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.

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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>&lt;strong&gt;Cytochrome oxidase staining&lt;/strong&gt;</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&#039;Diaminobenzidine tetrahydrochloride hydrate</td><td>Sigma-Aldrich</td><td>D5637</td><td></td></tr><tr><td>D(+)-Saccharose</td><td>Carl Roth&amp;nbsp;</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&amp;nbsp;</td><td>9105.4</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antigen retrieval&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Phosphate buffer/phosphate-buffered saline/prefix/PFA&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunohistochemistry&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mounting or freezing media&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Alcohol dehydration&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Devices/tools&lt;/strong&gt;</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

12.8K 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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

הדמיה כרונית של עכבר-Visual Cortex שימוש ההכנה סחוט הגולגולת

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

כיצד למדוד מתקפל קליפתיים מתמונות MR: מדריך שלב אחר שלב כדי לחשב מדד Gyrification מקומי

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Medicine

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

רב שכבתיים Ca קליפת המוח<sup&gt; 2+</sup&gt; הדמיה בחופשיות העברת עכברים עם בדיקות פריזמה מיקרוסקופית פלואורסצנטי מיניאטורי

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Behavior

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Multiphoton מיקרוסקופי של מוח העכבר הותר הבעת YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

תיוג ממוקד של תאי עצב בתחומים ספציפיים מייקר פונקציונליים של הניאוקורטקס ידי שילוב אותות פנימיים והדמית שני פוטונים

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

בתחום ההדמיה vivo של שכבות קליפתיים עמוק באמצעות Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Biology

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

שינוי של Colliculo-thalamocortical עכבר המוח פרוסה, שילוב הדפסת 3-D של לשכת רכיבים ודימות אופטי רב בקנה מידה

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

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) ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

הדמיה תלת מימדי בקנה מידה גדול של ארגון הסלולר קליפת העכבר

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Histochemical Imaging of Cortical Modules in Flattened Brain Sections

Source:&#160;Lauer, S. M.,&#160;et al.&#160;Visualization of Cortical Modules in Flattened Mammalian Cortices.&#160;J. Vis. Exp.&#160;(2018)&#160;This ...