עבודה זו נתמכה על ידי המכון הלאומי לבריאות נפש, והקרן הלאומית למדע. תודה מיוחדת לד&quot;ר ג&#39;ן Gutzman, ד&quot;ר אמנדה דיקינסון וחברי מעבדת סייב אחרות לדיונים שימושיים רבים וביקורת בונה, ואוליבייה Paugois לגידול בעלי דגי מומחה.

1. הכנת מחטי microinjection וחשמלי ניידים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מלא מנגנון אפנדורף CellTram שמן microinjector עם שמן מינרלים על פי הוראות היצרן. </li><li style=";text-align:right;direction:rtl"> הכן את מחטי microinjection ידי משייכת צינורות נימים באמצעות חולץ מחט מכשירי סאטר. </li><li style=";text-align:right;direction:rtl"> הר מחט על micromanipulator מחובר לאפנדורף CellTram. </li><li style=";text-align:right;direction:rtl"> זהירות לשבור את קצה המחט. לגודל קצה אחיד, למדוד עם מיקרומטר, או להשוות למחט הפניה. </li><li style=";text-align:right;direction:rtl"> מלא את אורך המחט עם שמן, שימוש CellTram לדחוף שמן לאורך, להיות זהיר, כדי למנוע בועות. </li></ol> 2. הכנת העוברים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חורים ב1% מנה מצופית agarose עם קצה פיפטה 1-200 μl, ולהסיר תקעי agarose. מלא צלחת עם תקשורת עובר (נעשה על פי 25 Westerfield). </li><li style=";text-align:right;direction:rtl"> שימוש במלקחי עוברים, dechorionate תחת stereomicroscope. </li><li style=";text-align:right;direction:rtl"> העברת עוברי dechorionatedלמנה מצופית agarose. </li><li style=";text-align:right;direction:rtl"> להרדים עוברים, להוסיף Tricaine (0.1 מ&quot;ג / מ&quot;ל) למנת agarose עד עוברים מפסיקים לנוע (שנעשה על פי 25 Westerfield). </li></ol> 3. ניקוז eCSF <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אוריינט את העוברים בעלי הזנבות באת החורים של agarose והצדדים האחוריים שלהם הקרובים לmicromanipulator, ומאפשר הדמיה של הצד הגבי של המוח. </li><li style=";text-align:right;direction:rtl"> מקם את המחט בrhombomere 0/1 ציר נקודה או נקודה הרחבה ביותר של מוח אחורי חדר לב (איור 1 א). </li><li style=";text-align:right;direction:rtl"> זהירות צלחת גג לנקב של חלל המוח האחורי להיות בטוח שלא לעבור את העומק של המוח לחלמון. </li><li style=";text-align:right;direction:rtl"> מסנן את eCSF באמצעות CellTram ולאסוף נוזלים במחט microinjection. להיות זהיר, כדי למנוע את כל תאים. </li></ol> 4. איסוף eCSF לניתוח רכב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ברגע eCSF נאסף במחט, להפסיק יניקה מCellTram זהירות. </li><li style=";text-align:right;direction:rtl"> מוve הצלחת מתחת למחט ולמקם את המחט בזהירות לתוך מבחנה המכילה את המאגר הרצוי. </li><li style=";text-align:right;direction:rtl"> שימוש CellTram, לנקז eCSF מתוך המחט לחיץ מנסה להימנע מלזהם את הפתרון בשמן. </li><li style=";text-align:right;direction:rtl"> על מנת להסיר את התאים שיש לך ונשאף יחד עם eCSF, ספין eCSF ב -10,000 x גרם. איסוף מזוהם eCSF (supernatant) ולאחסן ב -80 ° C עד מוכן לניתוח נוסף. </li></ol> 5. חידוש של גורמים נבחרים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מסנן את eCSF כל 2 שעות בפרק זמן רצוי, שכן הנוזל מתחדש בתוך תקופת זמן זו. בין ניקוז בזמן נקודות, הסר את המחט ולאחסן את העוברים ב28.5 מעלות צלזיוס או רצוי טמפרטורה. </li><li style=";text-align:right;direction:rtl"> טען מחט micropipette שנייה עם גורם מבחן. </li><li style=";text-align:right;direction:rtl"> מחט למקום micromanipulator מצורפת microinjector. </li><li style=";text-align:right;direction:rtl"> כיוון עוצמת זריקה עד 1 nl על ידי מדידת גודל הטיפה בזמן שמן והכוון הזרקה ו-pressure. </li><li style=";text-align:right;direction:rtl"> להזריק 1-2 nl לחדרים במוח כפי שתוארו קודם לכן 26. </li></ol>

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השימוש בטכניקה זו באופן ידני לניקוז eCSF מחדרים במוח דג זברה יהיה שימושי לקביעת הדרישה לeCSF במהלך פיתוח. בנוסף, טכניקה זו תאפשר תיאור של פרופיל חלבון eCSF במהלך התפתחות עוברית. זיהוי של חלבונים שונים בתקופה זו יאפשר חקירה נוספת לפונקציה של CSF והתפקיד הפוטנציאלי שלה במהלך ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td></tr><tr><td> אפנדורף CellTram שמן </td><td> אפנדורף </td><td> 516 000.025 </td></tr><tr><td> שמן מינרלים </td><td> סיגמא </td><td> M8410 </td></tr><tr><td> Tricaine אבקה </td><td> סיגמא </td><td> A5040 </td></tr><tr><td> צינורות נימים </td><td> Fhc בע&quot;מ </td><td> 30-30-1 </td></tr></tbody></table>

manual drainage of the zebrafish embryonic brain ventricles

נוזל השדרתי (CSF) הוא נוזל עשיר חלבון מכיל בתוך החדרים במוח. היא נמצאת במהלך ההתפתחות העוברית המוקדמת חוליות ונמשכת לאורך כל חיים. מבוגרי CSF הוא חשב לרפד את המוח, להסיר פסולת, ולשאת 1,2 מולקולות מופרשות. במבוגרים ומבוגרים העובר, רוב CSF נעשה על ידי דמה עין המקלעת, סדרה של אזורי הפרשת כלי דם מאוד ממוקמים סמוך למוח החללים 3-5. בדג הזברה, דמה עין המקלעת נוצרה במלואו בשעות שלאחר ההפריה 144 (hpf) 6. לפני זה, בעוברי החולייתנים הוא דג זברה ואחרים, כוללים עכבר, כמות משמעותית של עוברי CSF (eCSF) הוא הווה. נתונים אלה ומחקרים מראים כי בחומוס neuroepithelium היא הפרשה מוקדם בפיתוח ועלולה להיות המקור העיקרי של eCSF לפני דמה עין המקלעת 7 פיתוח. eCSF מכיל בערך פי שלושה לא יותר חלבוןהאן המבוגר CSF, המצביע על כך שזה יכול להיות תפקיד חשוב במהלך הפיתוח 8,9. מחקרים באפרוח ועכבר להוכיח כי גורמים מופרשים בeCSF, לחץ נוזל, או שילוב של אלה, הם חשובים לנוירוגנזה, ביטוי גנים, שגשוג תאים, ותא ההישרדות בneuroepithelium 10-20. מנתח proteomic של, עכברוש אדם, עכבר, ואפרוח eCSF זיהה חלבונים רבים שעשויה להיות נחוץ לתפקוד CSF. אלה כוללים מרכיבים תאיים מטריצה, apolipoproteins, חלבוני ויסות לחץ האוסמוטי, וחלבונים מעורבים במוות של תאים והתרבות 21-24. עם זאת, הפונקציות המורכבות של eCSF אינן ידועות. אנחנו פתחנו שיטה להסרת eCSF מחדרים במוח דג זברה, ובכך מאפשרים לזיהוי של רכיבי eCSF ולניתוח של דרישת eCSF במהלך פיתוח. למרות שיותר eCSF ניתן לאסוף מחוליות w מערכות האחרותעוברי ith גדולים, eCSF ניתן לאסוף מהשלבים המוקדמים של פיתוח דג זברה, ובתנאים גנטיים או סביבתיים המובילים למוח נורמלי נפח חלל או במורפולוגיה. הסרה ואוסף של eCSF מאפשרות ניתוח, חקירה המונית spectrometric תפקוד eCSF, וחידוש של גורמים נבחרים לתוך חדרי הלב לבדיקת תפקודם. לכן הנגישות של עובר דג הזברה מוקדם מאפשרת ניתוח מפורט של פונקצית eCSF במהלך פיתוח.

דוגמה לחדר לב מוח מרוקן מוצגת באיור 1 ב-C. חדרים במוח הם התמוטטו כהם חסרים eCSF (האיור 1B לעומת C). כפי שניתן לראות בתמונות גב (איור 1B-C, ואיור 2A-D) neuroepithelium המוח האחורי עושה לשמור מורפולוגיה האופיינית ונראה שהוא פתוח למרות חוסר eCSF הנראה בשל נקודתי צ?...

Watch this Scientific Journal Video about Manual Drainage of the Zebrafish Embryonic Brain Ventricles at JoVE.com

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

אנו מציגים שיטה לאיסוף נוזל שדרה (CSF) וליצור מערכת שאין בה CSF בתוך מערכת חדרית מוח דג הזברה העוברית. זה מאפשר לבדיקה נוספת של רכב CSF והדרישה שלה במהלך התפתחות מוח העוברית.

ניקוז ידני של חדרי המוח העוברי דג הזברה

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

manual-drainage-of-the-zebrafish-embryonic-brain-ventricles

Research

JoVE Journal

Neuroscience

9.9K Views.  Massachusetts Institute of Technology. אנו מציגים שיטה לאיסוף נוזל שדרה (CSF) וליצור מערכת שאין בה CSF בתוך מערכת חדרית מוח דג הזברה העוברית. זה מאפשר לבדיקה נוספת של רכב CSF והדרישה שלה במהלך התפתחות מוח העוברית.

Video: Manual Drainage of the Zebrafish Embryonic Brain Ventricles

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Assay לחדירות של Neuroepithelium עוברי דג הזברה

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

מיקרומניפולציה של ביטוי גנים במוח דג הזברה למבוגר באמצעות Microinjection Cerebroventricular של oligonucleotides morpholino

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

תיקון הקלטות קלאמפ מתאים עוברי דג הזברה מאוטנר

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

מקיף פרוטוקול לפילוח ידני של מבנים המדיאלי Temporal האונה

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

בידוד והתרבות של תאי האנדותל מהמוח הקדמי עוברי

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

הקלטת אלקטרו במוח של דג הזברה למבוגר שלמה

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

הדמיה חיה של מיטוזה בCortex עכבר עוברי פיתוח

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

מותנה הגנטי Transsynaptic איתור במוח העכבר עוברי

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

הדמיה חיה של צילה Ependymal בחדרים לרוחב של מוח העכבר

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.