This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

משפט ואתיקה: כל הנהלים הקשורים בנושאים בעלי חיים לעקוב אחר ההנחיות של טיפול בבעלי חיים ועדת האתיקה (ACEC) מאוניברסיטת ניו סאות &#39;ויילס (מספר ACEC שאושרה הינה 14 / 94a). 1. הכנה של חוט השדרה עכבר שקוף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת פתרון הידרוג&#39;ל קר קרח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת פתרון paraformaldehyde 16% (PFA) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף 16 גרם אבקת paraformaldehyde לתוך 70 מ&quot;ל מים מזוקקים מחומם מראש (50-55 מעלות צלזיוס) ומערבבים על בוחש מגנטי מחוממת עד paraformaldehyde נמס. הערה: אל תאפשר הפתרון לחמם מעל 55 מעלות צלזיוס ולהיות מודעים לכך paraformaldehyde הוא רעיל. </li><li style=";text-align:right;direction:rtl"> מעבירים את הפתרון paraformaldehyde כדי גליל ולהוסיף מים מזוקקים עד 100 מ&quot;ל. מצננים את הפתרון paraformaldehyde באמצעות מקרר 4 ° C.. </li></ol></li><li style=";text-align:right;direction:rtl"> ממיסים 125 מ&quot;ג יוזם VA-044 ב 26.25 מ&quot;ל מים מזוקקים ו לקרר את הפתרון בתוך 4° מקרר C. </li><li style=";text-align:right;direction:rtl"> פתרון acrylamide מגניב 5 מ&quot;ל (40%), 1.25 מ&quot;ל פתרון Bis (2%) (זהירות: Bis ופתרונות acrylamide רעילים, עלול לגרום למומים גנטיים או סרטן), 5 מ&quot;ל 10x PBS, 12.5 מ&quot;ל 16% פתרון PFA, ואת פתרון יוזם VA-044 על הקרח. </li><li style=";text-align:right;direction:rtl"> מערבבים את כל הפתרונות שהוצעו על הקרח. הערה: הנפח הכולל הוא 50 מ&quot;ל. </li></ol></li><li style=";text-align:right;direction:rtl"> זלוף וגבייה של קורד עכבר השדרה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לטשטש עכבר עם זריקה intraperitoneal של קטמין (80 מ&quot;ג / ק&quot;ג) ו xyzaline (5 מ&quot;ג / ק&quot;ג) מדולל מלוחים 0.9% נורמלי. המינון לעכבר יכול להיות נמוך מזה של חולדות. </li><li style=";text-align:right;direction:rtl"> על משטח פלסטיק מתאים במנדף, לתקן את הגפיים של העכבר מהגוף (סרט דביק יעיל) פעם העכבר אינו מגיב קמצוץ כדי עורו של כף הרגל. </li><li style=";text-align:right;direction:rtl"> חותכים את העור העכבר מעל החזה ואחר כך בחזה העצם כדי לחשוף את הלב עם מספריים. </li><li style=";text-align:right;direction:rtl"> באמצעות משאבת peristaltic, עם מחט 25 G מצורף thסוף הדואר של צינורות, הכנס את המחט לתוך החדר השמאלי של הלב, לגזור אטריום הזכות ליצור נקודת יציאה בדם ולאחר מכן להתחיל כביסה עם 40 מ&quot;ל 0.9% מלח רגיל בשיעור של כ -10 מ&quot;ל / דקה. </li><li style=";text-align:right;direction:rtl"> כשיושלם, perfuse את העכבר עם 35 פתרון הידרוג&#39;ל קר מ&quot;ל קרח. הערה: perfuse במהירות של 10 מ&quot;ל / דקה, כדי למנוע נפיחות של רקמת העצבים. </li><li style=";text-align:right;direction:rtl"> לחתוך את העור על הגב עם להב ולאחר מכן להסיר את השרירים ליד החוליות. לאחר חיתוך קשתות בחוליות בצד אחד מרפרף אותם לצד השני, לקחת חוט השדרה מתוך עמוד השדרה עם מספריים בסדר. </li></ol></li><li style=";text-align:right;direction:rtl"> ניקוי חוט השדרה עכבר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את חוט השדרה 15 מ&quot;ל של תמיסת הידרוג&#39;ל לילה ב 4 ° C.. </li><li style=";text-align:right;direction:rtl"> הסר 10 מ&quot;ל של פתרון הידרוג&#39;ל ויוצקים את שאר הפתרון עם מגזרים חוט השדרה אל צינור 5 מ&quot;ל. מלאו את צינור 5 מ&quot;ל עם הפתרון הידרוג&#39;ל עד שהוא לגמרי מלא. </li><li style=";text-align:right;direction:rtl"> למתוח חתיכה קטנה של parafilm על גבי הצינור 5 מ&quot;ל ולאחר מכן לעטוף parafilm סביב הצוואר של הצינור. הערה: ודא המגעים parafilm הפתרון הידרוג&#39;ל ואין בועות בין פתרון הידרוג&#39;ל ואת parafilm. </li><li style=";text-align:right;direction:rtl"> שים את הצינור 5 מ&quot;ל ב 37 &#160; מעלות צלזיוס בתנור למשך הלילה. שימו הפתרון הידרוג&#39;ל עד שזה הופך ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> קח את הצינור 5 מ&quot;ל מהתנור ולהסיר את הג&#39;ל מהצינור עם ברגים (כגון קצה פיפטה גרפו 1 מ&quot;ל). הסר את הג&#39;ל רקמה חוט השדרה על ידי דבק הרקמות הגסות הג&#39;ל ולאחר מכן לקחת את הרקמה הגסה משם (הג&#39;ל ידבק הרקמה ולכן יוסר על ידי הרקמה). </li><li style=";text-align:right;direction:rtl"> לאחר הסרת הג&#39;ל רקמת חוט השדרה, לשטוף את חוט השדרה 4 פעמים עם PBS 1x (pH 7.4) במשך 24 שעות על שייקר. </li><li style=";text-align:right;direction:rtl"> הכן את הפתרון סליקה מעוקב ידי המסת אוריאה 3.85 גרם ו 3.85 גרם N, N, N &#39;, N&#39;-tetrakis (2-Hydroxypropyl) ethylenediam<html> ine ב 5.38 מ&quot;ל מים מזוקקים. הערה: בוחש חם משמש. להוסיף פוליאתילן גליקול 2.31 גרם האתר מונו-p-isooctylphenyl / טריטון X-100 הפתרון פעם ברור מתקרר לטמפרטורת החדר. הערה: 10 גרם של שלושה כימיקלים אלה הם לעכבר אחד. </li><li style=";text-align:right;direction:rtl"> חותכים את חוט השדרה עכבר coronally לתוך 2-3 מקטעים ארוכים מ&quot;מ עם סכין גילוח ולשים אותם לתוך 5 מ&quot;ל של הפתרון סליקה מעוקב לעיל. מניחים על שייקר בתנור ב 37 &#160; ° צלזיוס במשך 3 ימים. </li><li style=";text-align:right;direction:rtl"> שלושה ימים לאחר מכן, לשנות את הפתרון וסליקה אחד טרי. הערה: הפתרון מעל מוכן לפני השימוש. </li><li style=";text-align:right;direction:rtl"> שניים עד שלושה ימים לאחר מכן לאחר רענון פתרון הסליקה מעוקב, לבדוק את השקיפות של הרקמה כנגד נייר עם גופן 8 אותיות. אם הוא שקוף, האותיות ניתן לראות דרך רקמת פינה. הסר את פתרון הסליקה ולהוסיף 4 מיליליטר של PBST (0.1% Triton-X100) כדי לשטוף את הרקמה 4 פעמים ביום (כל 6 שעות). </li></ol></li></ol>ילדה = &quot;jove_title&quot;&gt; 2. immunofluorescence מכתים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירת מגזרי חוט שדרת פתרון הנוגדן הראשוני (-סרוטונין אנטי, וגדל ארנב, מדולל 1: 100 ב PBST) במשך 3 ימים על שייקר בתנור 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את הפתרון נוגדן ולהוסיף 4 מ&quot;ל של PBST (0.1% Triton-X100) לשטוף את פלחי חוט השדרה 4 פעמים ביום (כל 6 שעות) בתנור 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את הפתרון PBST ולהוסיף הפתרון נוגדנים משני (594 IgG מצומדות עז נגד ארנב, מדולל 1: 100 ב PBST) כדי לדגור הרקמה במשך 3 ימים על שייקר בתנור 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את פתרון נוגדנים משני ולהוסיף 4 מיליליטר של PBST לשטוף מגזרי חוט השדרה 4 פעמים ביום (כל 6 שעות) על שייקר בתנור 37 מעלות צלזיוס. למחרת הרקמה מוכנה הדמיה. </li></ol> הדמיה 3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר את הפתרון PBST ולהוסיף 4 מ&quot;ל של גליצרול 85% על מנת להפוך את מקדם השבירה של הרקמה אפילו. </li><li&gt; בדקי את הבהירות של הרקמה. לאחר ברור נע בין 1 hr, לשים את הרקמה על גבי זכוכית נושאת גדולה, דקה אשר מתאימה את המסגרת החזקה של מיקרוסקופ פלואורסצנטי רב-צילום. </li><li style=";text-align:right;direction:rtl"> לשים כמה טיפות של גליצרול 85% ליד רקמת חוט השדרה ו coverslip אותו עם כוס coverslip 22 x 50 מ&quot;מ 2. הערה: הכיוון של חוט השדרה מחליט כיצד התמונה נראית. </li><li style=";text-align:right;direction:rtl"> שים את השקופית זכוכית על מסגרת ההחזקה של מיקרוסקופ פלואורסצנטי רב פוטון ולהעביר את הרקמה לתוך מסלול האור. </li><li style=";text-align:right;direction:rtl"> בחר את ננומטר לייזר נאון הליום קו 594 ולהתאים את עוצמת הלייזר לרמה אופטימלית על ידי בדיקת הבהירות של איתות חיובי בתמונה חיה. </li><li style=";text-align:right;direction:rtl"> בחר את אזור הסריקה של רקמת חוט השדרה באמצעות המטרה 20X (במים, NA 0.7) ולהכין ליצור Z- מחסנית, ואת העומק של כל שלב מוגדר 3 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> סרוק את הרקמה מלמעלה לתחתית הערימה-z תחת objec 20Xמגני אוזניים ואז המטרה 63X (בשמן, NA 1.4) (גודל הצעד היה 1 מיקרומטר), בהתאמה. לשחזר קטעי וידאו 3D באמצעות תוכנת 3D 28. </li></ol>

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A., Bhansali, P., Jurgens, C., Guido, W., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearT:+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140 (6), 1364-1368 (2013).</li><li>Ert&#252;rk, A., Bradke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+entire+organs+by+3-dimensional+imaging+of+solvent+cleared+organs+(3DISCO).">High-resolution imaging of entire organs by 3-dimensional imaging of solvent cleared organs (3DISCO).</a> Exp Neurol. 242, 57-64 (2013).</li><li>Kim, S. Y., Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+mapping+of+connections+in+the+intact+brain.">Light microscopy mapping of connections in the intact brain.</a> Trends Cogn Sci. 17 (12), 596-599 (2013).</li><li>Spence, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bringing+CLARITY+to+gray+matter+atrophy.">Bringing CLARITY to gray matter atrophy.</a> NeuroImage. 101, 625-632 (2014).</li><li>Ando, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inside+Alzheimer+brain+with+CLARITY:+senile+plaques,+neurofibrillary+tangles+and+axons+in+3-D.">Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.</a> Acta Neuropathol. 128 (3), 457-459 (2014).</li><li>Zhang, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Struct Funct. , (2015).</li><li>Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+application+of+the+electrophoretic+tissue+clearing+technology,+CLARITY,+to+intact+solid+organs+including+brain,+pancreas,+liver,+kidney,+lung,+and+intestine.">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 781 (2015).</li><li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Rosset, A., Spadola, L., Ratib, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OsiriX:+an+open-source+software+for+navigating+in+multidimensional+DICOM+images.">OsiriX: an open-source software for navigating in multidimensional DICOM images.</a> J Digit Imaging. 17, 205-216 (2004).</li></ol>

The authors have nothing to disclose.

הפרוטוקול המתואר מראה כיצד סיבי serotonergic תמונה בחוט השדרה עכבר עם CLARITY בשילוב וטכניקות מעוקב. הוא מציג תהליך סליקה מהר לעומת פרוטוקול הסליקה הפסיבי שפותח על ידי Cheung et al. 14 והתומר et al. 15 ומאפשר רקמת חוט השדרה כדי להיות נתמך היטב על ידי הידרוג&#39;ל במהל...

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

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			<td height="22" style="height:22px;width:495px;">Photoinitiator VA044</td>
			<td style="width:495px;">Wako</td>
			<td style="width:284px;">va-044/225-02111</td>
			<td style="width:807px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
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		<tr height="21">
			<td height="21" style="height:21px;">40% acrylamide solution</td>
			<td>Bio Rad</td>
			<td>161-0140</td>
			<td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% Bis Solution</td>
			<td>Bio Rad</td>
			<td>161-0142</td>
			<td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-879c-4d63-ba0b-82556a0ccf1d</td>
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			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td align="right">158127</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&#38;region=AU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">urea</td>
			<td>Merck Millipore</td>
			<td align="right">66612</td>
			<td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis (2-hydroxypropyl) ethylenediamine</td>
			<td>Merck Millipore</td>
			<td align="right">821940</td>
			<td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#39;,N&#39;-tetra-2-propanol,MDA_CHEM-821940</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X 100</td>
			<td>Merck Millipore</td>
			<td align="right">648462</td>
			<td>http://www.merckmillipore.com/AU/en/product/TRITON&#174;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&#38;region=AU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8217;,2&#8217;&#8217;- nitrilotriethanol</td>
			<td>Merck Millipore</td>
			<td align="right">137002</td>
			<td>http://www.merckmillipore.com/AU/en/product/Triethanolamine-(Trolamine),MDA_CHEM-137022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serotonin antibody</td>
			<td>Merck Millipore</td>
			<td>AB938</td>
			<td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 594 conjugate</td>
			<td>Life Technologies</td>
			<td>&#160;A-11012</td>
			<td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">multi-photon microscope</td>
			<td>Leica</td>
			<td>Leica TCS SP5 MP STED</td>
			<td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td>
		</tr>
	</tbody>
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imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

סיבי יורד ארוך אל חוט השדרה חיוני תנועה, תפיסת כאב, והתנהגויות אחרות. דפוס סיום סיבים בחוט השדרה של רוב מערכות סיבים אלה לא נחקר ביסודיות בכל מינים. סיבי serotonergic, אשר פרויקט אל חוט השדרה, נחקרו בחולדות ואופוסומים על סעיפים היסטולוגית המשמעות התפקודית שלהם כבר להסיק על סמך דפוס סיום סיבים שלהם בחוט השדרה. עם התפתחות CLARITY וטכניקות מעוקב, אפשר לחקור מערכת סיבים זה והפצתו בחוט השדרה, אשר צפוי לחשוף תכונות שלא היו ידועים קודם של מסלולים supraspinal serotonergic. כאן, אנו מספקים פרוטוקול מפורט הדמיה הסיבים serotonergic בחוט השדרה העכבר באמצעות CLARITY בשילוב וטכניקות מעוקב. השיטה כרוכה זלוף של עכבר עם פתרון הידרוג&#39;ל והבהרה של הרקמה עם COMBINation סליקת ריאגנטים. רקמת חוט שדרה נוקתה למשך קצת פחות משבועות, ואת מכתים immunofluorescent עוקב נגד סרוטונין הושלם בתוך פחות מעשרה ימים. עם מיקרוסקופ פלואורסצנטי רב פוטון, הרקמה נסרקה תמונת 3D שוחזרה באמצעות תוכנת Osirix.

סעיף זה מציג תוצאות מכתים נוגדן סרוטונין במוח שדרת העכבר השקוף באמצעות שילוב של CLARITY ופרוטוקולים מעוקבים. אנו מראים כי סיבי serotonergic נוכח בכל הרבדים של חוט השדרה עם דומיננטיות בחלק הגחוני של צופר הגחון (איור 1, ראו גם וידאו 1). רקמת הבי...

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

הדמית סיבי serotonergic בחוט שדרת עכבר שימוש CLARITY / טכניקה מעוקבת

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

imaging-serotonergic-fibers-mouse-spinal-cord-using-claritycubic

Research

JoVE Journal

Neuroscience

10.4K Views.  Neuroscience Research Australia. Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Video: Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

חוט השדרה Electrophysiology II: ייצור תאי אלקטרודה יניקה

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

בתחום ההדמיה vivo של חוט השדרה בעזרת עכבר דו פוטון מיקרוסקופית

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

חיתוך רוחב חוט השדרה בדג הזברה הזחל

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

נוהל השתלה קאמרי בעמוד השדרה לאורך<em&gt; In vivo</em&gt; הדמיה של כבל עכבר השדרה

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

ההדמיה דיפוזיה בחוט השדרה עכברוש צוואר הרחם

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

הדמיה שני פוטונים של Dynamics הסלולרי בחוט השדרה העכבר

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

הדמיה פעילות עצבית בתוך קליפת המגע הראשי באמצעות Thy1-העכברים הטרנסגניים GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

כריתת למינציה ואת חוט השדרה חלון השרשה בעכבר

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

דיפוזיה זנסור הדמיה מגנטית באמצעות דחיסת חוט השדרה כרונית

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

משלוח פנים-עורקי של תאי גזע עצביים למוח עכברוש ועכבר: יישום איסכמיה מוחין

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...