני (V) ציר של הפרוסה מצוין. Scalebars: 200 מיקרומטר.

<ol>
	<li>Tucker, K. L., Meyer, M., Barde, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+are+required+for+nerve+growth+during+development.">Neurotrophins are required for nerve growth during development.</a> Nat Neurosci. 4, 29-37 (2001).</li><li>Brachmann, I., Jakubick, V. C., Shaked, M., Unsicker, K., Tucker, K. L. <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+slice+culture+system+for+the+imaging+of+nerve+development+in+embryonic+mouse.">A simple slice culture system for the imaging of nerve development in embryonic mouse.</a> Dev Dyn. 236, 3514-3523 (2007).</li><li>Collingridge, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+slice+preparation:+a+tribute+to+the+pioneer+Henry+McIlwain.">The brain slice preparation: a tribute to the pioneer Henry McIlwain.</a> Journal of neuroscience methods. 59, 5-9 (1995).</li><li>Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+circuitry+of+identified+projection+neurons+in+cat+visual+cortex+brain+slices.">Local circuitry of identified projection neurons in cat visual cortex brain slices.</a> J Neurosci. 7, 1223-1249 (1987).</li><li>Hotary, K. B., Landmesser, L. T., Tosney, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+slices.">Embryo slices.</a> Methods Cell Biol. 51, 109-124 (1996).</li></ol>

לשם השוואה מקיפה של שיטות להכין תרבויות פרוסה עובריים של אמצע ההיריון עוברי עכברים (E10 - E12), ראינו כי vibratome מייצר ללא ספק את התוצאות הכי אמין לגבי הכדאיות הן הכוללת של תרבויות ושל שחזור של תוצאה העצב דפוסי. לעומת זאת, פרוסות מוכן באמצעות מסוק רקמות McIlwain 3 התבררה invia...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14180</td>
<td>&nbsp;</td>
<tr>
<td>Dissection tools</td>
<td>&nbsp;</td>
<td>FST</td>
<td>various</td>
<td>&nbsp;</td>
<tr>
<td>L.M.P. agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-022</td>
<td>&nbsp;</td>
<tr>
<td>Whatmann paper</td>
<td>&nbsp;</td>
<td>Whatman Int.</td>
<td>3030917</td>
<td>&nbsp;</td>
<tr>
<td>Shortened firepolished pipettes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>41966</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10270-106</td>
<td>&nbsp;</td>
<tr>
<td>Pen Strep</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine 100x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Microm International GmbH</td>
<td>HM 650 V</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX61WI</td>
<td>&nbsp;</td>
<tr>
<td>analySIS</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG 50</td>
<td>&nbsp;</td>
<tr>
<td>10 cm culture plates</td>
<td>&nbsp;</td>
<td>Greiner Bio-One</td>
<td>633171</td>
<td>&nbsp;</td>
<tr>
<td>LOCTITE 406</td>
<td>&nbsp;</td>
<td>Henkel</td>
<td>142580</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>none</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9105.2</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>x4 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>PL series</td>
<td>&nbsp;</td>
<tr>
<td>x10 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>UPLFL &#x2013;PH series</td>
<td>&nbsp;</td>
<tr>
<td>Filter</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>U-MNIBA2</td>
<td>&nbsp;</td>
<tr>
<td>CCD camera</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>SIS F-View II</td>
<td>&nbsp;</td>
<tr>
<td>Equipment for heated chamber</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>CTI-Controller 3700 and incubator S #11531171</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic slice culture of gfp-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth

למטרות רבות, טיפוח vivo עוברי עכבר לשעבר כמו פרוסות organotypic רצוי. לדוגמה, אנו מעסיקים קו עכבר מהונדס (tauGFP) שבו גרסה משופרת של חלבון פלואורסצנטי ירוק (EGFP) באה לידי ביטוי באופן בלעדי את כל הנוירונים של מערכת העצבים המרכזית לפיתוח היקפי 1, המאפשר את האפשרות לצלם גם את העצבוב של forelimb ו לתפעל את התהליך הזה עם טכניקות תרופתי ו גנטית 2. הפרמטר הקריטי ביותר לגידול מוצלח של תרבויות פרוסה כזו היא השיטה בה את פרוסות מוכנים. לאחר בדיקה מקיפה של מגוון שיטות, מצאנו כי vibratome הוא המכשיר הטוב ביותר הפרוסה עוברים כך שהם שגרתי תוצאה בתרבות מדגים יכולת הקיום על פני תקופה של מספר ימים, והכי חשוב, מפתחת בעידן באופן ספציפי. עבור אמצע ההריון עוברים, זה כולל את תוצאה נורמלית של העצבים בעמוד השדרה של חוט השדרה ואת הגרעינים השורש הגבי ליעדם בפריפריה וקביעת תקין של רקמת שריר השלד. בעבודה זו, אנו מציגים שיטה לעיבוד עוברים כל יום עובריים (E) E10 ל E12 לתוך 300-400 מיקרומטר פרוסות לגידול בתרבית רקמה רגילה החממה, אשר ניתן ללמוד עד יומיים לאחר הכנה פרוסה. קריטי להצלחה של גישה זו הוא השימוש vibratome על כל פרוסה העובר agarose-מוטבע. זה ואחריו הטיפוח של פרוסות על קרום Millicell מוסיף תרבות דגש על נפח קטן של המדיום, והתוצאה היא טכניקה תרבות ממשק. המלטה אחת עם ממוצע של 7 עוברים באופן שגרתי מייצר לפחות 14 פרוסות (2-3 פרוסות של האזור forelimb לכל העובר), אשר משתנה מעט עקב גיל העוברים כמו גם עובי הפרוסות. כ -80% את פרוסות תרבותי להראות תוצאה עצב, אשר ניתן למדוד througout 2 תקופת culturing. תוצאות נציג באמצעות שורת עכבר tauGFP הם הפגינו.

Watch this Scientific Journal Video about Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth at JoVE.com

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

אנו מציגים שיטה להכין פרוסות organotypic של אמצע ההיריון עוברי עכברים הדמיה טיפוח זמן לשגות תולדה של עצבים היקפיים.

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-slice-culture-gfp-expressing-mouse-embryos-for-real-time

Research

JoVE Journal

Neuroscience

16.1K Views.  University of Heidelberg. אנו מציגים שיטה להכין פרוסות organotypic של אמצע ההיריון עוברי עכברים הדמיה טיפוח זמן לשגות תולדה של עצבים היקפיים.

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

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 ...

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 ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

מרובה עכבר Neuroanatomical תהודה מגנטית

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

הקלטה נייד שלמים מן ההכנה פורסים Organotypic של הניאוקורטקס

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

אפיון אלקטרו של ה-GFP, הבעת אוכלוסיות תאים ברשתית אינטקט

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

פרוטוקול עבור fMRI ניהול בזמן אמת Neurofeedback אימונים

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

זמן לשגות Confocal הדמיה של נוירונים נודדים תרבות פרוסה Organotypic של המוח עכבר עובריים באמצעות<em&gt; באוטרו</em&gt; Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

. פשוט תאכל לשעבר אלקטרופורציה ותרבויות פרוסה Organotypic מוח עובריים בעכבר עבור Live-הדמיה של העברת Interneurons GABAergic

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

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

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

Immunostaining של Myelinating Organotypic פרוסה אסטרוציטומה תרבויות והכנה

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