מחקר זה נתמך על ידי מענק NIH (R15DA029863), מענק אוניברסיטת פלורידה אטלנטיק מחקר לתואר ראשון (M30014) ואוניברסיטת רוס של מענק מחקר לרפואת הווטרינרית. ברצוננו להודות למכון הלאומי לשימוש בסמים (Rockville, MD) למתן (±) 3,4-methylenedioxymethamphetamine (± MDMA) עבור עבודה זו.

נהלי שימוש בבעלי חיים מתוארים להלן אושרו על ידי הוועדה המוסדית הטיפול בבעלי חיים והשימוש (IACUC) באוניברסיטת רוס לרפואת וטרינרית ואוניברסיטת פלורידה אטלנטיק. למרות טכניקות וכפפות סטריליות נדרשות, הסביבה נטול RNase אין צורך בעת השימוש בפרוטוקול זה. 1. הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת רקמה וחתך <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הקצאת חולדות באופן אקראי לשלוש קבוצות: טרי / מלוח (SAL; 0.9% NaCl), ולפניה / SAL וקידומת / 3,4-methylenedioxymethamphetamine (MDMA; ראה פירוט בטבלת 1). </li><li style=";text-align:right;direction:rtl"> לנהל שלושה מינונים של 1 מיליליטר / קילוגרם SAL ו -10 מ&quot;ג / קילוגרם MDMA intraperitoneally, בהתאמה, לקבוצות SAL וMDMA במרווח 2 שעות. הרשה בעלי חיים כדי לשרוד את הטיפול במשך 6 ימים. </li><li style=";text-align:right;direction:rtl"> ביום 7, לנהל 100 מ&quot;ג / קילוגרם קטמין בשילוב של 5 מ&quot;ג / קילוגרם xylazine intraperitoneally לחולדות להרדמה עמוקה (כלומר, הפסד של מצמוץ בקרנית ות&quot;אil לצבוט רפלקסים). </li><li style=";text-align:right;direction:rtl"> לקבוצה הטרי / SAL, לערוף מהירות חולדות ולהסיר את המוח לבדיקת המוח הטרי (ראה פירוט בהתייחסות 5). </li><li style=";text-align:right;direction:rtl"> לקבוצות / MDMA קידומת / SAL וקידומת, לעשות חתך לאורך עצם החזה על 10 -12 סנטימטרים ולאחר מכן לחשוף את הסוף של עצם החזה. </li><li style=";text-align:right;direction:rtl"> החזק את הקצה של עצם החזה עם hemostat ולחתוך את הסרעפת רוחבית בשני הצדדים עם מספריים חדים ולאחר מכן לחתוך כלפי מעלה על פני הצלעות ומקבילות לריאות. </li><li style=";text-align:right;direction:rtl"> ביד אחת, מחזיק את קצה הגחון של הלב עם מלקחיים קטנים. ביד השנייה, פירס החדר השמאלי עם מחט מזרק G 18 (שים לב לצד המתאם של המחט מחוברת עם צינור סיליקון בצורת אות V למכולות SAL וparaformaldehyde קר כקרח ומשאבות peristaltic; ראה התייחסות 9). </li><li style=";text-align:right;direction:rtl"> הפעל את משאבת SAL ברמה בינונית של קצב הזרימה (~ 5 מיליליטר / דקה) ולנקב את אטריום ימין עם המלקחיים כדי לאפשר הבריחה של circ התמורהulation. לשמור זלוף SAL למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> הפעל את משאבת paraformaldehyde ברמה בינונית ולאפשר paraformaldehyde 4% קרים כקרח לינקב את החיה במשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> הסר מכשיר זלוף ולערוף את החיה. הפוך posteroanterior לחתוך את קו האמצע של הגולגולת, ואז לנפנף את הגולגולת כדי לחשוף את חלל גולגולת 10. </li><li style=";text-align:right;direction:rtl"> הסר את המוח עם מרית מחלל הגולגולת ולמקם את המוח בצינור צנטריפוגות 50 מיליליטר המכיל 25 מיליליטר של 4% paraformaldehyde. שמור את המוח בתוך הצינור המכיל paraformaldehyde ב 4 מעלות צלזיוס מקרר למשך 24 שעות. </li><li style=";text-align:right;direction:rtl"> הזז את המוח לתוך צינור צנטריפוגה 50 מיליליטר המכיל 25 מיליליטר של 30% תמיסת סוכרוז ב 4 מעלות צלזיוס במשך 3 ימים. לבסוף, לאחסן המוח בתוך שקיות פלסטיק ב -80 ° C עד שימוש. </li><li style=";text-align:right;direction:rtl"> הר המוח על צ&#39;אק עם תקשורת הטבעה ולהקפיא את תקשורת ההטבעה מכילה מוח בcryostat בטמפרטורה של -26 מעלות צלזיוס. חותך את קפא הטרימוח n ל -20 מיקרומטר חלקים וparaformaldehyde-התחילית מוח לסעיפים 10 מיקרומטר. העבר את הסעיף לשקופית מיקרוסקופ RT על ידי נגיעה בשקופית לסעיף. </li><li style=";text-align:right;direction:rtl"> אוויר יבש ב RT במשך 4 שעות. אחסן את שקופיות הסעיף בשקית אטומה הדוק ב -80 ° C עד שימוש. </li></ol></li><li style=";text-align:right;direction:rtl"> התייבשות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח שקופיות מהמקפיא -80 ° C ולהקצות חלקים לאחת משלוש בדיקות: HT2A, ppiB (ביקורת חיובית) או dapB (שליטה שלילית); סימן וחתכים תווית עם עט כדורי (לא להשתמש בעיפרון). לצלול שקופיות באלכוהול 100% ב RT במשך 5 דקות. ייבש את השקופיות במשך 5 דקות במנדף. </li></ol></li><li style=";text-align:right;direction:rtl"> טיפול מקדים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פיפטה 20 μl של המגיב לפני הטיפול-1 (איון של phosphatase אלקליין) בסעיפים בשקופיות. מניחים את השקופיות על רכבת מתלה במגש לחות (שים לב שהמגש מכוסה במכסה כדי לשמור על לחות גבוהה למניעת אידוי מsolutio התגובהn). </li><li style=";text-align:right;direction:rtl"> נער בעדינות את מגש הלחות על שייקר אופקי במהירות נמוכה במשך 10 דקות. שטוף את השקופיות פעמיים עם מים מזוקקים כפולים (DDH 2 O) במשך 2 דקות. להטביע את השקופיות בכוס 200 מיליליטר המכילה 150 מיליליטר של המגיב לפני הטיפול-2 (שבירה של הקשרים בין-הנגרמים על ידי קיבוע paraformaldehyde). מרתיחים את השקופיות עבור הסכום כולל של 5 דקות ב 100 מעלות צלזיוס (שיקום מושרה חום של מבנה RNA 11). </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם DDH 2 O 2 דקות. מניחים את השקופיות באלכוהול 100% במשך 5 דקות. ייבש את השקופיות במשך 5 דקות במנדף. השתמש בעט הידרופובי לצייר מעגל מסביב לאזור שנבחר בסעיף הרקמות (למשל, מבנים שאינן בקליפת המוח, כוללים התלמוס וההיפותלמוס כפי שמתוארים באיור 2). </li><li style=";text-align:right;direction:rtl"> פיפטה 10 μl של המגיב לפני הטיפול-3 (עלייה בחדירת בדיקה) לכל אזור שנבחר, ולכסות את מגש הלחות עם המכסה. מניחים את המגש בתנור ההכלאהמצויד בייקר האופקי ולהגדיר את טמפרטורת התנור ב -40 מעלות צלזיוס; נער בעדינות את המגש למשך 30 דקות. שטוף את השקופיות פעמיים עם DDH 2 O 2 דקות. </li></ol></li></ol> 2. הכלאה והגברה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פיפטה 10 μl של htr2a, ריאגנטים בדיקה ppiB וdapB, בהתאמה, באזורים הנבחרים. מכסה את מגש הלחות עם המכסה כדי למנוע אידוי של המגיב. נער בעדינות על שייקר האופקי להגדיר במהירות נמוכה. דגירה השקופיות ב 40 מעלות צלזיוס בתנור למשך 2 שעות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם חיץ כביסה למשך 2 דקות. פיפטה 10 μl של מגיב הגברה-1 על כל אזור שנבחר, לנער דגירה השקופיות ב 40 מעלות צלזיוס למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם חיץ כביסה למשך 2 דקות. פיפטה 10 μl של מגיב הגברה-2 על כל אזור שנבחר, לנער דגירה השקופיות ב 40 מעלות צלזיוס במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים wה- i כביסה חיץ במשך 2 דקות. פיפטה 10 μl של מגיב הגברה-3 בכל אזור שנבחר, לנער דגירה השקופיות ב 40 מעלות צלזיוס למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם חיץ כביסה למשך 2 דקות. פיפטה 10 μl של מגיב הגברה-4 בכל אזור שנבחר, לנער דגירה השקופיות ב 40 מעלות צלזיוס במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם חיץ כביסה למשך 2 דקות. פיפטה 10 μl של מגיב הגברה-5 בכל אזור שנבחר, לנער דגירה השקופיות ב RT למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם חיץ כביסה למשך 2 דקות. פיפטה 10 μl של מגיב הגברה-6 בכל אזור שנבחר, לנער דגירה השקופיות ב RT במשך 15 דקות. שטוף את השקופיות פעמיים עם חיץ כביסה למשך 2 דקות. </li></ol> 3. גילוי אותות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פיפטה 10 μl של המגיב האדום לכל אזור שנבחר (שים לב שמגיב היא תערובת של אדום-B ואדום-ביחס 1:60 ויש להשתמש ביmmediately). מניחים את השקף במגש הלחות על שייקר האופקי ב RT וללחוץ בעדינות במהירות נמוכה במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> שטוף את השקופיות פעמיים עם (DDH 2 O) במשך 10 דקות. ייבש את השקופיות על 60 מעלות צלזיוס בתנור במשך 15 דקות. מניחים את השקופיות בקסילן מתחת למכסת מנוע קטר במשך 5 דקות. פיפטה 20 μl של תקשורת הרכבה מבוססת קסילן בכל אזור שנבחר ולכסות עם coverslip. </li></ol> לכידת 4. תמונה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח microphotographs הדיגיטלי של האזורים הנבחרים (לדוגמא, ההיפותלמוס, איורים 1 ו 3) עם הגדלה של 4 × 20 × ועדשות אובייקטיביות. שמור את קובץ התמונה. </li></ol> ניתוח חלקיקים 5. תמונה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> גרור ושחרר את קובץ התמונה בחלון ImageJ העיקרי. עבור לשורת תפריטים ובחרו &#39;תמונה&#39;, הבא לבחור &quot;סוג&quot; מהתפריט הנפתח, ובחר &#39;8 סיביות &quot;. </li><li style=";text-align:right;direction:rtl"> בחירה &#39;התאם&#39;, ולאחר מכן בחר &#39;Threshold &#39;כדי לפתוח את תיבת הדו-שיח. התאם את ערך הסרגל התחתון בתיבת הדו שיח כדי שהרקע לא רצוי יוסר. עבור לשורת תפריטים ובחרו &#39;לנתח&#39;, ולאחר מכן בחר &#39;לנתח חלקיקים&#39; כדי לפתוח תיבה דו-שיח שנייה. </li><li style=";text-align:right;direction:rtl"> הגדר את גודל חלקיקים בגיל 10, הבא בחר &#39;הצג מתווה&#39; ובחר &#39;צג תוצאות &quot;ו&#39;קופסות&#39; לסכם. לבסוף, לחץ על &#39;אישור&#39; כדי להציג נתונים חלקיקים בתיבות דו-שיח. </li></ol> חישוב 6. גיליון אלקטרוני <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הזן את הנתונים לגיליון Excel וממוצע המספרים של חלקיקים בקבוצת SAL כבסיס. לחשב את האחוז של כל קטע ולעשות גרף בהתאם (איור 4). </li></ol>

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J., 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=Regional+and+cellular+distribution+of+serotonin+5-hydroxytryptamine2a+receptor+mRNA+in+the+nucleus+accumbens,+olfactory+tubercle,+and+caudate+putamen+of+the+rat.">Regional and cellular distribution of serotonin 5-hydroxytryptamine2a receptor mRNA in the nucleus accumbens, olfactory tubercle, and caudate putamen of the rat.</a> J Comp Neuro. 389, 1-11 (1997).</li><li>Li, Q., 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=Brain+region-specific+alterations+of+5-HT2A+and+5-HT2C+receptors+in+serotonin+transporter+knockout+mice.">Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice.</a> J Neuroche. 84, 1256-1265 (2003).</li><li>Horner, K. A., Gilbert, Y. E., Noble, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+5-HT2A+receptor+mRNA+expression+following+withdrawal+from+a+chronic+escalating+dose+regimen+of+D-amphetamine.">Differential regulation of 5-HT2A receptor mRNA expression following withdrawal from a chronic escalating dose regimen of D-amphetamine.</a> Brain Re. 1390, 10-20 (2011).</li><li>Mocci, G., Jimenez-Sanchez, L., Adell, A., Cortes, R., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+5-HT2A+receptors+in+prefrontal+cortex+pyramidal+neurons+projecting+to+nucleus+accumbens.+Potential+relevance+for+atypical+antipsychotic+action.">Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. Potential relevance for atypical antipsychotic action.</a> Neuropharmacolog. 79, 49-58 (2014).</li><li>Reneman, L., 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=The+acute+and+chronic+effects+of+MDMA+('Ecstasy')+on+cortical+5-HT2A+receptors+in+rat+and+human.">The acute and chronic effects of MDMA ('Ecstasy') on cortical 5-HT2A receptors in rat and human.</a> 26, 387-396 (2002).</li></ol>

המחברים מצהירים שאין להם אינטרסים כלכליים מתחרים.

One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution5,12. Although quick preservation is critical for hybridization using a riboprobe13, little is known about experimental conditions...

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants1, while also occurring in situations of MDMA use for recreational purposes2. Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment5,6. 
Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes7. Furthermore, the probes produce a low background signal without sacrificing specificity8. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.
Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr>
			<td>RNAscope Negative Control Probe-DapB</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310098</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Pretreat 4</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>320046</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - Red</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310036</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Probe - Rn-Ppib</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>313921</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Htr2a</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>300031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM 1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>VWR</td>
			<td>88032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Thermo Fisher Scientific</td>
			<td>222000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>Vector</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Provis AX70</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed brain of rats with serotonin syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

שימוש בבדיקות RNA oligonucleotide (&lt;25 NT), הכלאה יכולה להתגלות כנקודות אדומות בתאי ההיפותלמוס הוכנו מהמוח הקפוא-התחילית paraformaldehyde וטרי. מולקולות mRNA htr2a נמצאות בכמה תאים (המסומנים בחצים מוצקים), אך לא לאחרים (חיצים פתוחים). הבחנו כי אין הבדל בין-התחילית paraformaldehyde והרקמות קפוא...

Watch this Scientific Journal Video about Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome at JoVE.com

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

מהיר<em&gt; באתר</em&gt; הכלאה באמצעות oligonucleotide בדיקות במוח-התחילית Paraformaldehyde של חולדות עם סרוטונין תסמונת

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

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Research

JoVE Journal

Neuroscience

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

8.8K Views.  Ross University School of Veterinary Medicine. This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

Video: Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

פעמיים פלואורסצנטי באתרו הכלאה בסעיפים המוח טריים

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: שימוש בפרוטוקול תטא-Burst לחקור Mechasnism של פלסטיות של אנשים עם תסמונת X שביר אוטיזם ו

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

שילוב של צבע lipophilic, באתרו</emהכלאה>, אימונוהיסטוכימיה ו היסטולוגיה

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

חוט השדרה מלא פגיעה ומוח Dissection פרוטוקול Wholemount לאחר<em&gt; באתר</em&gt; הכלאה בזחל ים צמד ים

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

איתור של Axonally המותאם למקום mRNAs בסעיפי מוח באמצעות ברזולוציה גבוהה<em&gt; באתר</em&gt; הכלאה

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

שילוב קרינה זוגית<em&gt; באתרו</em&gt; כלאה עם Immunolabelling עבור איתור של הביטוי של שלושה גנים בסעיפי מוח עכבר

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

כימות של Endosome ו Lysosome Motilities ב נוירונים תרבותיים באמצעות בדיקות פלואורסצנטי

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

לוקליזציה של גנים קולטן Odorant ב אנטנות ארבה על ידי RNA<em&gt; באתרו</em&gt; הכלאה

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

אינדוקציה של פגיעה מוחית Axonal בחולדות מבוסס על האצת הסיבוב

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

שילוב של הכלאה פלואורסצנטית באתרו עם אימונוהיסטוכימיה פלואורסצנטית על חלקי מוח טריים של עכברים קפואים או קבועים

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...