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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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><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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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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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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicina

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn: um método fluorescente rápido e imparcial para detectar alterações na densidade da sinapse

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Bioquímica

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 ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identificação de Receptor de dopamina D1-alfa dentro roedores núcleo Accumbens por uma inovador RNA In Situ tecnologia da deteção

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Usando a fluorescência near-infrared e a exploração de alta resolução para medir a expressão da proteína no cérebro do roedor

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combinando Hibridização In Situ por Fluorescência Multiplex com Imunohistoquímica Fluorescente em Seções Cerebrais de Camundongos Frescos Congelados ou Fixos

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

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Source: Shokry, I. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53165">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

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