Este estudo foi apoiado pela concessão NIH (R15DA029863), a concessão Florida Atlantic University iniciação científica (M30014) e da Escola de Medicina Veterinária bolsa de investigação da Universidade Ross. Gostaríamos de agradecer ao Instituto Nacional sobre Abuso de Drogas (Rockville, MD) para fornecer (±) 3,4-metilenodioximetanfetamina (MDMA ±) para este trabalho.

Procedimentos de uso de animais descritos abaixo foram aprovados pelo o cuidado e uso Comitê Institucional Animal (IACUC) da Escola de Medicina Veterinária da Universidade Ross e Florida Atlantic University. Embora são necessárias técnicas e luvas estéreis, o ambiente livre de RNase não é necessário ao usar este protocolo. 1. Preparação <ol><li> Preparação de tecidos e de seccionamento <ol><li> Atribuir ratos aleatoriamente em três grupos: fresca / salina (SAL; 0,9% NaCl), prefixados / SAL e prefixados / 3,4-metilenodioximetanfetamina (MDMA; veja detalhes na Tabela 1). </li><li> Administrar três doses de 1 ml / kg de Sal e de 10 mg / kg por via intraperitoneal MDMA, respectivamente, para os grupos SAL e MDMA no intervalo de 2 h. Permitir que os animais sobrevivem ao tratamento durante 6 dias. </li><li> No dia 7, administrar 100 mg / kg de cetamina em combinação de 5 mg / kg de xilazina intraperitonealmente aos ratos para uma anestesia profunda (ou seja, perda de piscar da córnea e TAil beliscar reflexos). </li><li> Para o grupo fresco / SAL, decapitar ratos e remover rapidamente o cérebro para o teste de cérebro fresco (ver detalhes na referência 5). </li><li> Para os grupos / MDMA prefixados / SAL e prefixadas, faça um corte ao longo do esterno de cerca de 10 -12 cm e, em seguida, expor a extremidade do esterno. </li><li> Segurar o final do esterno com uma pinça hemostática e cortar o diafragma lateralmente em ambos os lados com uma tesoura afiada e em seguida cortado para cima entre as nervuras e paralelo para os pulmões. </li><li> Com uma mão, segure a ponta ventral do coração com pequenas pinças. Com a outra mão, perfurar o ventrículo esquerdo com 18 G agulha de seringa (observe o lado do adaptador da agulha está conectada com uma mangueira de silicone em forma de Y para o Sal e paraformaldeído recipientes geladas e bombas peristálticas, ver a referência 9). </li><li> Ligar a bomba de SAL ao nível médio do caudal (~ 5 ml / min) e punção do átrio direito com a pinça para permitir o escape de retorno circlação. Manter SAL perfusão durante 30 minutos. </li><li> Ligar a bomba de paraformaldeído a nível médio e permitir que o paraformaldeído a 4% em gelo para perfundir o animal durante 30 min. </li><li> Remover instrumento perfusão e decapitar o animal. Adicione uma póstero corte através da linha média do crânio, e, em seguida, a aba do crânio para expor a cavidade do crânio 10. </li><li> Remover o cérebro com uma espátula a partir da cavidade do crânio e colocar o cérebro em um tubo de centrífuga de 50 ml contendo 25 ml de paraformaldeído a 4%. Mantenha o cérebro dentro do tubo contendo paraformaldeído a 4 ºC geladeira por 24 horas. </li><li> Mover o cérebro para um tubo de centrífuga de 50 ml contendo 25 ml de solução de sacarose a 30% a 4 ° C durante 3 dias. Finalmente, armazenar o cérebro dentro de sacos de plástico a -80 ° C até utilização. </li><li> Monte o cérebro em um mandril com a mídia de incorporação e congelar os meios de comunicação incorporação contendo cérebro em um criostato à temperatura de -26 ºC. Corte o congelou frescoN cérebro em 20 um secções do cérebro e paraformaldeído prefixado em secções de 10 um. Transfira a seção para uma lâmina de microscópio RT tocando o slide para a seção. </li><li> Ar seco à TA durante 4 h. Armazenar as lâminas secção de um saco estanque selado a -80 ° C até utilização. </li></ol></li><li> Desidratação <ol><li> Tome slides da freezer -80 ° C e atribuir seções em um dos três testes: HT2A, PPIB (controle positivo) ou dapB (controle negativo); marca e seções de etiqueta com uma caneta esferográfica (não use um lápis). Submerge lâminas em 100% de álcool à temperatura ambiente durante 5 min. Secam-se as lâminas durante 5 min numa hotte. </li></ol></li><li> Pré-tratamento <ol><li> Pipetar 20 ul de reagente de Pré-tratamento (1-inactivação de fosfatase alcalina) para as secções em lâminas. Colocar as lâminas em um trilho da cremalheira num tabuleiro de humidade (note que o tabuleiro é coberto com uma tampa para manter uma elevada humidade para evitar a evaporação a partir da reacção solution). </li><li> Agite suavemente a bandeja de umidade num agitador horizontal a uma velocidade baixa para 10 min. Lavar as lâminas duas vezes com água bidestilada (ddH2O) durante 2 min. Mergulhe o slide em uma proveta de 200 ml contendo 150 ml da pré-tratamento-2 reagente (quebra das ligações cruzadas induzidas pela fixação paraformaldeído). Ferver as lâminas por um total de 5 min a 100 ° C (restauração induzida pelo calor da estrutura de ARN 11). </li><li> Lavar as lâminas duas vezes com ddH2O durante 2 min. Colocar as lâminas em álcool a 100% durante 5 min. Secam-se as lâminas durante 5 min no exaustor de fumos. Utilize uma caneta hidrofóbica para desenhar um círculo em torno da área seleccionada na secção de tecido (por exemplo, estruturas não-corticais, incluindo tálamo e hipotálamo, conforme descrito na Figura 2). </li><li> Pipeta 10 ml de reagente pré-tratamento-3 (aumentos na penetração de sonda) para cada área selecionada, e cobrir a bandeja de umidade com a tampa. Coloque a bandeja no forno de hibridaçãoequipado com o agitador horizontal e ajustar a temperatura do forno a 40 ° C; agitar suavemente a bandeja para 30 min. Lavar as lâminas duas vezes com ddH2O durante 2 min. </li></ol></li></ol> 2. A hibridação e amplificação <ol><li> Pipeta 10 ml de HTR2A, PPIB e dapB reagentes de sonda, respectivamente, nas áreas selecionadas. Cobrir a bandeja de humidade com a tampa para evitar a evaporação do reagente. Agite no agitador horizontal definida em uma velocidade baixa. Incubar as lâminas a 40 ° C no forno durante 2 horas. </li><li> Lavar as lâminas duas vezes com um tampão de lavagem durante 2 min. Pipetar 10 ul do reagente de amplificação-1 em cada zona seleccionada, agitar e incubar as lâminas a 40 ° C durante 30 min. </li><li> Lavar as lâminas duas vezes com um tampão de lavagem durante 2 min. Pipetar 10 ul de reagente de amplificação-2 em cada zona seleccionada, agitar e incubar as lâminas a 40 ° C durante 15 min. </li><li> Lavar as lâminas duas vezes wom um tampão de lavagem por 2 min. Pipetar 10 ul de reagente de amplificação-3 em cada zona seleccionada, agitar e incubar as lâminas a 40 ° C durante 30 min. </li><li> Lavar as lâminas duas vezes com um tampão de lavagem durante 2 min. Pipetar 10 ul da amplificação 4-reagente sobre cada zona seleccionada, agitar e incubar as lâminas a 40 ° C durante 15 min. </li><li> Lavar as lâminas duas vezes com um tampão de lavagem durante 2 min. Pipetar 10 ul da amplificação 5-reagente sobre cada zona seleccionada, agitar e incubar as lâminas à temperatura ambiente durante 30 min. </li><li> Lavar as lâminas duas vezes com um tampão de lavagem durante 2 min. Pipetar 10 ul de reagente de amplificação-6 em cada zona seleccionada, agitar e incubar as lâminas à temperatura ambiente durante 15 min. Lavar as lâminas duas vezes com um tampão de lavagem durante 2 min. </li></ol> 3. Detecção de Sinais <ol><li> Pipetar 10 ul do reagente Vermelho para cada área seleccionada (Note-se que o reagente é uma mistura de vermelho e vermelho-B-A com uma proporção de 1:60 e deve ser utilizado immediately). Coloque o slide na bandeja de umidade no agitador horizontal à TA e agitar suavemente a uma velocidade baixa para 10 min. </li><li> Lavar as lâminas duas vezes com (ddH2O), durante 10 min. Seque as lâminas a 60 ºC no forno por 15 min. Colocar as lâminas em xileno sob o exaustor durante 5 min. Pipetar 20 ul de meio de montagem à base de xileno em cada área seleccionada e cobrir com uma lamela. </li></ol> Captura 4. Imagem <ol><li> Tome microfotografias digitais das áreas selecionadas (por exemplo, o hipotálamo, Figuras 1 e 3) com uma ampliação de 4 × e 20 × lentes objetivas. Salve o arquivo de imagem. </li></ol> Análise de Partículas 5. Imagem <ol><li> Arraste e solte o arquivo de imagem para a janela ImageJ principal. Vá para a barra de menu e selecione &#39;Imagem&#39;, próxima escolha &#39;Tipo&#39; a partir do menu drop-down e selecione &#39;8-bit&#39;. </li><li> Selecione &#39;Ajustar&#39;, em seguida, selecione &#39;Threshold &#39;para abrir a caixa de diálogo. Ajuste o valor da barra inferior da caixa de diálogo para que o fundo não desejado é removido. Vá para a barra de menu e selecione &quot;Analisar&quot;, em seguida, selecione &quot;Analisar Partículas &#39;para abrir uma segunda caixa de diálogo. </li><li> Defina o tamanho de partícula de 10, ao lado selecione &quot;Show apresenta &#39;e selecione&#39; Mostrar resultados&quot; e &quot;caixas Resumir&quot;. Por fim, clique em &#39;OK&#39; para ver os dados de partículas nas caixas de diálogo. </li></ol> 6. Planilha de Cálculo <ol><li> Entre os dados para a folha de Excel e calcular a média dos números de partículas no grupo SAL como uma linha de base. Calcula-se a percentagem do nível de cada secção e fazer um gráfico em conformidade (Figura 4). </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+and+cellular+distribution+of+serotonin+5-hydroxytryptamine2a+receptor+mRNA+in+the+nucleus+accumbens,+olfactory+tubercle,+and+caudate+putamen+of+the+rat.">Regional and cellular distribution of serotonin 5-hydroxytryptamine2a receptor mRNA in the nucleus accumbens, olfactory tubercle, and caudate putamen of the rat.</a> J Comp Neuro. 389, 1-11 (1997).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+region-specific+alterations+of+5-HT2A+and+5-HT2C+receptors+in+serotonin+transporter+knockout+mice.">Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice.</a> J Neuroche. 84, 1256-1265 (2003).</li><li>Horner, K. A., Gilbert, Y. E., Noble, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+5-HT2A+receptor+mRNA+expression+following+withdrawal+from+a+chronic+escalating+dose+regimen+of+D-amphetamine.">Differential regulation of 5-HT2A receptor mRNA expression following withdrawal from a chronic escalating dose regimen of D-amphetamine.</a> Brain Re. 1390, 10-20 (2011).</li><li>Mocci, G., Jimenez-Sanchez, L., Adell, A., Cortes, R., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+5-HT2A+receptors+in+prefrontal+cortex+pyramidal+neurons+projecting+to+nucleus+accumbens.+Potential+relevance+for+atypical+antipsychotic+action.">Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. 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Os autores declaram que não têm interesses financeiros concorrentes.

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 as sondas de ARN de oligonucleótidos (&lt;25 NT), a hibridação pode ser detectada como pontos vermelhos em células hipotalâmicas preparados a partir dos cérebros congelados prefixado-paraformaldeído e frescas. As moléculas de mRNA HTR2A estão presentes em algumas células (indicadas pelas setas sólidas), mas não outros (setas abertas). Observou-se que não existe qualquer diferença entre o os tecidos congelados frescos-prefixo e paraformaldeído (Figura 1). Uma hibridização bem sucedida pode ser avaliada primeiro a olho nu (Figura 2). Descobrimos que a área seleccionada (marcados com círculos) hibridadas com a sonda dapB não mostram sinal visível de um a olho nu (Figuras 2A-B). Em contraste, a área hibridado com a sonda PPIB mostrou sinais homogeneamente distribuídas por toda a região examinada (Figuras 2C-D). Enquanto no teste HTR2A, os sinais estão presentes em certos núcleos (indicado pelas setas; <strong&gt; Figuras 2E-F), embora toda a área circulada foi hibridado com a sonda HTR2A. Curiosamente, os sinais em lâminas MDMA foram relativamente fraco em comparação com lâminas SAL. Detalhes substanciais de sinais de hibridação pode ser avaliada por um instrumento microscópica. Não há nenhum ponto vermelho (Figura 3A-B) nas células hibridizadas com a sonda dapB enquanto que os sinais pode ser visto ao longo do campo microscópico hibridado com a sonda PPIB (Figura 3C-D). Os sinais HTR2A estão localizados principalmente em certos núcleos do hipotálamo (Figura 3E-F). Observou-se uma redução nos sinais HTR2A nos tecidos cerebrais previamente tratados com MDMA sistémica a 10 mg / kg, que pode ser determinada quantitativamente utilizando o ImageJ análise. Como mostrado na Figura 4, encontramos uma redução de 20% na expressão HTR2A. Em conclusão, nossos resultados mostraram que o uso de oligosondas nucleotídicas é específico, e adequado para o tecido cerebral prefixado. Além disso, os procedimentos de protocolo, incluindo a hibridação in situ, análise de dados e detecção de sinal pode ser concluída dentro de dois dias. <img alt="figura 1" src="/files/ftp_upload/53165/53165fig1.jpg" /> Figura 1. Comparação dos tecidos congelados frescos e prefixados. Os procedimentos utilizados para cérebros congelados frescos foram descritos em outro cinco. Note-se que as secções congeladas frescas foram cortadas a 20 um de espessura, enquanto a secções congeladas fixadas a 10 uM. A sonda oligonucleotídica foi HTR2A que hibrida o ARNm do receptor 5-HT 2A, no hipotálamo. Setas sólidas indicam células contendo moléculas de mRNA HTR2A no fresco congelado (A) e tecidos pré-fixados (B). Setas abertas, não há moléculas de mRNA HTR2A detectado. Barra: 20 um. Usando NIHAnálise ImageJ, HTR2A foi contado. Números de contagem HTR2A não são diferentes entre os tecidos congelados frescos e prefixado-paraformaldeído (C). <a href="https://www.jove.com/files/ftp_upload/53165/53165fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53165/53165fig2.jpg" /> Figura 2. Uma visão do olho nu de sinais de hibridação. Os tecidos foram prefixados com paraformaldeído em ratos profundamente anestesiados previamente tratados com solução salina (SAL) e 3,4-metilenodioximetanfetamina (MDMA). As linhas brancas indicam a tracejado-região hibridada com as sondas de oligonucleótidos. A cor vermelha indica sinais de mRNA a olho nu. Nenhum sinal vermelho é observada durante o uso da sonda hibridizada para secções dapB de SAL-pré-tratados (A) ou ratos pré-tratados com MDMA (B </strong&gt;). Em contraste, os sinais vermelhos estão distribuídas homogeneamente ao longo das regiões do hipotálamo, enquanto utilizando a sonda PPIB (CD). Curiosamente, os sinais vermelhos produzidos pela sonda HTR2A são apenas algumas regiões (EF; As setas indicam a área do hipotálamo). Coordenadas Estereotáxicas: -3,50 -3,80 ~ em relação ao bregma. 3V, o terceiro ventrículo. <a href="https://www.jove.com/files/ftp_upload/53165/53165fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53165/53165fig3.jpg" /> Figura 3. Uma visão microscópica luz de sinais de hibridação. Os tecidos foram prefixados com paraformaldeído em ratos profundamente anestesiados previamente tratados com solução salina (SAL) e 3,4-metilenodioximetanfetamina (MDMA). Sondas de oligonucleotídeos são dapB (AB), ppiB (CD) e HTR2A (EF). Sinais de hibridação são identificados como pontos vermelhos usando poderes de ampliação objetivas variando de 4 × (Bares: 200 m). 20 × (microfotografias inserir) <a href="https://www.jove.com/files/ftp_upload/53165/53165fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53165/53165fig4.jpg" /> Figura 4. Efeito sobre a expressão HTR2A hipotalâmico em ratos previamente tratados com MDMA. As amostras foram ensaiadas em duplicado e repetidas três vezes. Os dados são expressos como uma percentagem média ± epm do grupo SAL (um total de 8 ratos utilizados neste estudo). * P &lt;0,05 vs. o grupo SAL pelo teste t de Student. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><t<html> d&gt; </td><td> Fresco </td><td></td><td colspan="2"> Prefixado-paraformaldeído um </td></tr><tr><td></td><td> SAL b </td><td></td><td> SAL b </td><td> MDMA c </td></tr><tr><td> Sonda alvo (HTR2A) </td><td> √ </td><td></td><td> √ </td><td> √ </td></tr><tr><td> Sonda positivo (PPIB) </td><td> - </td><td></td><td> √ </td><td> √ </td></tr><tr><td> Sonda negativa (dapB) </td><td> - </td><td></td><td> √ </td><td> √ </td></tr></tbody></table> √, indica o grupo examinado com uma sonda de oligonucleótidos no presente estudo. -, Não examinou; um, cada grupo contém 4 animais diferentes; b, o grupo de pré-tratados por via intraperitoneal com solução salina física (SAL; 0,9% de NaCl); C, pré-tratados intraperitonealmente com 10 mg / kg de MDMA. jove_content &quot;&gt; Tabela 1. Projeto 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; A hibridação usando sondas Oligonucleotide no cérebro prefixado-paraformaldeído de ratos com síndrome serotoninérgica

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 visualizações.  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.

Vídeo: 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.

Fluorescência dupla In situ Hibridação nas Seções cérebro 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 ...

Neurociência

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: Usando o protocolo Theta-Burst para Explorar Mechasnism de Plasticidade em Indivíduos com Síndrome do X Frágil 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.

Combinando corante lipofílico, In situ Hibridação, imunohistoquímica e histologia

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.

Spinal Cord Injury completo e Cérebro Dissection Protocolo para Wholemount Subsequente<em&gt; In Situ</em&gt; Hibridização em larval da lampreia 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.

Detecção de Axonally localizada mRNAs em seções do cérebro Usando de alta resolução<em&gt; In Situ</em&gt; Hibridização

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.

Combinando Duplo Fluorescência<em&gt; In Situ</em&gt; A hibridação com imunomarcação para a detecção da expressão de três genes em secções do cérebro do rato

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.

Gravação de EEG em Ratos Neonatais de Movimentação Livre Usando um Novo Método

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.

Localização de Genes de Receptor de Odorantes em Antenas de Locustídeo por RNA<em&gt; In Situ</em&gt; Hibridação

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Imagem latente avançada da difusão no Hippocampus dos ratos com ferimento de cérebro traumático suave

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

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.

Indução de lesão cerebral axonal difusa em ratos com base na aceleração 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 ...