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>
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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...

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 zuer...

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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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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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 ...

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 ...

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

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

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

Quantifizierung von Endosom- und Lysosom-Motilen in kultivierten Neuronen mit fluoreszierenden Sonden

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

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

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

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

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

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 ...

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.

Kombination von Multiplex-Fluoreszenz-In-situ-Hybridisierung mit fluoreszierender Immunhistochemie an frischen, gefrorenen oder fixierten Mäusehirnschnitten

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

Methods Article

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