NIH R01MH084894 participou no financiamento deste estudo. Nós gostaríamos de agradecer Drs. Yasmin Hurd e Scott Russo no Mount Sinai School of Medicine para a doação de ratos eo uso de suas instalações de cirurgia e comportamento durante as filmagens deste trabalho.

NOTA: Todos os procedimentos para a criação e dos cuidados dos animais foram realizados de acordo com o regulamento da Comissão de Cuidados e Uso de Animais Institucional (IACUC) de Icahn Faculdade de Medicina Mount Sinai. Certifique-se de usar luvas estéreis durante todo o procedimento. 1. Preparação de drogas e vírus <ol><li> Preparação da droga <ol><li> Prepare 15,0 ml de cetamina / xilazina anestésico por dissolução de 1,35 ml de 100 mg / ml de cetamina e 0,75 ml de 20 mg / ml de xilazina em 12,9 mL de 0,9% solução salina. Misture bem solução. </li></ol></li><li> Preparação de vírus <ol><li> Clone a mGlu2 e mGlu2ΔTM4N constrói num vector bicistrónico vírus do herpes simplex (HSV) seguindo protocolos convencionais anteriormente descrito 6. Compactar as partículas virais, como anteriormente descrito 6,13,14. A substituição de resíduos de Ala-677 4,40, Ala-681 4,44 e 4,48 em Ala685 mGlu2 para Ser686 4,40, PhE690 4,44 e Gli-694 4,48 em mGlu3 (HA-mGlu2ΔTM4N) foram descritos anteriormente 6. NOTA: foi previamente demonstrado que a construção quimérica HA-mGlu2ΔTM4N é expresso na membrana plasmática intacta com G dependente de proteína de sinalização 6. </li><li> Armazenar vectores virais em -80 ° C quando não estiver em uso. Descongelar vector viral em gelo, e, em seguida, aliquotar em alíquotas de 10 ul. Para o procedimento cirúrgico, manter em gelo. </li></ol></li></ol> 2. Cirurgia <ol><li> Preparação cirurgia <ol><li> Pesar do mouse e injetar rato com dose apropriada de cocktail de cetamina / xilazina (para detalhes, ver 1.1.1). </li><li> Verifique mouse para ver se devidamente anestesiados, espremer pé e cauda de resposta à dor, se não for possível obter uma resposta, o mouse está devidamente anestesiados. </li><li> Raspar a cabeça do rato a partir da base do crânio até a ponta do nariz usando cortadores. Aplique gel oftalmológico à af ratoterward para prevenir a cegueira do mouse. </li><li> Carregue cada seringa no quadro estereotáxico. Depois incline porção perpendicular de cada braço da armação estereotáxica de modo que eles estão 10 graus fora do normal. Certifique-se de que os braços são inclinadas, de modo que as agulhas estão enfrentando uns aos outros. </li><li> Limpar cada seringa, enchendo a agulha com 70% de etanol. Encher a agulha de, pelo menos, três vezes para garantir que a seringa é limpa. </li><li> Uma vez que a agulha foi limpa, lave a agulha, preenchendo a agulha com dupla H2O destilada Uma vez liberado, encha cada agulha com 1,3 l de dupla H2O destilada Torcer o êmbolo da seringa para libertar 0,3 ul de dupla H2O destilada Se contas de água na ponta da agulha, cuidadosamente limpar a água. Se nada sai da seringa, empurre o êmbolo completamente para baixo e, em seguida, repita a limpeza da seringa. </li><li> Depois de encher com água, em seguida, puxe a seringa encher a seringa com0,5 l de ar. </li><li> Uma vez que o ar e a água está na seringa, cuidadosamente encher a seringa, com 1,3 ml de solução de vírus. Neste ponto a que o volume total da seringa é de 2,8 ul. Novamente torcer a ponta da seringa para libertar 0,3 mL de vírus. Se os grânulos de líquido na extremidade da agulha, cuidadosamente enxugar líquido. Se nada sai da seringa, empurre o êmbolo completamente para baixo e, em seguida, repita a limpeza da seringa. </li></ol></li><li> Cirurgia <ol><li> Anexar o mouse para o quadro estereotáxico, certificando-se de ajustar o quadro estereotáxico para que o crânio é plana e na horizontal. Aplicar povodine-iodo para o couro cabeludo exposto. Utilizando um bisturi, fazer uma incisão sagital na linha média do crânio dentro da área rapada exposta. Em seguida, anexar os grampos buret para a pele no local da incisão para se certificar de que o crânio permanece exposta. </li><li> Use H 2 O 2 a dissolver o periósteo para expor as suturas docrânio. Agora que o bregma e suturas são visíveis, não se esqueça de ajustar o quadro estereotáxico para se certificar de que o crânio é nível. </li><li> Alinhar as pontas das agulhas das seringas com a bregma e gravar as coordenadas do bregma. Calcular as coordenadas do que as agulhas vão ser inseridas. <ol><li> Para o plano Rostral-Cauldal (RC), adicionar 1,6 mm para os gravados RC coordenadas bregma (+1,6 da bregma). Para o plano dorsal-ventral (DV), subtrair 2,4 mm a partir das coordenadas bregma DV gravadas (-2,4 partir de bregma). </li><li> Finalmente, para o plano Medial Lateral (ML), adicionar 2,6 mm para os gravados ML coordenadas bregma (+2,6 da bregma). Para todas as coordenadas certifique-se de gravar ambas as coordenadas esquerda e direita, como esta é uma injeção bilateral. </li></ol></li><li> Traga as agulhas para as coordenadas desejadas. Marque os locais de onde as agulhas estão indo para ser inserido e com uma broca, perfurar as áreas marcadas. </li><li> Com um aplicador de ponta de algodão wipe afastado qualquer fragmento de sangue ou osso excesso. </li><li> Traga as agulhas para o crânio onde as pontas das agulhas estão em contacto a superfície do cérebro. Em seguida, abaixe as agulhas para as coordenadas desejadas lentamente abaixá-los. </li><li> Uma vez que as agulhas são as coordenadas desejadas, lentamente injectar o conteúdo da seringa, rodando o êmbolo da agulha de 0,1 ul por minuto ao longo de 5 minutos (no total 0,5 mL). </li><li> Depois de a injecção ter sido feita sair da seringa no córtex durante mais 5 min. </li></ol></li><li> Fechando Up / Cuidados <ol><li> Retirar as agulhas a partir do córtex de rato de forma constante e lentamente. Em seguida, retire o mouse do quadro estereotáxico. </li><li> Aplicar cianoacrilato (adesivo dérmico) para as abas de base de pele a partir da incisão com uma pinça e, em seguida, agarrar as abas de pele e colocá-los em conjunto. </li><li> Permitir que o cianoacrilato para secar. Coloque o rato na gaiola sobre uma almofada de aquecimento (almofada de aquecimento é opcional se o surgsuíte iCal é mantida sob RT de 37 ° C - caso contrário, não é necessário). Certifique-se de colocar o mouse sobre uma toalha de papel para se certificar de que a cama não adere ao local da cirurgia. </li><li> Dependendo da duração do processo cirúrgico, assegurar que os ratos é de anestesia dentro de 30 - 60 min após o procedimento. Após a cirurgia, o animal é colocado na sua própria gaiola e monitorizado até que se torne consciente antes de retornar ao seu ambiente para recuperar. Nenhum analgésicos são usados ​​no pós-operatório, porque podem alterar os resultados de nossos experimentos, modificando alguns dos caminhos bioquímicos cerebrais. No entanto, os animais são monitorizados até recuperarem da anestesia no dia da cirurgia, e pós-operação diariamente para sinais de infecção e avaliação de dor / desconforto. </li></ol></li></ol> Experiência 3. Cabeça Twitch Response <ol><li> Configuração <ol><li> Realizar todos os testes comportamentais 10:00 - 14:00, 2 - 3 dias após stereotaCTIC injecção de partículas virais. </li><li> Dissolve-se (±) -1- (2,5-dimetoxi-4-iodofenil) -2-aminopropano (DOI) em uma solução salina a 0,9% para 2,0 mg / kg. Também prepare uma solução salina a 0,9%. </li><li> Prepare uma gaiola (28 x 18 x 15 cm), sem qualquer roupa de cama e usando um tri-pod, ajustar a câmera para que a vista da câmera de vídeo está diretamente acima da gaiola. </li><li> Habituar os ratos ao ambiente durante pelo menos 4 horas antes do início da experiência. </li><li> Configurar uma câmera de vídeo para gravar a contração cabeça. </li></ol></li><li> Experimentar <ol><li> Posicione a câmera de modo que é diretamente sobre uma gaiola. Calibrar a câmara para que toda a gaiola está no campo de visão. </li><li> Pesar ratinho e rato injectar por via intraperitoneal com a dose apropriada de qualquer de 0,9% de solução salina ou DOI (0,01 ml / g). NOTA: Se um rato pesa 25 gramas, administrar a dose para um volume total de 0,25 ml. </li><li> Coloque cada mouse de volta em sua gaiola casa foR 10 min. Após 10 min, rato lugar no centro da gaiola vazio e validar que não existem pontos cegos no campo de visão da câmara. Pressione gravar na câmara de vídeo. Deixe o quarto. NOTA: Movimentos do mouse e várias respostas comportamentais nele (... A cabeça contração muscular, zero ouvido, etc Por favor, consulte a tabela de dados suplementar 1 de Gonzalez-Maeso et al 2007 para a lista completa de respostas comportamentais induzidas por DOI) 3, será gravado por 30 minutos. Portanto, é importante não existem pontos cegos no campo de visão gravada. </li><li> Após 30 min parar a gravação no mouse câmara de vídeo e lugar de volta na gaiola lar original. Repita esse processo para cada rato. </li></ol></li><li> revisão <ol><li> Tem cada revisão árbitro as fitas cegar para as condições experimentais de rato (ie., Os medicamentos utilizados durante cabeça contração experiência ou vírus utilizado durante a injeção intracraniana)). registrar manualmente cada cabeça contração througHout o vídeo. NOTA: Head-contração é definido como um movimento rápido balançando a cabeça conduzida por um mouse (vídeo suplementar). </li><li> Para cada rato, em média, a resposta HTR final os três totais dos árbitros cegos. Em seguida, agrupar esses valores por condição experimental e realizar análises estatísticas (ie., T-test ou ANOVA). </li></ol></li></ol>

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The authors have nothing to disclose.

Juntamente com os resultados anteriores em ratinhos KO-mGlu2 5, os resultados com mGlu2 e mGlu2 / mGlu3 construções quiméricas que não formam o complexo receptor 2A -mGlu2 5-HT, em células cultivadas sugerem que o 5-HT 2A -mGlu2 complexo receptor heteromérico em rato córtex frontal é necessário para induzir o comportamento cabeça de contração por agonistas dos receptores 5-HT2A alucinógenos semelhantes ao LSD. Uma limitação deste método é que ele não mede proximidade molecular próximo a um nível subcelular no tecido nativo. Além disso, existem vários pontos críticos a serem observados. Uma vez que os ratinhos são injectados com um vector viral de HSV, o período de tempo que as experiências para ser realizado a 2 - 4 dias após a injecção. A localização e a expressão dos vectores virais deve ser verificada com coloração por imunofluorescência de cérebro seccionada não mais de 4 dias após a injecção inicial. Os ratinhos em que as coordenadas não corresponderem ou não expressam o vector viral deveser excluídos a partir dos dados experimentais como eles não expressam a mGlu2 ou mGlu2ΔTM4N. Cuidados após a cirurgia estereotáxica também é crucial, como fechamento inadequado do ferimento na cabeça pode levar à infecção que pode causar problemas em ambos os experimentos in vivo e a imunofluorescência. Por último, após a injecção estereotáxica qualquer paradigma comportamental (. Campo aberto, alternando t-teste, etc) podem ser usados ​​desde que esteja dentro do 2 - 4 após a injecção de partículas virais. Mais uma vez, coordenadas e expressão deve ser confirmada por imunofluorescência. O conceito de que a função de GPCR como homo- e / ou heterómeros em células vivas está agora bem estabelecida 16-18. No entanto, apesar de alguns progressos, são necessários mais estudos para definir o papel exato (s) de complexos heteroméricos GPCR em todo modelos animais 19. Abordagens como BRET e / ou de imagens FRET para investigar a proximidade física proteína-proteína nos tecidos profundos de pequenos animaismodelos podem fornecer meios para melhorar a compreensão do papel funcional da heterómeros GPCR em indivíduos de 20 a viver. Usando a expressão de HSV-mediada de GPCRs nativa ou com proteínas transmembranares modificados, proporcionam um modelo in vivo para avaliar a função e interacção de GPCRs. Também são necessários mais estudos em modelos de roedores para examinar a estabilidade e tempo de vida (formação e dissociação) de heterómeros GPCR 21-23, rearranjos estruturais entre seus componentes 24 e acoplamento potencial de proteína G após internalização do receptor 25,26. Dado o papel fundamental de GPCRs na sinalização celular e função, parece provável que esta área pode conduzir a interessantes estudos básicos e translacionais. Embora mais estudos são necessários para caracterizar quantitativamente a co-localização ultra-estrutural de ambos os receptores no SNC humano e rato, juntamente com stu anteriormorre que demonstram de maneira convincente o eletrofisiológico, celular e respostas comportamentais induzidas por alucinógenos em modelos do rato são intrínsecos à 5-HT que expressam o receptor A2A neurônios piramidais corticais 3,27,28, os resultados obtidos usando a abordagem de expressão HSV-mediada descritos aqui sugerem que a formação heteromérica entre os receptores A2A e mGlu2 5-HT no córtex frontal de rato é necessário para a psicose comportamento semelhante a cabeça de contração induzida por alucinogénios a 5-HT 2A agonista do receptor de DOI. Expressão de mGlu2 HSV-mediada, mas não mGlu2ΔTM4N, em neurônios corticais frontais de ratos mGlu2-KO resgata o comportamento cabeça de contração induzida pela alucinógena 5-HT agonista do receptor A2A DOI. Esta ferramenta de translação pode ser vantajoso para estudos pré-clínicos para avaliar os fenótipos comportamentais de heterómeros GPCR.

Alucinógenos, como o LSD, a psilocibina e mescalina causar mudanças significativas na consciência humana, cognição e emoção 7-9. Inactivação de sinalização do receptor de serotonina 5-HT2A por abordagens genéticas ou farmacológicas quer marcadamente atenuado provoca respostas comportamentais ao alucinógenos em ambos os modelos de roedores e seres humanos 3,10 11. Embora alucinógenos ligar outros subtipos de receptores 8, o receptor 5-HT 2A é considerado como necessário para a actividade comportamental única destes produtos químicos. Grupo II receptores de glutamato metabotrópicos (ie., MGlu2 e mGlu3) têm sido alvo de considerável atenção sobre o mecanismo molecular de alucinógenos e seu papel fundamental subjacente a psicose 12. Anteriormente, foi demonstrado que os ratinhos com nenhuma expressão de proteína de mGlu2 (ratinhos de mGlu2-KO) são insensíveis aos efeitos celulares e comportamentais de Hallucinogens 5. Também tem sido sugerido que a 5-HT 2A e os receptores de mGlu2 formar um complexo heteromérico específico através do qual a serotonina e glutamato ligandos modular o padrão de acoplamento de proteínas G em células vivas 1,2. Estruturalmente, transmembranares (TM) domínios 4 e 5 do mGlu2 desempenham um papel fundamental na formação heteromérica com o receptor 5-HT 2A 5. Adicionalmente, investigação adicional demonstrou que três resíduos localizados na extremidade intracelular de TM4 de mGlu2 são necessários para formar o heterocomplexo do receptor A2A -mGlu2 5-HT 6 em células vivas. Com base nestes resultados observados em sistemas de expressão heterólogos, aqui descreve-se o uso da expressão de HSV-mediada de tipo selvagem de mGlu2 e mGlu2 / mGlu3 construções quiméricas no córtex frontal de ratos mGlu2-KO para testar se a formação heteromérica entre 5-HT 2A e mGlu2 é necessário para ocomportamento cabeça de contração induzida por agonistas dos receptores 5-HT2A alucinógenas.

<table border="0" cellpadding="0" cellspacing="0" width="2329">
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">mGlu2 bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td style="width:1716px;">mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mGlu2&#916;TM4N bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">xylazine&#160;</td>
			<td style="width:71px;">Lloyd</td>
			<td style="width:119px;">List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">ketamine&#160;</td>
			<td style="width:71px;">Vedco</td>
			<td style="width:119px;">KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ophthalmic gel</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC0550805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">burret clips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9268369</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Feather surgical blade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9032736</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Hydrogen Peroxide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>19-898-919&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton syringe</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14815203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton&#8482; Small Hub Removable Needles (33 Ga)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14816206</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cordless Micro Drill</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9089241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dermabond Dermal Adhesive</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC0690470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">(&#177;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>42203-78-1</td>
			<td>Dissolved in .9% saline solution to the concentration of 2.0 mg/kg</td>
		</tr>
	</tbody>
</table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

Constatações anteriores demonstram que a cabeça de contração resposta comportamental murino é confiável e robusta provocada por alucinógenos, e é ausente em 5-HT 2A ratos -Ko 3. Além disso, demonstrou-se que a resposta da cabeça de contração induzida por alucinogénios os agonistas de 5-HT 2A e DOI LSD foi significativamente diminuída em ratinhos KO mGlu2-5. No entanto, apesar de resultados anteriores demonstram de forma convincente que a 5-HT 2A e mGlu2 são montadas como um complexo heteromérico in vitro em células transfectadas 1,2,15, se este arranjo estrutural comporta-se como tal em ratos vivos permaneceu por resolver. Para compreender totalmente a função do receptor A 2A heterocomplexo -mGlu2 5-HT nos efeitos psicoactivos semelhantes induzidas por alucinogénios agonistas do receptor 5-HT 2A, a expressão de qualquer um ou de mGlu2 mGlu2ΔTM4N em córtex frontal de ratos mGlu2-KO para examinar se essa manipulation regula o comportamento. Os ratos receberam injeções corticais intra-frontal de HSV bicistrï¿½ica expressando a proteína fluorescente verde (GFP) e quer mGlu2 ou mGlu2ΔTM4N, ou GFP sozinho. Em primeiro lugar, confirmou-se que o vírus da sobre-expressa mGlu2 mGlu2ΔTM4N ou no córtex frontal do rato (Figuras 1A e 1B). Como demonstrado anteriormente 5, o comportamento da cabeça de contração induzida por DOI estava ausente em ratinhos mGlu2-KO injectados com o vector vazio de HSV-GFP. Notavelmente, a resposta da cabeça de contração induzida pela alucinogénios agonista de 5-HT 2A DOI foi resgatado em ratinhos mGlu2-KO que sobre-expressam de mGlu2, mas não mGlu2ΔTM4N, no córtex frontal, tal como comparada com a observada em animais que expressam a GFP (Figura 1C). Juntos, estes resultados sugerem que o complexo de receptor 2A -mGlu2 5-HT no córtex frontal é crítico para a regulação estados psicose semelhantes. Figura 1. Expressão de um receptor de mGlu2 como heterocomplexo. A expressão de um receptor de mGlu2 como com 5-HT 2A é necessário para o comportamento psicose semelhante induzida por drogas alucinogénicas. (A) Imagem representativa da expressão do transgene HSV-mediada em córtex frontal. HSV-mGlu2, que também expressa a GFP, foi injectado no córtex frontal, e a expressão da GFP foi revelado por imunocitoquímica, barra de escala, 200-um. (B) de HSV-mediada expressão do transgene no córtex frontal do rato de ratos mGlu2-KO, e reatividade anti-mGlu2 foi medida por Western Blotting. Especificidade do anticorpo primário contra o receptor de mGlu2 foi previamente confirmada em murganhos nocaute 6. os receptores de glutamato metabotrópicos são GPCRs que formam homodímeros ligados covalentemente. Medimos a imunorreactividade de mGlu2 como um monómero (100 kDa) 6. (C </strong&gt;) expressão mediada por vírus de mGlu2, mas não mGlu2ΔTM4N, no córtex frontal de ratos KO mGlu2- resgata significativamente a resposta da cabeça de contração induzida por alucinogénios o agonista de 5-HT2A DOI (n = 4 por grupo). *** P &lt;0,001; ns, não significativo; teste post hoc de Bonferroni de one-way ANOVA. As barras de erro representam SEM figura foi modificado a partir de Moreno et al (2012) 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/53717fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/HeadTwitchResponse.wmv" target="_blank">Suplementar vídeo 1. Cabeça Twitch Response. (Clique direito a download).</a> Camundongos CD-1 WT foram injetados com 2,0 mg / kg DOI e colocado em uma gaiola (parede apaguei entre as duas gaiolas) para provocar resposta cabeça de contração (comportamento suscitou após *).

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Este artigo descreve como para injetar vetores virais no córtex frontal do rato para testar ensaios comportamentais que exigem formação heteromérica GPCR.

HSV-Mediated expressão do transgene da quiméricos Construções para estudar a função comportamental de GPCR heterómeros em Ratos

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.2K visualizações.  Icahn School of Medicine at Mount Sinai.  Este artigo descreve como para injetar vetores virais no córtex frontal do rato para testar ensaios comportamentais que exigem formação heteromérica GPCR. 

Vídeo: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Um método geral para a avaliação de Incubação de Craving Sacarose em Ratos

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neurociência

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Aplicação local de Drogas para estudar a função do receptor nicotínico de acetilcolina no cérebro do rato Fatias

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro e análise dos ritmos circadianos em Running rodas Atividade em Roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Um Ensaio Comportamental simples para Teste de Função Visual em<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Avaliação de dendríticas Arborização no giro dentado do hipocampo em Ratos Região

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantificação de filamentosas actina (F-actina) puncta no Rat neurônios corticais

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

Um método de fmRI de respiração livre para estudar a função olfativa humana

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Expressão do Transgene de mediada por vírus adeno-associado em geneticamente definidos neurônios da medula espinhal

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

O poder do intervalo de Interstimulus para a avaliação do processamento Temporal em roedores

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Rotulagem Balística de Neurônios Piramidais em Fatias Cerebrais e na Cultura Celular Primária

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...