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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	<li>Muzyk, A. J., Jakel, R. J., Preud'homme, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+syndrome+after+a+massive+overdose+of+controlled-release+paroxetine.">Serotonin syndrome after a massive overdose of controlled-release paroxetine.</a> Psychosomatics. 51, 437-442 (2010).</li><li>Davies, O., Batajoo-Shrestha, B., Sosa-Popoteur, J., Olibrice, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+recovery+after+severe+serotonin+syndrome,+severe+rhabdomyolysis,+multi-organ+failure+and+disseminated+intravascular+coagulopathy+from+MDMA.">Full recovery after severe serotonin syndrome, severe rhabdomyolysis, multi-organ failure and disseminated intravascular coagulopathy from MDMA.</a> Heart Lung. 43, 117-119 (2014).</li><li>Bosch, O. G., 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=Verbal+memory+deficits+are+correlated+with+prefrontal+hypometabolism+in+18FDG+PET+of+recreational+MDMA+users.">Verbal memory deficits are correlated with prefrontal hypometabolism in 18FDG PET of recreational MDMA users.</a> PLoS On. 8, e61234 (2013).</li><li>Smithies, V., Broadbear, J., Verdejo-Garcia, A., Conduit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunctional+overnight+memory+consolidation+in+ecstasy+users.">Dysfunctional overnight memory consolidation in ecstasy users.</a> J Psychopharmaco. 28, 751-762 (2014).</li><li>Winzer-Serhan, U. H., Broide, R. S., Chen, Y., Leslie, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+radioactive+in+situ+hybridization+using+full+length+hydrolyzed+riboprobes+to+detect+&#945;2+adrenoceptor+subtype+mRNAs+in+adult+and+developing+rat+brain.">Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect &#945;2 adrenoceptor subtype mRNAs in adult and developing rat brain.</a> Brain Res Brain Res Proto. 3, 229-241 (1999).</li><li>Zoeller, R. T., Fletcher, D. L., Butnariu, O., Lowry, C. A., Moore, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-ethylmaleimide+(NEM)+can+significantly+improve+in+situ+hybridization+results+using+35S-labeled+oligodeoxynucleotide+or+complementary+RNA+probes.">N-ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes.</a> J Histochem Cytoche. 45, 1035-1041 (1997).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diag. 14, 22-29 (2012).</li><li>Wang, H., 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=RNAscope+for+in+situ+detection+of+transcriptionally+active+human+papillomavirus+in+head+and+neck+squamous+cell+carcinoma.">RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.</a> J Vis E. , (2014).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis E. , (2012).</li><li>Mishra, A., 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=Imaging+pericytes+and+capillary+diameter+in+brain+slices+and+isolated+retinae.">Imaging pericytes and capillary diameter in brain slices and isolated retinae.</a> Nat Proto. 9, 323-336 (2014).</li><li>Shi, S. R., Cote, R. J., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+techniques:+current+perspectives.">Antigen retrieval techniques: current perspectives.</a> J Histochem Cytoche. 49, 931-937 (2001).</li><li>Qin, Y., Heine, V. M., Karst, H., Lucassen, P. J., Joels, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+patterns+in+rat+dentate+granule+cells:+comparison+between+fresh+and+fixed+tissue.">Gene expression patterns in rat dentate granule cells: comparison between fresh and fixed tissue.</a> J Neurosci Method. 131, 205-211 (2003).</li><li>Carter, B. S., Fletcher, J. S., Thompson, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+messenger+RNA+expression+by+in+situ+hybridization+using+RNA+probes+synthesized+via+in+vitro+transcription.">Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription.</a> Method. 52, 322-331 (2010).</li><li>Mijnster, M. 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><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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Universidad de Cartagena UDEC

Research

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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 ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

סמנים ביולוגיים במודל חיה לחשיפה קושרת עצבית, המטולוגית, והתנהגות של PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicine

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn: שיטה פלואורסצנטית מהירה ובלתי משוחדת לזיהוי שינויים בצפיפות הסינפסה

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Biochemistry

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 ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

זיהוי של דופמין קולטן D1-אלפא בתוך מכרסמים גרעין האקומבנס על ידי טכנולוגיה חדשנית RNA בחיי עיר זיהוי

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

שימוש בקרוב-אינפרא אדום פלואורסצנטית ברזולוציה גבוהה סריקה למדוד חלבון הביטוי במוח מכרסם

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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 ...

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Source: Shokry, I. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53165">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://appjove.unicartagenaproxy.elogim.com/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

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