עבודה זו נתמך על ידי Humboldt Universität זו ברלין, המרכז ברנשטיין לברלין Neuroscience חישובית, המרכז הגרמנית עבור מחלות ניווניות (DZNE), הגרמני הפדרלי במשרד החינוך, המחקר (BMBF, Förderkennzeichen 01GQ1001A), NeuroCure, של גוטפריד וילהלם לייבניץ פרס DFG. אנו מודים Shimpei אישיאמה לעיצוב גרפי מצוין, ניגן Diederichs לסיוע טכני מעולה.

<ol>
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M., Lenschow, C., Brecht, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+selected+size+differences+and+conserved+sexual+monomorphism+of+genital+cortex.">Sexually selected size differences and conserved sexual monomorphism of genital cortex.</a> J Comp Neurol. , (2017).</li><li>Fleischhauer, K., Petsche, H., Wittkowski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+bundles+of+dendrites+in+the+neocortex.">Vertical bundles of dendrites in the neocortex.</a> Anat Embryol. 136 (2), 213-223 (1972).</li><li>Bernardo, K. L., Woolsey, T. 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המחברים מצהירים כי המחקר בוצע בהיעדר יחסים מסחריים או פיננסי שיכול להחשב בתור ניגוד אינטרסים פוטנציאליים.

מודולריות ב קליפת התגלתה תוך שימוש במגוון טכניקות. המודולים המוקדם של קורטיקלית מחקרים בדרך כלל מזוהה גם דמיין תא אזורים צפופים או היעדרות של סיבים1. שיטות עוקבות נעזרו הנוכחות של חבילות דנדריטים24, afferents אזור מסוים25, או העשרה של נוירוטרנסמיטורים<sup cla...

חלק מהשינויים המשמעותיים במבנה המוח על פני האמיניות נראים גם קליפת המוח. למרות הבדלים משמעותיים, קליפת המוח של בעלי חיים עוקב אחר תבנית משותפת, ניתן לחלק בשתי דרכים נפרדות, על-ידי שכבות ואזורי1. שכבות קורטיקלית לשקר מקביל פני השטח של המוח, משתנים מספר 3 שכבות cortices הזוחלים2 על 6 שכבות cortices בתרבית של1. אזורים קורטיקליים לעומת אזורים הנבדלים זה מזה של קליפת המקבילים במידה רבה פונקציות שונות, למשל, קליפת המגע מעורב התחושה של מגע או קליפת הראיה בעיבוד חזותי תשומות. אזורים קורטיקליים אלה לעיתים קרובות נחלקים תיקונים או מודולים3, אשר נמצאים באופן קבוע חוזרות מבנים אנטומיים, בעיקרו של דבר מצא מקבילות אל פני השטח pial של המוח. מודולים בקליפת המוח עשוי להיות מוגבל רובד4, או להרחיבו על-גבי שכבות מספר5.
אופטים בשיטות הרגילות של המוח לערב מקטעים רגילים על פני השטח של המוח, כמו הילתית או הווריד. בעוד שיטות אלה ניתן להשתמש כדי להמחיש מודולים קורטיקלית, שפע של תכונות מעניינות יכול להתגלות כאשר המודולים בקליפת המוח הם דמיינו tangentially, במישור מקביל פני השטח של המוח. למשל, מודולים המגע במוח מכרסמים המייצג שפם, מופיעות כמו חביות כאשר visualized נורמלי השטח במוח, ולכן האזורים להפיק קליפת חבית שם. עם זאת, בלהמחיש את החביות בכיוון משיק, הם מגלים שפם-מפה, עם הקנים להיות אצלה כיוון טופוגרפית שיקוף את המבנה המדוייק של נכון על פני גוף חיצוני. במקרים מסוימים, אפילו ברח הסידור המודולרי זיהוי לתקופות ניכר, כאשר visualized באופן שאינו סוטה. קליפת entorhinal המדיאלי, ידועה הנוכחות של תאי הרשת, נוירונים אשר אש בתבנית משושה הרגילה כאשר חיים הוא וחוצה סביבה. למרות שזה אזור ובדוקים בכבדות, עד לאחרונה, הנוכחות של תיקונים או מודולים של תאים בקליפת entorhinal המדיאלי, אשר נפרסות פיזית בתוך תבנית משושה6, נמלט זיהוי. הנוכחות ואת הסידור של המודולים במוח עכברוש, היה בהנחייתם עושה קטעים וקולו של קליפת entorhinal המדיאלי, חוקרת את cytoarchitecture באופן layer-wise.
לאחר חלוקתה, ההיבט המסוים של ויזואליזציה של מודולים בקליפת המוח גם ניתן למימוש בדרכים רבות. בסגנון קלאסי, יש לימודי התחום מודולים מבוסס על התא צפיפות או סיבים פריסה1. גישה פופולרית נוספת היא השימוש ציטוכרום אוקסידאז להסטולוגיה, אשר חושף את תחומי פעילות גבוהה יותר8. גישות חדשות יותר כוללים מסתכל על סוגי תאים נקבע גנטית, מכובד על בסיס שלהם חלבון ביטוי פרופילים6,8.
במחקר זה, אנו מדגישים שיטות כדי לבודד את קליפת המוח של המוח יונקים, להשיג חלקים וצורניים שעברו שיטוח להמחיש מודולים קורטיקלית המבוסס על להסטולוגיה אוקסידאז ציטוכרום אימונוהיסטוכימיה של חלבונים מסוימים סוג התא.

<table>
	<tbody>
		<tr>
			<td>Cytochrome oxidase staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;Diaminobenzidine tetrahydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5637</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Saccharose</td>
			<td>Carl Roth&#160;</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium nickel(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A1827</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antigen retrieval</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phosphate buffer/phosphate-buffered saline/prefix/PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-Sodium hydrogen phosphate dihydrate</td>
			<td>Carl Roth</td>
			<td>4984.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON-X 100</td>
			<td>Carl Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k puriefied from chicken gut, Mouse monoclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000347</td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k from recombinant rat calbindin D-28k, Rabbit polyclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000340</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V, biotin free</td>
			<td>Carl Roth</td>
			<td>0163.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mounting or freezing media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount (immunofluorescence)</td>
			<td>Sigma-Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt (histochemistry)</td>
			<td>Sigma-Aldrich</td>
			<td>03989</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium</td>
			<td>Leica Biosystems</td>
			<td>NC0696746</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Alcohol dehydration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Carl Roth</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth</td>
			<td>P075.3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth</td>
			<td>6752.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>Fluka</td>
			<td>78475</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Devices/tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microm HM 650V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jung RM2035</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps - Inox</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Inox Biology Tip</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps - Inox Super Fine Tip</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Shears - 24 cm</td>
			<td>Fine Science Tools</td>
			<td>16150-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16000-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Scissors</td>
			<td>Fine Science Tools</td>
			<td>14000-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Large Loops</td>
			<td>Fine Science Tools</td>
			<td>14101-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of cortical modules in flattened mammalian cortices

קליפת בתרבית של מוח הוא parcellated לתוך substructures ברורים או מודולים. מודולים בקליפת המוח בדרך כלל לשקר מקבילה לגליון קורטיקלית, יכול להיות המותווית על ידי מסוימים פתולוגיה ושיטות immunohistochemical. במחקר זה, אנו מדגישים שיטה כדי לבודד את קליפת המוח של המוח יונקים ולשטח אותם לקבל במקביל סעיפים בגיליון קורטיקלית. אנחנו עוד גולת הכותרת שנבחרה פתולוגיה, immunohistochemical שיטות לעיבוד אלה משוטחים מקטעים וצורניים להמחיש מודולים קורטיקלית. בתוך קליפת המגע של יונקים שונים, אנו מבצעים להסטולוגיה אוקסידאז ציטוכרום כדי לחשוף את הגוף מפות או מודולים קורטיקלית המייצגים חלקים שונים של הגוף של החיה. בתוך קליפת entorhinal המדיאלי, אזור שבו נוצרים תאי הרשת, אנו מנצלים immunohistochemical שיטות לסימון מודולים של נוירונים נקבע גנטית אשר מסודרים בתבנית-רשת בגליון בקליפת המוח על פני מספר מינים. בסך הכל, אנו מספקים מסגרת כדי לבודד ולהכין layer-wise משוטחים חלקים קורטיקליים, ותנסו לדמיין את המודולים בקליפת המוח באמצעות פתולוגיה ושיטות immunohistochemical במגוון רחב של יונקים את המוח.

אנו להשיג חלקים קורטיקליים שעברו שיטוח של קליפת המגע במגוון של המוח, לעבד אותם להסטולוגיה אוקסידאז ציטוכרום להמחיש את המודולים somatotopic המייצגים את חלקי הגוף השונים. גישה השוואתית זו מאפשרת לימוד כוחות האבולוציוני הזה cortex צורה, למשל, מראה מאוד שנשמרת ייצוג mystacial vibrissae ...

Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

Visualization of Cortical Modules in Flattened Mammalian Cortices

מאמר זה מתאר מתודולוגיה מפורט כדי להשיג מקטעים וצורניים שעברו שיטוח של יונקים cortices והמחש מודולים בקליפת המוח באמצעות פתולוגיה ושיטות immunohistochemical.

ויזואליזציה של מודולי קורטיקליים Cortices יונקים שעברו שיטוח

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

visualization-of-cortical-modules-in-flattened-mammalian-cortices

Research

JoVE Journal

Neuroscience

12.7K Views.  Humboldt University of Berlin. מאמר זה מתאר מתודולוגיה מפורט כדי להשיג מקטעים וצורניים שעברו שיטוח של יונקים cortices והמחש מודולים בקליפת המוח באמצעות פתולוגיה ושיטות immunohistochemical.

Video: Visualization of Cortical Modules in Flattened Mammalian Cortices

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

ויזואליזציה של עצבים המגזרי הזחל ב 3 Rd Instar תסיסנית הכנות הזחל

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

בשנת vivo ויזואליזציה של שלפוחית ​​Synaptic בתוך האקסונים תסיסנית המגזרי הזחל

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

ניגון אוטומטי של וידאו אינטראקטיבי ללימודי תקשורת של בעלי חיים

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

סידן הדמיה תפקודית בפיתוח רשתות קליפתיים

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

ויזואליזציה של Proprioceptors ב<em&gt; תסיסנית</em&gt; זחלים וגלמים

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

בידוד ותרבות של האסטרוציטים עכבר קליפתיים

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo שני פוטוני הדמיה של שינויים מולקולריים ניסיון תלוי בנוירונים בקליפת מוח

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Subtype-selective Electroporation of Cortical Interneurons

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

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

Electroporation תת סוג סלקטיבי של בקליפת המוח interneurons

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

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

כימות פילמנטיות אקטין (F- אקטין) puncta ב עכברוש נוירונים קליפתיים

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

ויזואליזציה של האקסון Thalamocortical הסתעפות, סינפסה-צורה Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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ויזואליזציה של מודולי קורטיקליים Cortices יונקים שעברו שיטוח

Summary

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Chapters in this video

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ניגון אוטומטי של וידאו אינטראקטיבי ללימודי תקשורת של בעלי חיים

סידן הדמיה תפקודית בפיתוח רשתות קליפתיים

ויזואליזציה של Proprioceptors ב

בידוד ותרבות של האסטרוציטים עכבר קליפתיים

In vivo שני פוטוני הדמיה של שינויים מולקולריים ניסיון תלוי בנוירונים בקליפת מוח

Electroporation תת סוג סלקטיבי של בקליפת המוח interneurons

כימות פילמנטיות אקטין (F- אקטין) puncta ב עכברוש נוירונים קליפתיים

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