Diese Studie wurde von den NIH (R15DA029863) unterstützt, der Florida Atlantic University Undergraduate-Forschungsstipendium (M30014) und die Ross University School of Veterinary Medicine Forschungsstipendium. Wir möchten das National Institute on Drug Abuse (Rockville, MD) für die Bereitstellung von (±) 3,4-methylenedioxymethamphetamine (± MDMA) für diese Arbeit danken.

Unten beschriebenen Tiernutzung Verfahren wurden von der Institutional Animal Care und Verwenden Committee (IACUC) bei Ross University School of Veterinary Medicine und Florida Atlantic University zugelassen. Obwohl sterile Techniken und Handschuhe erforderlich sind, ist die RNase-freie Umgebung nicht notwendig, während Sie dieses Protokoll. 1. Vorbereitung <ol><li> Gewebepräparation und Schneiden <ol><li> Vergeben Ratten zufällig in drei Gruppen: frisch / Salzlösung (SAL; 0,9% NaCl), vorangestellt ist / SAL und voran / 3,4-methylenedioxymethamphetamine (MDMA, siehe Angaben in Tabelle 1). </li><li> Verwalten drei Dosen von 1 ml / kg SAL und 10 mg / kg intraperitoneal MDMA, die jeweils mit den SAL und MDMA Gruppen 2 Stunden-Intervall. Lassen Sie Tiere, die Behandlung für 6 Tage zu überleben. </li><li> Am 7. Tag verabreichen 100 mg / kg Ketamin in Kombination von 5 mg / kg Xylazin intraperitoneal den Ratten nach einem tiefe Narkose (dh Verlust an Hornhaut blinken und tail kneifen Reflexe). </li><li> Für die Frisch / SAL-Gruppe, schnell zu enthaupten Ratten und entfernen Sie das Gehirn für die frischen Gehirn-Test (siehe Details in der Referenz 5). </li><li> Für die voran / SAL und voran / MDMA Gruppen, machen einen Schnitt entlang des Brustbeins etwa 10 bis 12 cm und dann setzen die Ende des Brustbeins. </li><li> Halten Sie das Ende des Brustbeins mit einer Gefäßklemme und schneiden Sie die Membran seitlich auf beiden Seiten mit einer scharfen Schere und dann nach oben geschnitten auf den Rippen und parallel zu den Lungen. </li><li> Mit einer Hand halten Sie die ventralen Spitze der Herzen mit kleinen Zange. Mit der anderen Hand, durchbohren die linke Herzkammer mit einer 18 G Injektionsnadel (beachten Sie den Adapter Seite der Nadel ist mit einem Y-förmigen Silizium-Schlauch an eiskalten SAL und Para Behälter und Schlauchpumpen verbunden, siehe die Referenz 9). </li><li> Schalten Sie den SAL Pumpe an der mittleren Ebene der Flussrate (~ 5 ml / min) und Punktion der rechten Vorhof mit der Zange, um das Entweichen der Rückkehr circ ermöglichenlation. Aufrechtzuerhalten SAL Perfusion für 30 min. </li><li> Schalten Sie den Parapumpe auf mittlerem Niveau und lassen Sie das eiskalte 4% Paraformaldehyd, das Tier für 30 Minuten durchströmen. </li><li> Entfernen Perfusion Instrument und enthaupten das Tier. Einen posteroanterior durch die Mittellinie des Schädels geschnitten und dann flattern den Schädel, um die Schädelhöhle 10 freizulegen. </li><li> Entfernen des Gehirns mit einem Spatel aus der Schädelhöhle und legen das Gehirn in ein 50 ml Zentrifugenröhrchen, das 25 ml von 4% igem Paraformaldehyd. Halten das Gehirn innerhalb des Parahaltigen Röhrchen bei 4 ºC Kühlschrank für 24 Stunden. </li><li> Bewegen das Gehirn in ein 50 ml Zentrifugenröhrchen, das 25 ml 30% Saccharose-Lösung bei 4 ° C für 3 Tage. Schließlich speichert das Gehirn in Plastiktüten bei -80 ° C bis Gebrauch. </li><li> Montieren Sie das Gehirn auf einem Spannfutter mit Einbettungsmittel und frieren die Einbettungsmittel Gehirn enthält, in einem Kryostaten bei einer Temperatur von -26 ºC. Schneiden Sie den frisch eingefrorenn Gehirn in 20 um Abschnitte und Para-Präfix Gehirn in 10 &amp; mgr; m-Abschnitte. Übertragen Sie den Abschnitt zu einem RT Mikroskop-Objektträger, indem Sie den Schieber auf den Abschnitt. </li><li> Luft trocknen bei Raumtemperatur für 4 Stunden. Bewahren Sie die Sektion Folien in einer dicht verschlossenen Beutel bei -80 ° C bis Gebrauch. </li></ol></li><li> Entwässerung <ol><li> Nehmen Objektträger aus dem -80 ° C Gefrierschrank und weisen Abschnitte in eine von drei Tests: HT2A, PPIB (positive Kontrolle) oder dapB (Negativkontrolle); Zeichen und Etikettenabschnitte mit einem Kugelschreiber (nicht mit einem Bleistift). Tauchen Sie Folien in 100% Alkohol bei Raumtemperatur für 5 min. Trocknen Sie die Objektträger für 5 min in einer Abzugshaube. </li></ol></li><li> Vorbehandlung <ol><li> Je 20 ul der Vorbehandlung-1-Reagens (Inaktivierung von alkalischer Phosphatase) zu den Abschnitten auf Objektträger. Objektträger auf einem Rack-Schiene in einer feuchtigkeitsFach (beachten Sie, dass die Schale mit einem Deckel abgedeckt, um eine hohe Luftfeuchtigkeit zu verhindern, daß Verdampfung aus dem Reaktions solutio pflegenn). </li><li> Schütteln des Feuchtigkeits Tablett auf einem Horizontalschüttler bei niedriger Geschwindigkeit für 10 min. Zweimal für 2 min Waschen Sie die Objektträger mit doppelt destilliertem Wasser (ddH 2 O). Tauchen die Folie in einem 200 ml Becherglas, das 150 ml der Vorbehandlungs-2-Reagenz (Brechen der Querverbindungen von Paraformaldehyd Fixierung induziert). Man kocht die Folien für eine Gesamtzahl von 5 min bei 100 ºC (wärmeinduzierte Wiederherstellung der RNA-Struktur 11). </li><li> Zweimal für 2 min Waschen Sie die Objektträger mit ddH 2 O. Objektträger in 100% Alkohol für 5 min. Trocknen Sie die Objektträger für 5 Minuten im Abzug. Verwenden Sie einen hydrophoben Stift, um einen Kreis um den ausgewählten Bereich im Gewebeschnitt zu ziehen (zB nicht-kortikalen Strukturen, einschließlich Thalamus und Hypothalamus, wie in 2 beschrieben). </li><li> Je 10 ul der Vorbehandlung-3-Reagenz (Anstieg der Sondenpenetration) zu jedem ausgewählten Bereich, und decken den Feuchtefach mit dem Deckel. Legen Sie das Fach in der Hybridisierungsofenmit dem Horizontalschüttler ausgestattet und stellen Sie die Ofentemperatur bei 40 ºC; schütteln Sie das Fach für 30 min. Zweimal für 2 min Waschen Sie die Objektträger mit ddH 2 O. </li></ol></li></ol> 2. Die Hybridisierung und Amplifikation <ol><li> Pipette 10 ul HTR2A, PPIB und dapB Sonde Reagenzien jeweils auf den ausgewählten Bereichen. Decken den Feuchtigkeitsschale mit dem Deckel um eine Verdampfung des Reagens zu verhindern. Schütteln Sie auf dem Horizontalschüttler bei niedriger Geschwindigkeit eingestellt. Die Folien bei 40 ºC inkubieren in den Ofen für 2 Stunden. </li><li> Zweimal mit einem Waschpuffer für 2 min Waschen der Objektträger. Je 10 ul der Amplification-1 Reagenz auf jedem ausgewählten Bereich, schütteln und Inkubation der Objektträger bei 40 ° C für 30 min. </li><li> Zweimal mit einem Waschpuffer für 2 min Waschen der Objektträger. Je 10 ul der Amplification-2-Reagenz auf jedem ausgewählten Bereich, schütteln und Inkubation der Objektträger bei 40 ° C für 15 min. </li><li> Zweimal w Waschen Sie die Objektträgerit einem Waschpuffer für 2 min. Je 10 ul der Amplification-3 Reagenz auf jedem ausgewählten Bereich, schütteln und Inkubation der Objektträger bei 40 ° C für 30 min. </li><li> Zweimal mit einem Waschpuffer für 2 min Waschen der Objektträger. Je 10 ul der Amplification-4-Reagenz auf jedem ausgewählten Bereich, schütteln und Inkubation der Objektträger bei 40 ° C für 15 min. </li><li> Zweimal mit einem Waschpuffer für 2 min Waschen der Objektträger. Je 10 ul der Amplification-5-Reagenz auf jedem ausgewählten Bereich, schütteln und Inkubation der Objektträger bei Raumtemperatur 30 min. </li><li> Zweimal mit einem Waschpuffer für 2 min Waschen der Objektträger. Je 10 ul der Amplification-6-Reagenz auf jedem ausgewählten Bereich, schütteln und Inkubation der Objektträger bei Raumtemperatur für 15 min. Zweimal mit einem Waschpuffer für 2 min Waschen der Objektträger. </li></ol> 3. Signalerkennung <ol><li> Je 10 ul der Red-Reagenz zu jedem ausgewählten Bereich (Beachten Sie, dass das Reagenz ist eine Mischung aus Rot-B und Red-A bei einem 1:60 Verhältnis und sollte ich nutzennmittelbar). Legen Sie die Folie in der Feuchtigkeit Tablett auf dem Horizontalschüttler bei RT und leicht schütteln bei niedriger Geschwindigkeit für 10 min. </li><li> Zweimal für 10 min Waschen Sie die Objektträger mit (ddH 2 O). Trocknen Sie die Objektträger bei 60 ° C in den Ofen für 15 Minuten. Objektträger in Xylol unter dem Abzug für 5 min. Je 20 &amp; mgr; l Xylol-basierten Montagemedium auf jeder ausgewählten Bereichs und mit einem Deckglas. </li></ol> 4. Bildaufnahme <ol><li> Nehmen digitale Mikrofotografien von den ausgewählten Bereichen (z, Hypothalamus, Figuren 1 und 3) mit einer Vergrößerung von 4 × 20 × und Objektivlinsen. Speichern Sie die Bilddatei. </li></ol> 5. Teilchenbildanalyse <ol><li> Ziehen Sie die Bilddatei in den Haupt ImageJ Fenster. Gehen Sie auf die Menüleiste und wählen Sie &#39;Bild&#39;, neben auswählen &#39;Type&#39; aus dem Dropdown-Menü, und wählen Sie &quot;8-Bit&quot;. </li><li> Wählen Sie &quot;Anpassen&quot;, und wählen Sie &quot;Threshold &#39;, um das Dialogfeld zu öffnen. Stellen Sie die untere Stange Wert im Dialogfeld, so dass die unerwünschte Hintergrund wird entfernt. Gehen Sie in der Menüleiste und wählen Sie &quot;Analysieren&quot;, wählen Sie dann &quot;Analyze Particles&quot;, um ein zweites Dialogfeld zu öffnen. </li><li> Stellen Partikelgröße bei 10, nächste Option &quot;umreißt anzeigen&quot; und wählen Sie &quot;Ergebnis anzeigen&quot; und &quot;Zusammenfassen&quot; Boxen. Schließlich klicken Sie auf &quot;OK&quot;, um die Partikeldaten in den Dialogfeldern anzuzeigen. </li></ol> 6. Tabellenkalkulation <ol><li> Geben Sie die Daten in die Excel-Tabelle und der Mittelwert der Anzahl der Partikel in der SAL-Gruppe als Grundlinie. Berechnen Sie die prozentuale Höhe der einzelnen Abschnitte und machen einen Graphen entsprechend (Abbildung 4). </li></ol>

<ol>
	<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>

Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

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 for oligonucleotide probes. In this study using oligonucleotide probes, we found that there was no difference between the paraformaldehyde-prefixed and fresh brains quickly preserved in the ice/alcohol solution. This suggests that brain RNA prefixed by paraformaldehyde had no significant effect on the quality of hybridization using the oligonucleotide probe. Since the prefixed brain is easily sectioned compared to the fresh brain, this protocol provides an alternative tissue preparation for the hybridization test. 
We believe that keeping the brain tissue sections inside the sealed moisture tray with constant shaking on the horizontal shaker is essential for successful hybridization. Failure to follow those steps may result in uneven hybridization or high background. To eliminate possibilities of false-negative or false-positive results, tests should include necessary control slides. We found that the dapB probe did not show a visible signal. This is not surprising since dapB is a bacterial gene that is unlikely found in mammalian cells. In contrast, the area hybridized with the ppiB probe showed signals homogenously distributed throughout the region examined. This is predictable since the housekeeping gene should be present in any kind of mammalian cells. It has been suggested that 5-HT2A mRNA is distributed heterogeneously in the brain nuclei, including the hypothalamus14,15. As expected, htr2a signals are present in certain nuclei in the region of the hypothalamus although the probe has been applied to all of the selected areas. It should be kept in mind that the use of alcohol should be avoided after the Red reagent (step 3.1) since red signals can be easily decolored by alcohol. We found that, after cellular RNA stained with the Red reagent, cells cannot be counterstained with hematoxylin or crystal violet although the exact cause of counterstaining failure is unknown. In summary, a characteristic distribution of htr2a mRNA supports the idea that these oligonucleotide probes are gene-specific despite the shorter sequence relative to other probes16,17. 
Background signals are another major concern, which likely affects the quality and faithfulness of data analysis. Unlike the high background produced by riboprobes13, oligonucleotide probes normally show a very low background. Thus, image can be easily and reasonably quantified using publicly-available NIH ImageJ software. In this study, we showed an example of this technique by examining changes in htr2a expression in response to systemic MDMA at 10 mg/kg. We demonstrated that MDMA caused a 20% reduction in htr2a expression, supporting previous reports18. 
In addition to the three genes outlined above, this protocol has been validated with two other gene probes (trh, thyrotropin-release hormone gene, and trhr, thyrotropin-releasing hormone receptor gene; data not shown), demonstrating a high sensitivity and accuracy in detecting mRNA levels. Since most reactions require only 10 &#181;l of reagents, the cost of an assay is also significantly reduced, compared to radioactive riboprobes. Furthermore, it is important to note that this protocol does not require the RNase-free environment. In conclusion, this protocol improves the speed of in situ hybridization assay, and increases reproducibility with relatively low cost.

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.

Unter Verwendung der Oligonukleotid-RNA-Sonden (&lt;25 nt), kann die Hybridisierung als rote Punkte im Hypothalamus Zellen von den Para-Präfix und frisch gefrorenes Hirn hergestellt nachgewiesen werden. Die HTR2A mRNA-Moleküle sind in einigen Zellen (durch durchgezogene Pfeile) vorhanden, aber andere nicht (offene Pfeile). Wir beobachteten, dass es keinen Unterschied zwischen der Paraformaldehyd-Präfix und die frisch gefrorenes Gewebe (Abbildung 1). Eine erfolgreiche Hybridisierung kann zuerst mit dem bloßen Auge (2) ausgewertet werden. Wir fanden, dass der ausgewählte Bereich (mit Kreisen markiert) mit dem dapB-Sonde hybridisiert nicht ein sichtbares Signal für das bloße Auge (2A-B) zeigen. Im Gegensatz dazu zeigte der Bereich mit der Sonde hybridisiert PPIB Signale homogen in der gesamten Region sucht (2C-D) verteilt sind. Während in der HTR2A Test, in bestimmten Kernen (durch Pfeile angedeutet vorhanden sind Signale; <strong&gt; 2E-F), obwohl nicht alle eingekreisten Bereich mit der HTR2A Sonde hybridisiert worden. Interessanterweise waren die Signale in MDMA gleitet relativ schwach im Vergleich zu SAL Folien. Wesentliche Details des Hybridisierungssignale können unter einem mikroskopischen Instrument bewertet werden. Es gibt kein roter Punkt (3A-B) in den Zellen mit dem dapB Sonde hybridisiert während Signale können in der gesamten mikroskopischen Feld mit dem PPIB Sonde (3C-D) hybridisiert gesehen werden. Die HTR2A Signale hauptsächlich in bestimmten Kernen des Hypothalamus (3E-F) angeordnet ist. Wir beobachteten eine Verringerung HTR2A Signale in den zuvor eine systemische MDMA bei 10 mg / kg behandelten Gehirngeweben, die quantitativ unter Verwendung des ImageJ Analyse bestimmt werden kann. Wie in Abbildung 4 dargestellt, fanden wir eine 20% ige Verringerung HTR2A Ausdruck. Zusammenfassend zeigten unsere Ergebnisse, dass unter Verwendung von OligoNukleotidsonden spezifisch ist und geeignet für die voranHirnGewebe. Weiterhin Protokollverfahren, einschließlich in-situ-Hybridisierung, Signalerfassung und der Datenanalyse können innerhalb von zwei Tagen durchgeführt werden. <img alt="Abbildung 1" src="/files/ftp_upload/53165/53165fig1.jpg" /> Abbildung 1. Vergleich der frischen und gefrorenen Gewebe vorangestellt. Die Verfahren für frisch gefrorenes Hirn benutzt habe an anderer Stelle 5 beschrieben worden. Beachten Sie, dass die frische Gefrierschnitte wurden bei 20 &amp; mgr; m in der Dicke geschnitten, während der feste Gefrierschnitten bei 10 &amp; mgr; m. Die Oligonukleotidsonde wurde HTR2A, die die 5-HT 2A -Rezeptor-mRNA im Hypothalamus hybridisiert. Solide Pfeile zeigen Zellen, die HTR2A mRNA-Moleküle in der frisch gefroren (A) und dem Präfix Gewebe (B). Offene Pfeile, keine HTR2A mRNA-Molekülen detektiert. Bar: 20 &amp; mgr; m. Verwenden von NIHImageJ Analyse wurde HTR2A gezählt. Zahl der HTR2A zählt, sind keine Unterschiede zwischen frischen und Para-Präfix gefrorenen Gewebe (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53165/53165fig2.jpg" /> Abbildung 2. Ein nackter Perspektive von Hybridisierungssignale. Die Gewebe wurden mit Paraformaldehyd in tief narkotisierten Ratten zuvor mit Kochsalzlösung (SAL) und 3,4-methylenedioxymethamphetamine (MDMA) behandelt vorangestellt. Die weiß gestrichelten Linien die Region mit Oligonukleotidsonden hybridisiert. Die rote Farbe bezeichnet mRNA-Signale mit dem bloßen Auge. Keine roten Signal beobachtet, während Sie das dapB-Sonde, um Abschnitte des SAL-vorbehandelt (A) oder MDMA-vorbehandelten Ratten hybridisiert (B </strong&gt;). Im Gegensatz dazu werden über Infrarotsignale homogen im Hypothalamus Regionen verteilt während der Verwendung der Sonde PPIB (CD). Interessant ist, rote Signale von der HTR2A Sonde hergestellt werden, sind nur in einigen Regionen (EF; Pfeile zeigen die Hypothalamus-Bereich). Stereotaktischen Koordinaten: -3,50 -3,80 ~ relativ zum Bregma. 3V, die dritte Kammer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/53165/53165fig3.jpg" /> Abbildung 3. Eine lichtmikroskopische Ansicht der Hybridisierungssignale. Die Gewebe wurden mit Paraformaldehyd in tief narkotisierten Ratten zuvor mit Kochsalzlösung (SAL) und 3,4-methylenedioxymethamphetamine (MDMA) behandelt vorangestellt. Oligonukleotidsonden dapB (AB), ppiB (CD) und HTR2A (EF). (Bars: 200 &amp; mgr; m) Hybridisierungssignale werden als rote Punkte mit Objektivvergrößerung Leistungen von 4 × identifiziert. Bis 20 x (Einschub Mikrofotografien) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 4" src="/files/ftp_upload/53165/53165fig4.jpg" /> Abbildung 4. Auswirkung auf den Hypothalamus HTR2A Expression bei Ratten zuvor mit MDMA behandelt. Die Proben wurden in Duplikaten dreimal getestet und wiederholt. Daten werden als Mittelwert ± SEM in Prozent der SAL Gruppe (insgesamt 8 Ratten in dieser Studie verwendet) ausgedrückt. * P &lt;0,05 vs. die SAL-Gruppe unter Verwendung von Student t-Test. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><t<html> d&gt; </td><td> Frische </td><td></td><td colspan="2"> Para-Präfix ein </td></tr><tr><td></td><td> SAL b </td><td></td><td> SAL b </td><td> MDMA c </td></tr><tr><td> Ziel-Sonde (HTR2A) </td><td> √ </td><td></td><td> √ </td><td> √ </td></tr><tr><td> Positive Sonde (PPIB) </td><td> - </td><td></td><td> √ </td><td> √ </td></tr><tr><td> Negative Sonde (dapB) </td><td> - </td><td></td><td> √ </td><td> √ </td></tr></tbody></table> √, zeigt die Gruppe mit Oligonukleotid-Sonde, die in dieser Studie untersucht. -, Nicht untersucht; a, enthält jede Gruppe 4 verschiedene Tieren; b, die Gruppe intraperitoneal mit physischen Kochsalzlösung vorbehandelt (SAL; 0,9% NaCl); c, intraperitoneal mit 10 mg / kg MDMA vorbehandelt. jove_content &quot;&gt; Tabelle 1 Experimentelles Design

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.

Schnelle<em&gt; In-situ-</em&gt; Hybridisierung unter Verwendung von Oligonukleotidsonden auf Paraformaldehyd-Präfix Gehirn von Ratten mit Serotonin-Syndrom

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

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

8.8K Aufrufe.  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.

Serie: 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.

Doppel-Fluoreszenz- In situ Hybridisierung in Fresh Hirnschnitten

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

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Neurowissenschaften

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: Mit dem Theta-Burst-Protokoll zu Mechasnism der Plastizität in Individuen sich mit Fragile X-Syndrom und Autismus

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.

Die Kombination lipophiler Farbstoff, In situ Hybridisierung, Immunhistochemie und Histologie

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

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Schnelle Bestimmung der Wärmeleistung und nozizeptiven Grenzwerts bei diabetischen Ratten

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

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.

Komplette Spinal Cord Injury and Brain Dissection Protokoll für spätere Wholemount<em&gt; In-situ</em&gt; Hybridisierung in Larven Seeneunauge

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.

Nachweis von Axonally lokalisierten mRNAs in Gehirnschnitten mittels hochauflösender<em&gt; In-situ-</em&gt; Hybridization

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.

Die Kombination von Doppel-Fluoreszenz<em&gt; In Situ</em&gt; Die Hybridisierung mit Immunmarkierung für Nachweis der Expression von drei Gene in Mäusehirnschnitten

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

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Aufzeichnung von EEG in frei bewegten neonatalen Ratten mit einer neuartigen Methode

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

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.

Lokalisierung von Odorant-Rezeptor-Genen in Locust-Antennen durch RNA<em&gt; In Situ</em&gt; Hybridisierung

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.

Induktion von diffusen axonalen Hirnverletzungen bei Ratten basierend auf Rotationsbeschleunigung

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 ...