Questo studio è stato sostenuto dalla concessione NIH (R15DA029863), la concessione Florida Atlantic University di ricerca triennale (M30014) e la Scuola di Medicina Veterinaria assegno di ricerca Ross University. Vorremmo ringraziare il National Institute on Drug Abuse (Rockville, MD) per la fornitura di (±) 3,4-metilene (± MDMA) per questo lavoro.

Procedure per l&#39;uso degli animali descritti di seguito sono stati approvati dalla cura e l&#39;uso Comitato istituzionale animali (IACUC) Ross University School di Medicina Veterinaria e Florida Atlantic University. Anche se sono necessarie tecniche sterili e guanti, l&#39;ambiente privo di RNasi non è necessario durante l&#39;utilizzo di questo protocollo. 1. Preparazione <ol><li> Preparazione dei tessuti e sezionamento <ol><li> Assegnare i ratti a caso in tre gruppi: fresco / salina (SAL; 0,9% NaCl), il prefisso / SAL e prefissati / 3,4-metilene (MDMA; vedi dettagli nella tabella 1). </li><li> Somministrare tre dosi di 1 ml / kg SAL e 10 mg / kg per via intraperitoneale MDMA, rispettivamente, ai gruppi SAL e MDMA a intervalli 2 ore. Lasciare gli animali a sopravvivere il trattamento per 6 giorni. </li><li> Il giorno 7, somministrare 100 mg / kg di ketamina in combinazione di 5 mg / kg per via intraperitoneale xilazina ai ratti per una anestesia profonda (cioè, perdita di lampeggio corneale e tail pizzico riflessi). </li><li> Per il gruppo fresco / SAL, decapitate rapidamente ratti e rimuovere il cervello per il test del cervello fresco (vedi dettagli nel riferimento 5). </li><li> Per i gruppi / MDMA prefissati / SAL e prefissati, effettuare un taglio lungo lo sterno circa 10 -12 cm e quindi esporre l&#39;estremità dello sterno. </li><li> Tenere l&#39;estremità dello sterno con una pinza emostatica e tagliare il diaframma lateralmente da entrambi i lati con forbici affilate e poi tagliare verso l&#39;alto attraverso le costole e paralleli ai polmoni. </li><li> Con una mano, tenere la punta ventrale del cuore con piccole pinze. Con l&#39;altra mano, perforare il ventricolo sinistro con una G siringa 18 (notare il lato dell&#39;adattatore dell&#39;ago è collegato con un tubo flessibile di silicio a forma di Y per SAL e paraformaldeide contenitori ghiacciate e pompe peristaltiche, vedere il riferimento 9). </li><li> Accendere la pompa SAL al livello medio della portata (~ 5 ml / min) e forare l&#39;atrio destro con la pinza per consentire la fuoriuscita di ritorno circlamento. Mantenere SAL perfusione per 30 min. </li><li> Accendere la pompa paraformaldeide al livello medio e consentire il ghiacciata 4% paraformaldeide per defluire in all&#39;animale per 30 min. </li><li> Rimuovere strumento perfusione e decapitare il animali. Fare un postero tagliare la linea mediana del cranio, e poi sbattere il cranio per esporre la cavità del cranio 10. </li><li> Rimuovere il cervello con una spatola dalla cavità cranio e posizionare il cervello in una provetta da centrifuga da 50 ml contenente 25 ml di 4% paraformaldeide. Mantenere il cervello all&#39;interno del tubo-paraformaldeide contenente 4 ºC frigorifero per 24 ore. </li><li> Spostare il cervello in una provetta da centrifuga da 50 ml contenente 25 ml di soluzione di saccarosio al 30% a 4 ° C per 3 giorni. Infine, conservare il cervello in sacchetti di plastica a -80 ° C fino all&#39;uso. </li><li> Montare il cervello su un mandrino con i media embedding e congelare i media incorporamento contenenti cervello in un criostato ad una temperatura di -26 ° C. Tagliare il congelato frescon cervello in 20 sezioni micron e il cervello paraformaldeide-preceduti in sezioni di 10 micron. Trasferire la sezione di un vetrino da microscopio RT toccando la slitta alla sezione. </li><li> Asciugare a temperatura ambiente per 4 ore. Conservare i vetrini sezione in un sacchetto stretto sigillato a -80 ° C fino all&#39;uso. </li></ol></li><li> Disidratazione <ol><li> Prendere diapositive dal -80 ° C freezer e assegnare sezioni in una delle tre prove: HT2A, ppiB (controllo positivo) o dapB (controllo negativo); marchio e le sezioni di etichette con una penna a sfera (non usare una matita). Immergere i vetrini in 100% di alcol a temperatura ambiente per 5 minuti. Asciugare i vetrini per 5 minuti in una cappa aspirante. </li></ol></li><li> Pretrattamento <ol><li> Pipettare 20 ml di pretrattamento-1 (reagente inattivazione della fosfatasi alcalina), alle sezioni su vetrini. Porre i vetrini su una cremagliera in un vassoio di umidità (notare che il vassoio è coperta con un coperchio per mantenere una elevata umidità per prevenire evaporazione dal solutio reazionen). </li><li> Agitare delicatamente il vassoio di umidità su un agitatore orizzontale a bassa velocità per 10 min. Lavare i vetrini due volte con acqua bidistillata (DDH 2 O) per 2 min. Immergere il vetrino in un becher da 200 ml contenente 150 ml del pretrattamento-2 reagente (rottura dei legami trasversali indotte da fissazione paraformaldeide). Lessare i vetrini per un totale di 5 min a 100 ° C (ripristino indotta dal calore della struttura dell&#39;RNA 11). </li><li> Lavare i vetrini due volte con DDH 2 O per 2 min. Porre i vetrini in 100% di alcol per 5 min. Essiccare i vetrini per 5 min in cappa. Utilizzare una penna idrofobico per disegnare un cerchio attorno all&#39;area selezionata nella sezione di tessuto (ad esempio, strutture non corticali, tra talamo e ipotalamo come delineate nella figura 2). </li><li> Pipettare 10 ml di pretrattamento-3 reagente (aumenti di penetrazione della sonda) per ogni area selezionata, e coprire il vassoio di umidità con il coperchio. Posizionare il vassoio nel forno di ibridazioneequipaggiato con lo shaker orizzontale e impostare la temperatura del forno a 40 ° C; scuotere delicatamente il cassetto per 30 min. Lavare i vetrini due volte con DDH 2 O per 2 min. </li></ol></li></ol> 2. Ibridazione e Amplificazione <ol><li> Pipettare 10 ml di HTR2A, ppiB e dapB reagenti sonda, rispettivamente, sulle aree selezionate. Coprire il vassoio di umidità con il coperchio per evitare l&#39;evaporazione del reagente. Agitare delicatamente l&#39;agitatore orizzontale impostato a bassa velocità. Incubare i vetrini a 40 ° C in forno per 2 ore. </li><li> Lavare i vetrini due volte con un tampone di lavaggio per 2 min. Pipettare 10 ml di amplificazione-1 reattivo su ogni area selezionata, agitare e incubare i vetrini a 40 ° C per 30 min. </li><li> Lavare i vetrini due volte con un tampone di lavaggio per 2 min. Pipettare 10 ml di reagente di amplificazione-2 per ogni area selezionata, agitare e incubare i vetrini a 40 ° C per 15 minuti. </li><li> Lavare i vetrini due volte with un tampone di lavaggio per 2 min. Pipettare 10 ml di amplificazione-3 reattivo su ogni area selezionata, agitare e incubare i vetrini a 40 ° C per 30 min. </li><li> Lavare i vetrini due volte con un tampone di lavaggio per 2 min. Pipettare 10 ml di reagente di amplificazione-4 per ogni area selezionata, agitare e incubare i vetrini a 40 ° C per 15 minuti. </li><li> Lavare i vetrini due volte con un tampone di lavaggio per 2 min. Pipettare 10 ml di reagente di amplificazione-5 per ogni area selezionata, agitare e incubare i vetrini a temperatura ambiente per 30 min. </li><li> Lavare i vetrini due volte con un tampone di lavaggio per 2 min. Pipettare 10 ml di reagente di amplificazione-6 su ogni area selezionata, agitare e incubare i vetrini a temperatura ambiente per 15 min. Lavare i vetrini due volte con un tampone di lavaggio per 2 min. </li></ol> 3. Rilevamento segnale <ol><li> Pipettare 10 ml di reagente Red a ciascuna area selezionata (Si noti che il reagente è una miscela di rosso-B e Red-A con un rapporto 1:60 e deve essere utilizzato imediatamente). Posizionare il vetrino nel cassetto umidità sulla agitatore orizzontale a temperatura ambiente e agitare delicatamente a bassa velocità per 10 min. </li><li> Lavare i vetrini due volte con (DDH 2 O) per 10 min. Essiccare i vetrini a 60 ° C in forno per 15 min. Porre i vetrini in xilene sotto la cappa per 5 min. Dispensare 20 ml di mezzo di montaggio a base di xilene, su ogni area selezionata e coprire con un coprioggetto. </li></ol> 4. Acquisizione Immagine <ol><li> Prendere microfotografie digitali delle aree selezionate (ad esempio, ipotalamo, le figure 1 e 3) con un ingrandimento di 4 × 20 × e lenti dell&#39;obiettivo. Salvare il file di immagine. </li></ol> Analisi Particle 5. Immagine <ol><li> Trascinare e rilasciare il file immagine nella finestra di ImageJ principale. Vai alla barra dei menu e selezionare &#39;Immagine&#39;, successiva selezionare &#39;Tipo&#39; dal menu a discesa, quindi selezionare &#39;8-bit&#39;. </li><li> Seleziona &#39;Regola&#39;, quindi selezionare &#39;Threshold &#39;per aprire la finestra di dialogo. Regolare il valore barra inferiore della finestra di dialogo in modo che il fondo indesiderato viene rimosso. Vai alla barra dei menu e selezionare &#39;Analizza&#39;, quindi selezionare &#39;Analizza Particelle&#39; per aprire una seconda finestra di dialogo. </li><li> Impostare la dimensione delle particelle a 10, prossimo selezionare &#39;Mostra delinea&#39; e selezionare &#39;Mostra risultati &quot;e&quot; scatole Riassumere&#39;. Infine, fare clic su &#39;OK&#39; per visualizzare i dati delle particelle nelle finestre di dialogo. </li></ol> 6. Foglio di calcolo <ol><li> Inserire i dati nel foglio Excel e la media dei numeri di particelle nel gruppo SAL come linea di base. Calcolare il livello percentuale di ogni sezione e fare un grafico di conseguenza (Figura 4). </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

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.

Usando le sonde oligonucleotidi RNA (&lt;25 nt), ibridazione può essere rilevato come punti rossi nelle cellule dell&#39;ipotalamo preparate dal cervello paraformaldeide-prefissato e freschi congelati. Le molecole di mRNA HTR2A sono presenti in alcune cellule (indicati dalle frecce solidi), ma non altri (frecce aperti). Abbiamo osservato che non vi è alcuna differenza tra la paraformaldeide-prefissato e tessuti freschi congelati (Figura 1). Un ibridazione successo può essere valutato per pri...

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.

Rapido<em&gt; In Situ</em&gt; Ibridazione utilizzando oligonucleotidi sonde su Paraformaldeide prefissa cervello di ratti con Sindrome serotoninergica

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 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.

Doppia fluorescenza In situ</emIbridazione> nelle sezioni cervello fresco

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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Neuroscienze

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: Utilizzo del Theta Burst protocollo Esplora Mechasnism di plasticità in individui con Sindrome X Fragile e autismo

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.

La combinazione di coloranti lipofili, In situ Ibridazione, Immunoistochimica e Istologia

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.

Completa lesioni del midollo spinale e del cervello dissezione Protocollo per Wholemount successiva<em&gt; In Situ</em&gt; Ibridazione in larvale 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 ...

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.

Rilevazione di assoni localizzato mRNA nelle sezioni del cervello Utilizzando ad alta risoluzione<em&gt; In Situ</em&gt; Ibridazione

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.

La combinazione di doppio Fluorescenza<em&gt; In Situ</em&gt; Ibridazione con Immunolabelling per il rilevamento della Manifestazione di tre geni nel cervello di topo Sezioni

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.

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati ​​utilizzando sonde fluorescenti

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.

Localizzazione dei geni del recettore degli odoranti nell&#39;antenna di Locust da RNA<em&gt; In Situ</em&gt; Ibridazione

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.

Induzione di lesioni cerebrali assonali diffuse nei ratti in base all'accelerazione rotazionale

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.

Combinazione dell'ibridazione in situ a fluorescenza multiplex con immunoistochimica fluorescente su sezioni cerebrali di topo fresche, congelate o fisse

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