Agradecemos ao Dr. Toshio Terashima, da Universidade de Kobe, no Japão, para o ensino da técnica de cirurgia de laringe, e Dr. Lynn Enquist da Universidade de Princeton para o fornecimento de PRV-Bartha. A pesquisa foi financiado pelo NIH prêmio pioneiro DP1 OD000448 para Erich D. Jarvis e um prêmio NSF Graduate Research Fellowship para Gustavo Arriaga. Dados do adequadamente creditado trabalho anterior são usadas sob a PLoS ONE acesso aberto licença Creative Commons (CC-BY), em conformidade com as políticas editoriais da revista.

NOTA: Todos os procedimentos com animais foram revistos e aprovados pelo Comitê Animal Care Institucional e Uso da Universidade Duke. 1. Armazenando vírus da pseudo- <ol><li> Obtemos vírus vivo (PRV152) a partir do laboratório do Dr. Lynn Enquist na Universidade de Princeton com uma concentração de 1 x 10 9 pfu / m. O protocolo para gerar o vírus foi publicada 2. </li><li> Alíquota do vírus em 20 ul por tubo dentro de um gabinete de biossegurança BSL-2 e armazenar a -80 ° C em condições de biossegurança adequadas. </li><li> Descongelar uma alíquota de PRV imediatamente antes da injecção. </li></ol> 2. Preparação cirúrgica para injecções no músculo <ol><li> Induzir a anestesia geral por injeco intramuscular de cetamina-xilazina (100 mg / kg de cetamina, 10 mg / kg xilazina) e manter uma plano anestésico apropriado utilizando isofluorano. </li><li> Proteja as córneas com uma pomada oftálmica. </li><li> Prepare tele sítio cirúrgico de acordo com a técnica asséptica por aparar o cabelo e desinfecção do local cirúrgico com a alternância de scrubs de Betadine e álcool a 70% (mínimo de 3 ciclos). Certifique-se de usar campos estéreis para cobrir áreas cirúrgicas. Certifique-se a aderir às técnicas estéreis durante os procedimentos. </li><li> Faça uma incisão na pele e revelar o músculo de interesse. Por exemplo, para acessar a musculatura laríngea cricothyroid é necessário primeiro remover a parte sobreposta do músculo esterno. </li><li> Selar qualquer músculos transeccionados com adesivo de tecido Vetbond. </li></ol> 3. Injecção de PRV em músculo <ol><li> Carregue a 10 ul sistema de micro Nanofil com solução PRV recém-descongelado, coloque uma agulha de aço inoxidável 34 G, e montá-lo cuidadosamente no dispositivo estereotáxico. </li><li> Lentamente limpar o espaço morto e verifique se solução saia a ponta micro. Descarte fluido como resíduos bioharzard. </li><li> Usando um microp estereotáxicodispositivo ositioning coloque cuidadosamente a ponta da microsseringa no músculo de interesse e lentamente encher o músculo até um ligeiro inchaço é visível. A taxa de injecção variará dependendo do tamanho do músculo e o volume a ser injectado. Por exemplo, de cinco injecções de 200 nl (1 mL no total) a uma taxa de 4 nl / s deve ser feito para além de 1 min no mesmo local para preencher o músculo crico. Mova a seringa para o próximo músculo de interesse (cricoaritenóideo lateral, neste caso) e repita o procedimento de injeção. Apenas perfurar cada músculo uma vez. </li><li> Retrair o micro depois de cinco minutos. </li><li> Selar a ruptura na fáscia usando adesivo de tecido Vetbond. </li><li> Depois de todas as injecções foram concluídos, fechar a ferida utilizando adesivo de tecido Vetbond. Dependendo de suas diretrizes locais institional uso de animais, suturas ou clipes ferida pode ser usado. </li><li> Monitorar o animal até decúbito esternal e fornecer analgesia, comida, água e cuidados como exigido por ynossas diretrizes de uso de animais institucionais. </li><li> Após o tempo de sobrevivência determinados experimentalmente (neste caso, 90 h para rotular 2a ordem neurónios corticais), sacrifício do animal por dose excessiva de pentobarbital e perfundir transcardíaca com solução salina a 0,9%, seguido por 4% de formaldeído em PBS a 0,1 M. </li><li> Retire e pós-fixar o cérebro em formaldeído 4% durante 24 horas. </li><li> Cryoprotect o cérebro em tampão de fosfato contendo 30% de sacarose pelo menos durante 48 horas. </li></ol> 4. detecção imunoquímica de eGFP <ol><li> Cortar secções de 40 um em um micrótomo deslizante e salvar seções flutuantes em 0,1 M PBS. Cortes mais finos, por exemplo de 30 um, pode ser utilizado quando a coloração secções montadas. </li><li> Extingue-se secções durante 30 minutos em 0,3% de H 2 O 2 em PBS protegidas da luz. </li><li> Bloquear antigénios não específicos nas secções durante 30 minutos em PBS contendo 0,3% de Tween 20 com soro de cabra normal a partir do kit Vectastain Elite (VE kit). </li><li&gt; Incubar secções bloqueados durante 3,5 horas em coelho anti-EGFP (1: 1.000), à TA. </li><li> Lave os cortes três vezes em PBS durante 5 min, em seguida incubar durante 1 h em anticorpo secundário de cabra anti-coelho a partir do kit de VE, à TA. </li><li> Preparar a solução ABC do VE kit de acordo com as instruções do fabricante. </li><li> Lave os cortes três vezes em PBS durante 5 min, em seguida, eles reagem, durante 1 h em solução ABC do kit de VE, à TA. </li><li> Lave os cortes três vezes em tampão de fosfato durante 10 min, e desenvolver durante 8 minutos em tampão de fosfato, pH 7,4, contendo 0,05% de DAB (3, 3&#39;-diaminobenzidina) e 0,015% de H 2 O 2. </li><li> Seções de montagem em lâminas SuperFrost Bonés e desidratar-los através de uma série álcool graduado (70%, 95%, 100% e 100% durante 5 minutos cada). </li><li> Desmarque as secções através de duas lavagens xileno (5 min cada) e lamela as lâminas com Permount meio de montagem. </li><li> Imagem as seções montadas em um microscópio. O produto da reacção DAB inside as células devem aparecer marrom. Imagens digitalizadas podem ser cor invertida para realçar processos finas. </li></ol> 5. Preparação cirúrgica para a injeção de Tracers em regiões do cérebro Descoberto por PRV Tracing <ol><li> Induzir a anestesia geral por injeco intramuscular de cetamina-xilazina (100 mg / kg de cetamina, 10 mg / kg xilazina) e manter uma plano anestésico apropriado utilizando isofluorano. </li><li> Proteja as córneas com uma pomada oftálmica. </li><li> Fixe a cabeça em um quadro estereotáxico apropriado. </li><li> Prepare o local cirúrgico de acordo com a técnica asséptica por aparar o cabelo e desinfecção do local com a alternância de scrubs de Betadine e álcool a 70% (mínimo de 3 ciclos). </li><li> Faça uma incisão no couro cabeludo e retrair a pele sobre a região do cérebro de interesse. </li><li> Realize uma pequena craniotomia nas coordenadas estereotáxica adequadas. Por exemplo, as coordenadas para córtex motor laryngeally conectado identificado por PRV rastreamento em umn rato adulto são 1,2 milímetros lateral e 0,2 mm rostral para Bregma. </li></ol> 6. Injecção da biotinilados Dextran Aminas em Cérebro <ol><li> Prepare a 7,5% biotinilados aminas dextrano (BDA) por dissolução de 25 mg de BDA (MW 10.000) em 333 ul de solução salina estéril. </li><li> Carregar o sistema micropipeta Nanoject II com solução BDA suficiente para as injeções planejadas. </li><li> Lentamente limpar o espaço morto e verificar que a solução é sair da ponta da micropipeta. </li><li> Usando um dispositivo estereotáxico microposicionamento cuidadosamente abaixe a ponta da micropipeta para a região do cérebro de juros e injetar lentamente BDA. Ajustar a velocidade de injecção, dependendo do tamanho da região a ser marcado. Por exemplo, 12 injeções de 4,6 nl deve ser fez em quatro locais diferentes 0,2 milímetros distante (sentido rostro-caudal) para cobrir o córtex motor laryngeally conectado em ratos adultos. O volume final de injecção deve ser ajustada de acordo com o tamanho da região do cérebro de interesse, Tendo em mente que a etiqueta pode difundir a partir do núcleo de injecção por difusão através do tecido cerebral e ao longo dos processos de neurónios marcados. </li><li> Selar a craniotomia com cimento dental, e fechar o ferimento no couro cabeludo usando adesivo de tecido Vetbond. </li><li> Monitorar o animal até decúbito esternal e fornecer analgesia, comida, água e cuidados como exigido por suas diretrizes de uso de animais institucionais. </li></ol> 7. Injecção da toxina da cólera Subunidade b no músculo <ol><li> Prepare 1% Toxina da Cólera subunidade b (CTB) por dissolução de 1 mg de CTB em 100 ul de solução salina estéril. </li><li> Seis dias após a injecção BDA no cérebro, realizar a preparação cirúrgica, como acima descrito para injecções PRV no músculo. </li><li> Carregue a 10 ul sistema de micro Nanofil com solução CTb, anexar uma agulha de aço inoxidável 34 G, e cuidadosamente montá-lo no dispositivo estereotáxico. </li><li> Execute as microinjeções no músculo (s) de interesse como anteriormente descrito para PRV. </li><li> Monitorar o animal até decúbito esternal e fornecer analgesia, comida, água e cuidados como exigido por suas diretrizes de uso de animais institucionais. </li><li> Três dias após a injecção de CTB, sacrifício do animal por dose excessiva de pentobarbital e perfundir transcardíaca com solução salina a 0,9%, seguido por 4% de formaldeído em PBS a 0,1 M. </li><li> Retire e pós-fixar o cérebro em formaldeído 4% durante 24 horas. </li><li> Cryoprotect o cérebro em tampão de fosfato contendo 30% de sacarose pelo menos durante 48 horas. </li></ol> 8. Detecção de BDA e CTb nas mesmas seções <ol><li> Cortar secções de 40 um em um micrótomo e salvar seções flutuantes em 0,1 M PBS. </li><li> Extingue-se secções durante 30 minutos em 0,3% de H 2 O 2 em PBS protegidas da luz. </li><li> Preparar a solução ABC do VE kit de acordo com as instruções do fabricante. </li><li> Lave os cortes três vezes em PBS durante 5 min, em seguida, eles reagem, durante 1 h em solução ABC à TA. </li&gt; <li> Lave os cortes três vezes em tampão de fosfato durante 10 min, e desenvolver durante 8 minutos em tampão de fosfato, pH 7,4, contendo 0,05% de DAB (3, 3&#39;-diaminobenzidina), 0,015% de H 2 O 2, e 0,05% de cloreto de níquel. O produto da reacção DAB no interior das células deve aparecer preto. </li><li> Secções de blocos de 30 min em PBS contendo 0,3% de Tween 20 com soro normal de coelho a partir do kit Vectastain Elite. </li><li> Incubar secções bloqueados durante 2 horas em cabra anti-CTB (1: 10.000), à TA. </li><li> Lave os cortes três vezes em PBS durante 5 min, depois incubar durante 1 h em anticorpo secundário de coelho anti-cabra a partir do kit de VE, à TA. </li><li> Preparar a solução ABC do VE kit de acordo com as instruções do fabricante. </li><li> Lave os cortes três vezes em PBS durante 5 min, em seguida, eles reagem durante 30 min em solução ABC à TA. </li><li> Lave os cortes três vezes em tampão de fosfato durante 10 min, e para desenvolver até 8 min em tampão de fosfato, pH 7,4, contendo 0,05% de DAB (3, 3&#39;-diaminobenzidine) e 0,015% de H 2 O 2. O produto da reacção de DAB no interior das células deve aparecer castanho. </li><li> Seções de montagem em lâminas SuperFrost Bonés e desidratar-los através de uma série álcool graduado (70%, 95%, 100% e 100% durante 5 minutos cada). </li><li> Desmarque as secções através de duas lavagens xileno (5 min cada) e lamela as lâminas com Permount meio de montagem. </li><li> Imagem as seções montadas em um microscópio. </li></ol>

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No conflicts of interest declared.

Há uma série de questões que devem ser levadas em consideração ao planejar uma experiência usando PRV152 4,21. Mais importante ainda, vírus da pseudo é letal. Como mencionado anteriormente, grandes macacos, incluindo os seres humanos não são suscetíveis à infecção, mas cuidado apropriado deve ser exercido para proteger outros animais. Ratos adultos normalmente sobreviver cinco a sete dias após a inoculação com a estirpe atenuada PRV152. Portanto, PRV152 não é adequado para experiências que...

Rastreamento transsináptica tornou-se uma poderosa ferramenta utilizada para analisar eferentes centrais que regulam alvos periféricos através de circuitos multi-sinápticos. Esta abordagem tem sido o mais amplamente utilizado no cérebro de roedor, utilizando o vírus da pseudo-raiva de agentes patogénicos suína (PRV), especialmente a estirpe atenuada de PRV-Bartha descrita pela primeira vez em 1961 12. A seguir, apresentamos um protocolo para a identificação da representação cortical motora dos músculos específicos ou grupos musculares utilizando uma estirpe do vírus da pseudo-raiva recombinantes (PRV152) que expressa o reforçada proteína verde fluorescente (eGFP) gene repórter 2. O método descrito explora o comportamento de vírus neurotrópico, que produzem progénie infeccioso que sinapses cruzadas para infectar outros neurónios dentro de um circuito funcional 3,4,13. PRV152, que é isogênico com PRV-Bartha, única atravessa sinapses retrogradamente através da seqüência hierárquica das conexões sinápticas longe do local de infecção 3,5 &lt;/ sup&gt;. Ao controlar com precisão o local periférico de infecção é possível limitar a entrada do vírus para o cérebro através de um subconjunto específico de neurónios motores. À medida que o vírus infecta sequencialmente cadeias de neurónios ligados, o padrão resultante de sinal eGFP em todo o cérebro, em seguida resolver a rede de neurónios que estão ligados às células inicialmente infectadas. Uma vantagem adicional da utilização de vírus, para um rastreio neural é a amplificação da proteína repórter (eGFP, neste caso) no interior das células infectadas. Esta amplificação de sinal fornece um nível de sensibilidade que permite a detecção de projeções ainda esparsas. Por exemplo, uma projecção a partir de córtex motor esparso vibrissa para os neurónios motores faciais que controlam os bigodes foi encontrada em ratos utilizando viralmente expressa a proteína fluorescente verde 14; Estudos anteriores não conseguiram encontrar essa projeção usando marcadores clássicos sem amplificação do gene repórter 11,15. Infelizmente, Muitos vectores virais de rastreio, tal como o utilizado no estudo citado, não atravessam sinapses, limitando assim o seu uso para o rastreamento de circuitos multi-sinápticos. Ao apresentar vantagens distintas para identificar a rede de células que participam em um circuito de motor, a natureza distribuída da transsináptica rastreamento com PRV-152 torna a interpretação de conexões específicas dentro do circuito difícil. Por isso, apresentamos um método simples para a validação de conexões específicas dentro de circuitos identificados usando PRV-152 by-dupla rotulagem utilizando aminas biotinilados dextrano (BDA) e toxina da cólera subunidade b (CTB). O uso combinado do BDA e CTb é uma abordagem bem estabelecido para rastreamento de conexões entre conjuntos específicos de neurônios 6-8,11. Quando utilizados em conjunto, estes dois marcadores podem ser visualizados na mesma secção utilizando um DAB duas cores (3, 3&#39;-diaminobenzidina) Procedimento 16. Alto peso molecular BDA (BDA10kDa) foi selecionada para este protocolo porque yfields rotulagem detalhada dos processos neuronais 6,7,9. As vantagens adicionais da BDA10kDa incluem o seguinte: ele é preferencialmente transportados na direcção anterógrada 6-8; que podem ser entregues por iontoforese ou de injecção a pressão 6-8; que pode ser visualizada por uma simples HRP (ABC) Procedimento 17 biotinilada-avidina; e pode ser fotografada pela luz ou microscopia eletrônica 6,7,18. Detecção imunoquímica da CTB com peroxidase e DAB foi escolhido para a marcação retrógrada de neurónios motores, pois é eficaz em processos celulares, incluindo revelando dendritos distais 10,19. Nós recentemente utilizado esta abordagem para identificar a via motora vocal em camundongos e para revelar uma conexão escasso de córtex motor primário para os neurônios motores da laringe, que foi previamente assumidos estar ausente 20.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NanoFil Microinjection System</td>
			<td>World Precision Instruments</td>
			<td>IO-Kit</td>
			<td>34 gauge option</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>Leica</td>
			<td>SM2010 R</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>VetBond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane (Forane)</td>
			<td>Baxter&#160;</td>
			<td>1001936060</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine Swab Stick</td>
			<td>Cardinal Health</td>
			<td>2130-01</td>
			<td>200 count</td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated dextran amines</td>
			<td>Invitrogen</td>
			<td>D-1956</td>
			<td>10,000 MW</td>
		</tr>
		<tr>
			<td>Pseudorabies virus</td>
			<td>Laboratory of Dr. Lynn Enquist (Princeton University)</td>
			<td>PRV152</td>
			<td>Titer &#62; 1 x 107</td>
		</tr>
		<tr>
			<td>Anti-Cholera Toxin B Subunit (Goat)</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin B Subunit</td>
			<td>List Biological Laboratories</td>
			<td>103B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-eGFP</td>
			<td>Open Biosystems</td>
			<td>ABS4528</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;-diaminobenzidine</td>
			<td>Sigma-Aldrich</td>
			<td>D5905</td>
			<td>10 mg tablets</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>H3410</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Ketamine HCl &#38; Xylazine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>K4138</td>
			<td>80 mg/mL &#38; 6 mg/mL</td>
		</tr>
		<tr>
			<td>Nickel chloride</td>
			<td>Sigma-Aldrich</td>
			<td>339350</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>P3619</td>
			<td>1.0 M; pH 7.4</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X; pH 7.4</td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>50 mg/mL dose</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>Histological grade</td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6101 (rabbit); PK-6105 (goat)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optixcare opthalmic ointment</td>
			<td>Vet Depot</td>
			<td>1017992</td>
			<td></td>
		</tr>
	</tbody>
</table>

transsynaptic tracing from peripheral targets with pseudorabies virus followed by cholera toxin and biotinylated dextran amines double labeling

Rastreamento transsináptica tornou-se uma poderosa ferramenta utilizada para analisar eferentes centrais que regulam alvos periféricos através de circuitos multi-sinápticos. Esta abordagem tem sido mais amplamente utilizado no cérebro, utilizando o vírus da pseudo-raiva de agentes patogénicos suína (PRV) 1. O PRV não infectar grandes macacos, incluindo os seres humanos, por isso é mais comumente usado em estudos sobre pequenos mamíferos, especialmente roedores. O PRV152 pseudorabies estirpe expressa a proteína fluorescente (eGFP) gene repórter verde reforçada e só atravessa sinapses funcionais retrogradamente através da seqüência hierárquica das conexões sinápticas de distância do local da infecção 2,3. Outras estirpes de PRV têm propriedades microbiológicas distintas e pode ser transportado em ambas as direcções (PRV-Becker e PRV-Kaplan) 4,5. Este protocolo tratará exclusivamente com PRV152. Ao fornecer o vírus num sítio periférico, tais como o músculo, é possível limitar a entrada do vírus em tele cérebro através de um conjunto específico de neurônios. O padrão resultante de sinal eGFP em todo o cérebro, em seguida, resolve os neurónios que são ligados às células inicialmente infectadas. Como a natureza distribuída de rastreamento transsináptica com o vírus da pseudo-raiva faz interpretar conexões específicas dentro de uma rede identificada difícil, apresentamos um método sensível e confiável que emprega aminas biotinilados dextrano (BDA) e toxina da cólera subunidade b (CTB) para confirmar as conexões entre as células identificadas usando PRV152. Detecção imunoquímica de BDA e CTB e com peroxidase de DAB (3, 3&#39;-diaminobenzidina) foi escolhida, porque eles são eficazes em processos celulares, incluindo revelando dendritos distais 6-11.

A coloração para eGFP deve começar mostrando sinal fraco em neurônios motores primários de aproximadamente 72 horas após a injecção de PRV152 em músculo. A replicação e transporte transsináptica de vírus são titer- e 4 dependente do tempo. Aproximadamente 90 horas após a injecção, coloração eGFP irá revelar sinal robusto em células infectadas 2ª ordem. Tempos mais longos de sobrevivência irá revelar 3 e células de ordem superior, mas o tempo de sobrevivência sã...

Watch this Scientific Journal Video about Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling at JoVE.com

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsináptica Rastreamento de Alvos periféricos com vírus da pseudo-Seguido por cólera toxina e Biotinilado Dextran aminas Duplo Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

transsynaptic-tracing-from-peripheral-targets-with-pseudorabies-virus

Research

JoVE Journal

Neuroscience

12.6K visualizações.  Duke University Medical Center. Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Vídeo: Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

No útero Eletroporação seguido por Cultura Neuronal primário para estudar a função do gene no subconjunto dos neurônios corticais

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Neurociência

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Marcação retrógrada de células ganglionares da retina em Zebrafish Adulto com corantes fluorescentes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

A detecção do genoma e Transcrições de um vírus DNA persistente em tecidos neuronais por fluorescentes Em situ Hibridização Combinado com Imunocoloração

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Rápido genotipagem dos animais Seguido por Estabelecer culturas primárias de neurônios do cérebro

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Condicional Genetic transsináptica Tracing no Embryonic do cérebro do rato

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

A imuno-histoquímica e Rotulagem múltipla com anticorpos da espécie hospedeira Mesmas para Estudar Adulto Hippocampal Neurogênese

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Neuronal Tracing em explantes cérebro guiadas a laser

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

Melhorar a aplicação de alto Peso Molecular Biotinylated dextrano amina para projeção Thalamocortical rastreamento em ratos

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Viver a imagem, seguida por uma única célula de rastreamento para biologia celular de Monitor e a progressão da linhagem de várias populações neurais

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Isolamento e cultivo de progenitores neurais seguido de imunoprecipitação de cromatina da marcação de dimetilação da lisina 9 da histona 3

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...