Este estudio fue apoyado por el NIH subvención (R15DA029863), la concesión de la Universidad Florida Atlantic investigación de pregrado (M30014) y la Escuela de Medicina Veterinaria de la beca de investigación de la Universidad de Ross. Nos gustaría dar las gracias al Instituto Nacional sobre el Abuso de Drogas (Rockville, MD) para proporcionar (±) 3,4-metilendioximetanfetamina (MDMA ±) para este trabajo.

Procedimientos de uso de animales se describen a continuación fueron aprobados por el Cuidado y Uso Comité Institucional Animal (IACUC) de la Facultad de Medicina Veterinaria de la Universidad de Ross y la Florida Atlantic University. Aunque se requieren técnicas y guantes estériles, el medio ambiente RNasa libre no es necesario durante el uso de este protocolo. 1. Preparación <ol><li> Preparación del tejido y seccionando <ol><li> Asignar ratas al azar en tres grupos: fresco / solución salina (SAL; 0,9% NaCl), el prefijo / SAL y el prefijo / 3,4-metilendioximetanfetamina (MDMA; véanse los detalles en el cuadro 1). </li><li> Administrar tres dosis de 1 ml / kg SAL y 10 mg / kg por vía intraperitoneal MDMA, respectivamente, a los grupos SAL y MDMA en el intervalo de 2 hr. Permitir a los animales para sobrevivir el tratamiento durante 6 días. </li><li> El día 7, administrar 100 mg / kg de ketamina en combinación de 5 mg / kg de xilazina por vía intraperitoneal a las ratas para una anestesia profunda (es decir, la pérdida de abrir y cerrar la córnea y tail pellizcar reflejos). </li><li> Para el grupo fresca / SAL, decapitar rápidamente ratas y retire el cerebro para la prueba cerebral fresco (ver detalles en la referencia 5). </li><li> Para los grupos / MDMA prefijo / SAL y prefijados, hacer un corte a lo largo del esternón unos 10 -12 cm y luego exponer la punta del esternón. </li><li> Mantenga el extremo del esternón con una pinza hemostática y cortar el diafragma lateralmente en ambos lados con unas tijeras afiladas y luego se corta hacia arriba a través de las costillas y paralelo a los pulmones. </li><li> Con una mano, sostenga la punta ventral del corazón con pequeñas pinzas. Con la otra mano, perforar el ventrículo izquierdo con una G aguja de la jeringa 18 (nótese el lado del adaptador de la aguja está conectado con una manguera de silicio en forma de Y a SAL y paraformaldehído contenedores enfriado con hielo y bombas peristálticas; ver la referencia 9). </li><li> Encienda la bomba de SAL en el nivel medio de la velocidad de flujo (~ 5 ml / min) y la punción de la aurícula derecha con las pinzas para permitir el escape de retorno circulación. Mantener SAL perfusión durante 30 min. </li><li> Encienda la bomba de paraformaldehído en el nivel medio y deje que el helado de paraformaldehído al 4% a perfundir el animal durante 30 minutos. </li><li> Retire instrumento perfusión y decapitar al animal. Hacer un posteroanterior cortar a través de la línea media del cráneo, y luego la solapa del cráneo para exponer la cavidad 10 del cráneo. </li><li> Retire el cerebro con una espátula de la cavidad del cráneo y el cerebro colocar en un tubo de centrífuga de 50 ml que contiene 25 ml de paraformaldehído al 4%. Mantener el cerebro dentro del tubo que contiene paraformaldehído-4 ºC en refrigerador durante 24 hr. </li><li> Mueva el cerebro en un tubo de centrífuga de 50 ml que contiene 25 ml de solución de sacarosa al 30% a 4 ºC durante 3 días. Por último, guarde el cerebro dentro de bolsas de plástico a -80 ° C hasta su uso. </li><li> Montar el cerebro en un plato con los medios de incrustación y congelar los medios de incrustación contienen cerebral en un criostato a una temperatura de -26 ºC. Cortar el congeló frescan cerebral en 20 micras secciones y el cerebro paraformaldehído-prefijo en secciones de 10 micras. La transferencia de la sección a un portaobjetos de microscopio RT tocando la diapositiva a la sección. </li><li> Aire seco a temperatura ambiente durante 4 hr. Guarde la sección diapositivas en una bolsa cerrada sellada a -80 ° C hasta su uso. </li></ol></li><li> Deshidratación <ol><li> Tome diapositivas de la -80 ° C congelador y asignar secciones en una de tres pruebas: HT2A, PPIB (control positivo) o dapB (control negativo); marca y las secciones de la etiqueta con un bolígrafo (no utilizan un lápiz). Sumergir los portaobjetos en 100% de alcohol a temperatura ambiente durante 5 min. Se secan los portaobjetos durante 5 minutos en una campana de humos. </li></ol></li><li> Pretratamiento <ol><li> Pipeta 20 l de reactivo de pretratamiento-1 (inactivación de la fosfatasa alcalina) a las secciones en las diapositivas. Colocar los portaobjetos en un carril de bastidor en una bandeja de humedad (nótese que la bandeja se cubre con una tapa para mantener una alta humedad para prevenir la evaporación de la reacción solution). </li><li> Agite suavemente la bandeja de la humedad en un agitador horizontal a una velocidad baja durante 10 minutos. Lavar los portaobjetos dos veces con agua doblemente destilada (ddH 2 O) durante 2 min. Sumergir el portaobjetos en un vaso de 200 ml que contiene 150 ml de la Pretratamiento-2 reactivo (ruptura de los enlaces cruzados inducidos por la fijación de paraformaldehído). Hervir las diapositivas para un total de 5 min a 100 ºC (restauración inducida por calor de la estructura del ARN 11). </li><li> Lavar los portaobjetos dos veces con ddH2O durante 2 min. Colocar los portaobjetos en 100% de alcohol durante 5 min. Secar los portaobjetos durante 5 minutos en la campana de humos. Use un lápiz hidrofóbico para dibujar un círculo alrededor del área seleccionada en la sección de tejido (por ejemplo, las estructuras no-corticales, incluyendo el tálamo y el hipotálamo como se indica en la figura 2). </li><li> Pipeta 10 l de la Pretratamiento-3 reactivo (aumentos en la penetración de la sonda) a cada área seleccionada, y cubrir la bandeja de humedad con la tapa. Colocar la bandeja en el horno de hibridaciónequipado con el agitador horizontal y fijar la temperatura del horno a 40 ºC; agite suavemente la bandeja durante 30 minutos. Lavar los portaobjetos dos veces con ddH2O durante 2 min. </li></ol></li></ol> 2. Hibridación y Amplificación <ol><li> Pipeta 10 l de HTR2A, PPIB y dapB reactivos de sonda, respectivamente, en las áreas seleccionadas. Cubrir la bandeja de la humedad con la tapa para evitar la evaporación del reactivo. Agite suavemente en el agitador horizontal fijado a una velocidad baja. Incubar los portaobjetos a 40 ºC en el horno durante 2 horas. </li><li> Lavar los portaobjetos dos veces con un tampón de lavado durante 2 min. Pipeta 10 l de la amplificación-1 reactivo en cada área seleccionada, agitar e incubar los portaobjetos a 40 ºC durante 30 minutos. </li><li> Lavar los portaobjetos dos veces con un tampón de lavado durante 2 min. Pipeta 10 l de reactivo de amplificación-2 en cada área seleccionada, agitar e incubar los portaobjetos a 40 ºC durante 15 minutos. </li><li> Lavar los portaobjetos dos veces won un tampón de lavado durante 2 min. Pipeta 10 l de reactivo de amplificación-3 en cada área seleccionada, agitar e incubar los portaobjetos a 40 ºC durante 30 minutos. </li><li> Lavar los portaobjetos dos veces con un tampón de lavado durante 2 min. Pipeta 10 l de la amplificación-4 de reactivo en cada área seleccionada, agitar e incubar los portaobjetos a 40 ºC durante 15 minutos. </li><li> Lavar los portaobjetos dos veces con un tampón de lavado durante 2 min. Pipeta 10 l de la amplificación-5 de reactivo en cada área seleccionada, agitar e incubar los portaobjetos a temperatura ambiente durante 30 min. </li><li> Lavar los portaobjetos dos veces con un tampón de lavado durante 2 min. Pipetear 10 l de la amplificación-6 reactivo en cada área seleccionada, agitar e incubar los portaobjetos a TA durante 15 min. Lavar los portaobjetos dos veces con un tampón de lavado durante 2 min. </li></ol> 3. Detección de la señal <ol><li> Pipeta 10 l de reactivo Roja para cada área seleccionada (Tenga en cuenta que el reactivo es una mezcla de rojo-B y Rojo-A en una proporción de 1:60 y se debe utilizar immediately). Colocar el portaobjetos en la bandeja de humedad en el agitador horizontal a temperatura ambiente y agitar suavemente a baja velocidad durante 10 minutos. </li><li> Lavar los portaobjetos dos veces con (ddH 2 O) durante 10 min. Secar los portaobjetos a 60 ºC en el horno durante 15 minutos. Colocar los portaobjetos en xileno bajo la campana de humos durante 5 min. Pipeta 20 l de los medios de montaje a base de xileno en cada área seleccionada y cubrir con un cubreobjetos. </li></ol> Capture 4. Imagen <ol><li> Tome microfotografías digitales de las áreas seleccionadas (por ejemplo, el hipotálamo, las Figuras 1 y 3) con un aumento de 4 × 20 × y lentes del objetivo. Guarde el archivo de imagen. </li></ol> Análisis de partículas 5. Imagen <ol><li> Arrastre y suelte el archivo de imagen en la ventana principal de ImageJ. Ir a la barra de menú y seleccione &#39;Imagen&#39;, al lado seleccione &#39;Tipo&#39; en el menú desplegable y seleccione &quot;8 bits&quot;. </li><li> Seleccione &#39;Ajuste&#39;, a continuación, seleccione &#39;Threshold &#39;para abrir el cuadro de diálogo. Ajuste el valor de la barra inferior del cuadro de diálogo de manera que se elimina el fondo no deseado. Ir a la barra de menú y seleccione &quot;Analizar&quot; y seleccione &quot;Analizar Partículas &#39;para abrir un segundo cuadro de diálogo. </li><li> Ajuste del tamaño de partícula en el 10, al lado seleccione &#39;Mostrar esboza &quot;y seleccione&quot; Mostrar resultados &quot;y&quot; cajas Resumir&#39;. Por último, haga clic en &#39;Aceptar&#39; para ver los datos de partículas en los cuadros de diálogo. </li></ol> Cálculo 6. Hoja de cálculo <ol><li> Introduzca los datos en la hoja de Excel y un promedio de la cantidad de partículas en el grupo SAL como línea de base. Calcule el nivel de porcentaje de cada sección y hacer una gráfica en consecuencia (Figura 4). </li></ol>

<ol>
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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. 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Los autores declaran que no tienen intereses financieros en competencia.

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.

Utilizando las sondas de ARN oligonucleótido (&lt;25 nt), la hibridación se puede detectar como puntos rojos en las células hipotalámicas preparados a partir de los cerebros congelados paraformaldehído prefijado y frescas. Las moléculas de ARNm HTR2A están presentes en algunas células (indicados por flechas continuas), pero no en otros (flechas abiertas). Hemos observado que no hay diferencia entre el paraformaldehído-prefijado y los tejidos congelados frescos (Figura 1). A la hibridación exitosa puede ser evaluado primero por el ojo desnudo (Figura 2). Se encontró que el área seleccionada (marcada con círculos) hibridado con la sonda dapB no mostró una señal visible al ojo desnudo (Figuras 2A-B). En contraste, el área hibridado con la sonda PPIB mostró señales homogéneamente distribuidos por toda la región examinada (Figuras 2C-D). Mientras que en la prueba HTR2A, las señales están presentes en ciertos núcleos (indicada por las flechas; <strong&gt; Figuras 2E-F), aunque toda el área de un círculo se ha hibridado con la sonda HTR2A. Curiosamente, las señales en las diapositivas de MDMA fueron relativamente débil en comparación con diapositivas SAL. Detalles sustanciales de las señales de hibridación pueden ser evaluados por un instrumento microscópico. No hay punto rojo (Figura 3A-B) en las células hibridadas con la sonda dapB mientras que las señales se puede ver en todo el campo microscópico se hibridaron con la sonda PPIB (Figura 3C-D). Las señales HTR2A se encuentran principalmente en ciertos núcleos del hipotálamo (Figura 3E-F). Se observó una reducción en las señales de HTR2A en los tejidos cerebrales previamente tratados con MDMA sistémica a 10 mg / kg, que se puede determinar cuantitativamente utilizando el análisis ImageJ. Como se muestra en la Figura 4, se encontró una reducción del 20% en la expresión HTR2A. En conclusión, nuestros resultados mostraron que el uso de oligosondas de nucleótidos es específico, y adecuado para el tejido cerebral prefijado. Además, los procedimientos de protocolo, incluyendo la hibridación in situ, la detección de señales y análisis de datos se pueden completar en dos días. <img alt="Figura 1" src="/files/ftp_upload/53165/53165fig1.jpg" /> Figura 1. Comparación de los tejidos congelados frescos y prefijados. Los procedimientos utilizados para los cerebros congelados frescos se han descrito en otra parte 5. Tenga en cuenta que los cortes congelados frescos se redujeron a 20 micras de espesor, mientras que el fijo secciones congeladas a las 10 micras. La sonda de oligonucleótido se hibrida HTR2A que el ARNm del receptor 5-HT 2A en el hipotálamo. Las flechas continuas indican las células que contienen moléculas de ARNm en el HTR2A fresco congelado (A) y tejidos con el prefijo (B). Las flechas abiertas, no hay moléculas de ARNm HTR2A detectados. Bar: 20 micras. El uso de los NIHAnálisis ImageJ, se contó HTR2A. El número de recuentos HTR2A no son diferentes entre los tejidos congelados frescos y paraformaldehído prefijada (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53165/53165fig2.jpg" /> Figura 2. Una vista a simple vista de las señales de hibridación. Los tejidos se prefija con paraformaldehído en ratas profundamente anestesiados previamente tratados con solución salina (SAL) y 3,4-metilendioximetanfetamina (MDMA). Las líneas y puntos blancos indican la región hibridada con sondas de oligonucleótidos. El color rojo indica señales de ARNm para el ojo desnudo. No se observa ninguna señal roja durante el uso de la sonda hibridada dapB a las secciones de SAL-pretratado (A) o ratas pretratadas con MDMA (B </strong&gt;). En contraste, las señales de color rojo se distribuyen homogéneamente a lo largo de las regiones hipotalámicas durante el uso de la sonda PPIB (CD). Curiosamente, las señales rojas producidas por la sonda HTR2A están sólo en algunas regiones (EF; Las flechas indican el área hipotalámica). Estereotáxica coordenadas: -3,50 ~ -3,80 relación al bregma. 3V, el tercer ventrículo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig2large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53165/53165fig3.jpg" /> Figura 3. Una vista microscópica luz de señales de hibridación. Los tejidos se prefija con paraformaldehído en ratas profundamente anestesiados previamente tratados con solución salina (SAL) y 3,4-metilendioximetanfetamina (MDMA). Las sondas de oligonucleótidos son dapB (AB), ppiB (CD) y HTR2A (EF). Las señales de hibridación se identifican como puntos rojos usando poderes de magnificación objetivos que van desde 4 × (Bares: 200 m). 20 × (microfotografías inserción) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53165/53165fig4.jpg" /> Figura 4. Efecto sobre la expresión hipotalámica HTR2A en ratas tratadas previamente con MDMA. Las muestras se ensayaron por duplicado y se repitieron tres veces. Los datos se expresan como un porcentaje media ± SEM del grupo SAL (un total de 8 ratas utilizadas en este estudio). * P &lt;0,05 vs. el grupo SAL mediante la prueba t de Student. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><t<html> d&gt; </td><td> Fresh </td><td></td><td colspan="2"> Paraformaldehído-prefijado un </td></tr><tr><td></td><td> SAL b </td><td></td><td> SAL b </td><td> MDMA c </td></tr><tr><td> Sonda Target (HTR2A) </td><td> √ </td><td></td><td> √ </td><td> √ </td></tr><tr><td> Sonda positiva (PPIB) </td><td> - </td><td></td><td> √ </td><td> √ </td></tr><tr><td> Sonda Negativo (dapB) </td><td> - </td><td></td><td> √ </td><td> √ </td></tr></tbody></table> √, indica el grupo examinó con sonda de oligonucleótido en este estudio. -, No examinada; una, cada grupo contiene 4 animales diferentes; b, el grupo tratado previamente por vía intraperitoneal con solución salina física (SAL; NaCl al 0,9%); c, pretratados por vía intraperitoneal con 10 mg / kg MDMA. jove_content &quot;&gt; Tabla 1. Diseño experimental

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.

Rápido<em&gt; In Situ</em&gt; La hibridación usando sondas de oligonucleótidos en Cerebro paraformaldehído-prefijo de ratas con síndrome de serotonina

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

rapid-situ-hybridization-using-oligonucleotide-probes-on

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 vistas.  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.

Doble fluorescencia In situ La hibridación en las secciones del cerebro 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 ...

Investigación

Neurociencias

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: Uso del protocolo Theta-Burst para explorar Mechasnism de plasticidad en los individuos con el síndrome de X frágil y el 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 combinación de colorantes lipofílicos, In situ</emHibridación>, inmunohistoquímica y la histología

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.

Determinación rápida del umbral nociceptivo térmico en ratas diabéticas

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.

Lesiones de la Médula Espinal completo y Protocolo Disección del cerebro por Wholemount posterior<em&gt; In Situ</em&gt; La hibridación en larvas lamprea de mar

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.

La detección de ARNm axón localizado en las secciones cerebrales Uso de alta resolución<em&gt; In Situ</em&gt; La hibridación

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 combinación de doble fluorescencia<em&gt; In Situ</em&gt; La hibridación con Immunolabelling para la detección de la expresión de tres genes en las secciones de cerebro de ratón

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.

Grabación de EEG en Ratas Neonatales que se mueven libremente usando un método nuevo

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.

Localización de Genes Receptor Odorante en Antenas Locustinas por RNA<em&gt; In Situ</em&gt; Hibridación

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.

Inducción de lesión cerebral axonal difusa en ratas basada en aceleración rotacional

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 ...