We thank Andrea Moran together with members of Lab Animal Service Facility (LASF) at Stowers Institute for their excellent support on animal husbandry and technical services. This work is supported by funding from Stowers Institute and the NIH (NIDCD 008003) to CRY. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Deafness and Other Communication Disorders or the National Institutes of Health. U.S. patent pending for the tetO-G-CaMP2 mice for Stowers Institute, CRY and LM.

1. Preparação da solução <ol><li> Prepare 10X R1, R2 10X e 10X soluções R3 acordo com a tabela. R1 <table border="1"><tbody><tr><td> Produtos químicos </td><td> MW (g / mol) </td><td> mM (1X) </td><td> Estoque de 10X (g / L) </td></tr><tr><td> NaCl </td><td> 58,44 </td><td> 125 </td><td> 73,05 </td></tr><tr><td> KCl </td><td> 74,55 </td><td> 2,5 </td><td> 1,86 </td></tr><tr><td> MgCl 2 </td><td> Um estoque M </td><td> 1 </td><td> 10 ml </td></tr><tr><td> CaCl 2 · 2H 2 O </td><td> 147,02 </td><td> 2 </td><td> 2,94 </td></tr><tr><td> NaH 2 PO 4 · H 2 O </td><td> 137,99 </td><td> 1,25 </td><td> 1,72 </td></tr></tbody></table> R2 <table border="1"><tbody><tr><td> Químico </td><td> MW (g / mol) </td><td> mM (1X) </td><td> Estoque de 10X (g / L) </td&gt; </tr><tr><td> NaHCO 3 </td><td> 84,01 </td><td> 25 </td><td> 21 </td></tr></tbody></table> R3 <table border="1"><tbody><tr><td> Produtos químicos </td><td> MW (g / mol) </td><td> mM (1X) </td><td> Estoque de 10X (g / L) </td></tr><tr><td> NaCl </td><td> 58,44 </td><td> 125 </td><td> 73,05 </td></tr><tr><td> KCl </td><td> 74,55 </td><td> 2,5 </td><td> 1,86 </td></tr><tr><td> MgCl 2 </td><td> Um estoque M </td><td> 2 </td><td> 20 ml </td></tr><tr><td> CaCl 2 · 2H 2 O </td><td> 147,02 </td><td> 2 </td><td> 2,94 </td></tr><tr><td> HEPES </td><td> Um estoque M </td><td> 5 </td><td> 50 ml </td></tr></tbody></table></li><li> Fazer 1 litro do mouse fluido cérebro-espinhal artificial (mACSF) no dia do experimento, misturando 100 ml de R1 10X e 100 ml de R2 10X em água destilada duas vezes (DDW) e add 1,8 g de dextrose (final 10 mm). Este método de preparação impede a precipitação de carbonato de cálcio em pH elevado. A osmolaridade do mACSF é de cerca de 310-315 mOsm / L. Ajustar a osmolaridade com dextrose ou água se necessário. Aerar a solução com Carboxygen de gás (95% de oxigênio e dióxido de carbono 5%) para pelo menos 30 minutos antes de ajustar o pH da solução para 7,2-7,4. </li><li> Fazer 1 litro de solução de Ringer no dia do experimento, misturando 100 ml de R2 10X e 100 ml de R3 10X no DDW e adicionar 1,8 g de dextrose (10 mM). A osmolaridade e pH da solução de Ringer devem ser semelhantes aos do mACSF. Aerar a solução com gás Carboxygen. </li><li> Prepare 4% baixa temperatura de fusão de agarose (LMA) em qualquer mACSF ou solução de Ringer. Alíquota a agarose derretida em tubos eppendorf e armazenar a 4 ° C até o uso. LMA, que é composto de polissacarídeo purificado, permanece fluido a 37 ° C e solidifica rapidamente em temperatura abaixo de 25 ° C. Estas propriedades o tornam ideal para a incorporação de VNO e fisiologia do tecido vivo. Materiais impuros, tais como agar, não deve ser a escolha para o experimento tecido vivo como componentes descaracterizados pode interferir com a resposta neuronal. </li><li> Prepare estímulos feromônio em solução de Ringer. Para a urina do rato, diluição 1:100 dar origem a respostas robusto. </li></ol> 2. Preparação de fatia VNO <ol><li> Antes de sacrificar o animal, colocar dois tubos de LMA em um bloco de aquecimento e ajuste a temperatura&gt; 60 ° C para derreter a agarose. Uma vez que o gel se liquefaz, os tubos de transferência para um bloco de aquecimento 37 ° C. </li><li> Decapitar um rato G-CaMP2 seguintes CO 2 a eutanásia. Cortar os ossos da mandíbula com uma tesoura para retirar o maxilar inferior. Descolar o tecido palato ridged superior para expor a cavidade nasal (Figura 1A). Separar os maxilares através da inserção de uma lâmina cirúrgica entre os dois incisivos da frente. Remova cuidadosamente os maxilares para expor VNO. Neste ponto, o VNO todo pode ser levantado da ncavidade Asal, segurando no osso da cauda (Figura 1B). Transferir o VNO ao frio solução mACSF oxigenado imediatamente. </li><li> Em âmbito dissecção, separar os dois VNOs usando a ponta do # 5 fórceps deslize suavemente ao longo da parede septal do osso (Figura 1C). Descascar o osso vômer que envolve o tecido VNO. Levantar delicadamente o tecido VNO inteiro a partir da cavidade óssea. Extremo cuidado deve ser tomado nesta etapa para não danificar o neuroepitélio. Pequenos fragmentos ósseos deixados na superfície do tecido deve ser removido completamente antes de incorporação. Fragmentos deixados no tecido pode ser capturado pela lâmina de corte para puxar o tecido fora do bloco de agarose. </li><li> Use a pinça para segurar a extremidade posterior do tecido VNO e delicadamente mergulhe-a na agarose derretida (Figura 1D). Rapidamente esfriar o tubo em gelo para solidificar a agarose. O processo de incorporação e de refrigeração deve demorar menos de 2 minutos. </li><li> VF300 slicer tecido (Instr Precisionarytos, Greenville, NC) é usado para fazer seções. Cortar o bloco de agarose contendo o VNO em forma e cola-lo ao titular do tecido (Figura 1E e F). Insira o suporte de tecido para dentro do cilindro de metal e encha a câmara restante com LMA adicionais. Uma vez que a LMA solidifica em gelo, vá para a secção imediatamente. Fornecimento mACSF oxigenado frio na câmara de corte e começar a cortar na espessura de 180-200 mM por fatia. Ajustar a velocidade de avanço e freqüência de vibração, para que o tecido não é comprimida durante o corte e VNO não cair da agarose. Coletar e transferir as fatias seccionadas para mACSF câmara de incubação (Figura 2A). As fatias são viáveis ​​por 6-8 horas em mACSF oxigenada em temperatura ambiente. Figura 2B e C mostram imagens DIC e 2-fótons de uma fatia VNO G-CaMP2, respectivamente. </li></ol> 3. Imagens da câmara configurado <ol><li> Coloque a fatia VNO no meio da perfusãocâmara (Siskiyou, Grants Pass, OR) e segure a fatia para baixo com uma âncora fatia (Warner Instruments, Hamden, CT) (Figura 3A). Os fios da âncora só deve pressionar contra a parte da fatia de LMA, mas não o tecido VNO. Oxigenado mACSF é entregue à câmara de perfusão através da porta de entrada em ~ 100 l / seg eo líquido é drenado através de uma agulha de sucção através da porta de saída para fornecer um fluxo contínuo de mACSF fresco (Figura 3A e B). </li><li> Encher uma seringa de 30 ml com solução de Ringer e apertá-lo para a bomba de seringa (Sistemas Bomba New Era, Farmingdale, NY). Definir a velocidade da bomba de 300-600 mL / min para fornecer um fluxo contínuo de solução de Ringer sobre a fatia (Figura 3C). </li><li> Conecte a saída do da campainha para uma injeção HPLC loop (Chromtech, Apple Valley, MN) (Figura 3D). Tamanho do loop diferentes podem ser selecionados para controle preciso do volume a ser entregue. Usamos um loop 20 l em oexperimentos ur. O controle de loop de injeção possui duas rotas de fluxo (Figura 3E). No &quot;load&quot; de posição, as amostras podem ser carregados para o loop de amostra com uma seringa de precisão Hamilton. Excesso de saídas de amostra do loop de amostra através da saída de resíduos. Solução de Ringer injetada pela bomba de seringa ignora o circuito de amostra e vai diretamente à tomada, que é conectado à ponta de perfusão (Figura 3A e E). Em &quot;injeção&quot; de posição, a solução da bomba flui através do circuito de amostra e empurre os estímulos à tomada (Figura 3E). Antes do experimento de imagem, bolhas de ar devem ser expulsos para garantir fluidez do líquido de perfusão. Outros comerciais ou de sistemas de injeção personalizado feito também pode ser implementado para entrega de estímulo. </li><li> Ajuste a ponta de perfusão sob uma lente de 5x ou 10x de modo que a ponta é cerca de 1 mm de distância da fatia VNO. </li><li> Quando um sistema de perfusão nova configuração, é aconselhável para medir o tempo de atraso de amostra e durração com corante fluorescente. Carga corante rodamina 6G 0,1% para o loop de amostra e mudar a válvula para injetar posição menos 5 segundos após o início da aquisição da imagem (Figura 3F). O sinal fluorescente é detectado entre 10-30 segundos. A curva de sinal fluorescente também indica que há pouca turbulência gerada sob a lente de imersão e que a perfusão adequada dos mACSF oxigenado atinge a fatia. </li></ol> 4. Lapso de tempo de imagem <ol><li> Nós usamos o microscópio Zeiss AxioSkope FS2 com uma lente de 10X ou 20X de água-dipping para imagens lapso de tempo. Padrão bandpass GFP filtro (450-490nm) é usado para G-CaMP2 sinais. As imagens de epifluorescência são adquiridas por uma câmera CCD (Zeiss HRM) com 1X1 ou 2x2 binning dependendo do nível de expressão de G-CaMP2. </li><li> Definir a velocidade de aquisição de 1 frame por segundo. Ajustar a intensidade da luz para minimizar os sinais de branqueamento de G-CaMP2 foto e danos às células. Para cada exmentam, que normalmente adquirem uma pilha de imagens de 60 quadros (~ 60-70 segundos). Interruptor da válvula para a posição de injeção em um ponto de tempo específico (por exemplo, 5 segundo na Figura 3F) para um conjunto de experiência para obter tempo de atraso consistente em todos os ensaios. </li><li> Realizar um ensaio utilizando solução de Ringer como o estímulo. Re-ajustar a configuração de perfusão se artefato movimento é introduzido durante a injeção da amostra. </li><li> Realizar um controlo positivo correr com urina do rato diluído a 1:100 em solução de Ringer. O típico valor de ΔF / F máximo de G-CaMP2 resposta a urina do rato é de cerca de 20-40%. Se necessário, pode-se confirmar a viabilidade da fatia, oferecendo 10 mM KCl em solução de Ringer para estimular as fatias no final dos experimentos. </li><li> Lavar a seringa Hamilton em solução de Ringer com pelo menos três vezes depois de carregar um estímulo. Lavar o circuito da amostra com solução de Ringer, pelo menos, três vezes entre diferentes estímulos. Estes passos evitar a contaminação cruzada entre os difereamostras nt. </li><li> Espere por 40-10 min para o VSNs para recuperar antes de aplicar o estímulo seguinte. </li></ol> 5. A análise dos dados <ol><li> Realizar o registro de imagem de todas as imagens adquiridas a partir de uma fatia. Nós usamos uma custom-script VBA escrito em AxioVision (Carl Zeiss, América do Norte) para automatizar esse processo. Todos os quadros de imagem dentro do mesmo experimento são registradas contra um quadro de referência comum escolhido com elástico registo (Figura 4A). Elástica de registro, também conhecido como registro não-linear, é uma categoria de técnica de registro de imagem com ênfase na transformação de uma imagem-alvo não rigidamente a uma imagem de referência. Aqui usamos a implementação de AxioVision. Este quadro de referência é escolhido arbitrariamente das pilhas de imagem. </li><li> Executar a subtração de imagem para identificar as células responder. Nós usamos macros personalizadas escrito em ImageJ v1.42 ( <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> , NIH, Bethesda, MD) para automatizar esse processo. A imagem de projeção mínima é gerado para cada pilha. Células que respondem surgir após a projeção mínima é subtraído do stacks-primas (Figura 4B). </li><li> Identificar regiões de interesse (ROI) das pilhas subtraído e obter o ROI utilizando coordenadas Multi-Measure plugin de ImageJ. Processo de todas as pilhas de um experimento e salvar todos os ROI coordenadas em uma lista mestra ROI (Figura 4C). </li><li> Use a lista principal ROI para medir as respostas de células a partir de pilhas de imagens brutas com custom-written macro e Multi-Measure plugin do ImageJ (Figura 4D). Plot curvas de resposta e heatmap em Matlab (Mathworks Inc., Natick, MA). </li></ol> 6. Resultados representante Um exemplo da experiência de imagem VNO utilizando amostras de urina coletadas de quatro camundongos indivíduo é mostrado na Figura 5. A fatia responde à estimulação de urina com diversas patterns de ativação. Cerca de 80 células são identificadas mostrando resposta a pelo menos um dos estímulos de urina e sua resposta ΔF / F valores são plotados no mapa de calor (Figura 5A). Os traços resposta das células 1, 2 e 3 são plotados em Figura 5B mostra a ativação curso de tempo por estímulos urina. Célula 1 exibe resposta a ambas as amostras de urina feminina, mas não as amostras do sexo masculino, enquanto célula 2 mostra os padrões de ativação oposto e responde a ambas as amostras do sexo masculino. Célula 3 é ativado por ambas as amostras individuais masculino e feminino. Células 1 e 2 mostram resposta característica ao sexo sugestões específicas na urina 15. <img alt="Figura 1" src="/files/ftp_upload/3404/3404fig1.jpg" /> Figura 1 Ilustração esquemática da VNO processo de dissecação. A. A localização anatômica do VNO na cabeça de rato. A cabeça de um rato G-CaMP2 foi dissecado e é colocada de cabeça para baixo com o t molesquestão removido do palato para expor o VNO. De entrada mostra uma imagem fluorescente do mesmo quadro. O neuroepithelia do VNO são verdes brilhantes. B. A vista lateral do VNO isolado que é fechado no osso vômer (em cima) ea imagem fluorescente do VNO mesmo (baixo). C. Uma visão coronal do VNO e dissecção processo. Um VNO é separada do septo eo osso vômer pode então ser removido para livrar o neuroepitélio. D. VNO é incorporado LMA. E. O bloco embutido é colado ao titular de tecido para corte. O titular tecido é avançada no 180-200 mM por fatia empurrando o bloco de agarose para fora do cano de metal para corte. A lâmina de corte é posicionada perto do barril de metal. F. Vista lateral do sistema slicer VF300 tecido. BV, dos vasos sanguíneos. NE, neuroepitélio. <img alt="Figura 2" src="/files/ftp_upload/3404/3404fig2.jpg" /> <strong&gt; Figura 2 fatias VNO. fatias A. VNO são mantidos em mACSF oxigenada em temperatura ambiente. B. Uma imagem DIC da fatia VNO. C. A imagem 2-fótons do VNO de G-CaMP2 mouse. Barra de escala, 50 mm. <img alt="Figura 3" src="/files/ftp_upload/3404/3404fig3.jpg" /> Figura 3 Ilustração da configuração do sistema de perfusão. A. A câmara de perfusão típico, com entrada e saída. Fatia VNO é posicionado no centro da câmara e pressionou para baixo com uma âncora de tecido. Os fios são removidos do meio da âncora do tecido de modo que o tecido VNO não está pressionado. B. A câmara de perfusão é colocado no palco microscópio no âmbito do objectivo de imersão. A entrada mACSF tomada de aspiração, e ponta de perfusão são indicados. C. A bomba de seringa barril proporciona um fluxo contínuo de Ringer com a ponta de perfusão. D. A inje HPLCsistema de circuito ction é adotada para carregamento conveniente estímulo modelo e injeção. E. Ilustração esquemática das direções de fluxo em carga e posições de injeção. Ringer, verde. Amostra de estímulo, magenta. Setas indicam o sentido do fluxo. F. Delay e lave medição do tempo do sistema de perfusão. Rodamina 6G corante fluorescente é carregado para o loop de amostra ea válvula é comutada para a posição de injeção na segunda 5, que é indicado com uma seta. A mudança de sinal fluorescente é detectado entre 10-30 segundos e diminui depois. <img alt="Figura 4" src="/files/ftp_upload/3404/3404fig4.jpg" /> Figura 4 pipeline de processamento de imagem. A. Todos os quadros de imagem de uma fatia são registradas contra um quadro de referência. B. A subtração da projeção mínima de uma pilha de seus quadros-primas. Responder células tornam-se proeminentes após a subtração. C. ROIs são selecionados e suas coordenadas são compilados em lista principal. D. Medição de células que respondem usando ROI lista mestra da pilha-primas. <img alt="Figura 5" src="/files/ftp_upload/3404/3404fig5.jpg" /> Figura 5 Um representante do G-CaMP2 VNO experiência de imagem. A. O heatmap resposta ΔF / F de uma fatia estimulado com urina masculino e feminino individual. Amostras de urina são coletadas de indivíduo camundongos machos e fêmeas. Eixo X, respondendo as células identificadas a partir da fatia. Y-eixo, amostras de urina utilizadas no experimento. Barra de cores indica o valor de ΔF / F. B. claro Tempo de resposta de 3 células representante marcado na Arrows A. indicam o tempo de aplicação do estímulo. F-FVB, fêmea FVB; F-CBA, do sexo feminino CBA; M-FVB, do sexo masculino FVB; M-BL6, do sexo masculino C57BL / 6. Tabela de reagentes e equipamentos específicos: <table border="1"><tbody><tr><td> Nome doreagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> câmara de perfusão </td><td> Siskiyou </td><td> PC-H </td><td> horizontal </td></tr><tr><td> fatia de fixação </td><td> Warner Instruments </td><td> SHD-22CL </td><td></td></tr><tr><td> titular porta perfusão </td><td> ALA científica, Inc. </td><td> MPIOH-S </td><td></td></tr><tr><td> bomba de seringa </td><td> Sistemas de bomba New Era, Inc. </td><td> NE-300 </td><td></td></tr><tr><td> válvula de injeção de baixa pressão </td><td> Chromtech </td><td> V-451 </td><td></td></tr><tr><td> Tubulação PEEK </td><td> Chromtech </td><td> 1531 </td><td> 1 / 16 &quot;OD, 0,25 milímetros ID </td></tr><tr><td> PEEK loop de amostra </td><td> Chromtech </td><td> 1803 </td><td> 20 mL </td></tr><tr><td> Hamilton seringa </td><td> Chromtech </td><td> 80630 </td><td> 100 L </td></tr></tbody></table>

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E., Dulac, C., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+vomeronasal+neurons+to+natural+stimuli.">Responses of vomeronasal neurons to natural stimuli.</a> Science. 289, 1569-1572 (2000).</li><li>Chamero, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+pheromones+that+promote+aggressive+behaviour.">Identification of protein pheromones that promote aggressive behaviour.</a> Nature. 450, 899-902 (2007).</li><li>Leinders-Zufall, T., Ishii, T., Mombaerts, P., Zufall, F., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+requirements+for+the+activation+of+vomeronasal+sensory+neurons+by+MHC+peptides.">Structural requirements for the activation of vomeronasal sensory neurons by MHC peptides.</a> Nat. Neurosci. 12, 1551-1558 (2009).</li><li>Kimoto, H., Haga, S., Sato, K., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+peptides+from+exocrine+glands+stimulate+mouse+vomeronasal+sensory+neurons.">Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons.</a> Nature. 437, 898-901 (2005).</li><li>Holekamp, T. F., Turaga, D., Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+three-dimensional+fluorescence+imaging+of+activity+in+neural+populations+by+objective-coupled+planar+illumination+microscopy.">Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy.</a> Neuron. 57, 661-672 (2008).</li><li>Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I+peptides+as+chemosensory+signals+in+the+vomeronasal+organ.">MHC class I peptides as chemosensory signals in the vomeronasal organ.</a> Science. 306, 1033-1037 (2004).</li><li>He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+signals+conveyed+by+pheromone+concentrations+to+the+mouse+vomeronasal+organ.">Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ.</a> J. Neurosci. 30, 7473-7483 (2010).</li><li>Haga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+male+mouse+pheromone+ESP1+enhances+female+sexual+receptive+behaviour+through+a+specific+vomeronasal+receptor.">The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor.</a> Nature. 466, 118-122 (2010).</li><li>Boschat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+detection+mediated+by+a+V1r+vomeronasal+receptor.">Pheromone detection mediated by a V1r vomeronasal receptor.</a> Nat. Neurosci. 5, 1261-1262 (2002).</li><li>Hendel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+changes+of+genetic+calcium+indicators+and+OGB-1+correlated+with+neural+activity+and+calcium+in+vivo+and+in.">Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in.</a> 28, 7399-7411 (2008).</li><li>Pologruto, T. A., Yasuda, R., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+neural+activity+and+[Ca2+]+with+genetically+encoded+Ca2++indicators.">Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators.</a> J. Neurosci. 24, 9572-9579 (2004).</li><li>Jayaraman, V., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+a+genetically+encoded+optical+sensor+of+neural+activity+using+electrophysiology+in+intact+adult+fruit+flies.">Evaluating a genetically encoded optical sensor of neural activity using electrophysiology in intact adult fruit flies.</a> Front Neural Circuits. 1 (3), (2007).</li><li>Tian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li></ol>

No conflicts of interest declared.

The majority of the vomeronasal receptors (VRs) remain as orphan receptors since their discovery by Dulac and Axel 5. The pheromone ligands for these chemosensory receptors and their roles in mediating animal behaviors are not well understood. Until now, only one pair of ligand/receptor, the ESP1 peptide and its cognate receptor, Vmn2r116 (V2Rp5), has been identified and shown to convey specific social information 19,23. Another receptor, V1rb2, has been shown to respond to 2-heptanone, which presum...

imaging neuronal responses in slice preparations of vomeronasal organ expressing a genetically encoded calcium sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

Watch this Scientific Journal Video about Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor at JoVE.com

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Responses imagem Neuronal em Preparações Slice of Organ Vomeronasal Expressando um sensor de cálcio geneticamente codificado

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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Neuroscience

10.7K visualizações.  Stowers Institute for Medical Research. The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Vídeo: Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Imagens de cálcio de odor-provocou respostas no Drosophila Lobo antenal

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Neurociência

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imagem Pheromone Sensing em um Preparação Slice rato Vomeronasal aguda do tecido

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Na imagem cálcio vivo Neuronal em C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo preparações da Intacta Vomeronasal Órgão e acessórios bulbo olfatório

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imagens intracelular Ca<sup&gt; 2+</sup&gt; Sinais em estriado astrócitos de ratos adultos, através de indicadores de cálcio geneticamente codificados

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Gravação Light-provocou respostas pós-sinápticos nos neurônios em Dark-adaptados, Preparações da retina do rato fatia usando técnicas Grampo patch

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Membrana de imagem Potencial com dois tipos de sensores de tensão fluorescentes geneticamente codificados

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controlo óptico de uma proteína neuronal utilizando um ácido Unnatural Amino geneticamente codificados em neurónios

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

A incubação prolongada de aguda Neuronal Tissue de Eletrofisiologia e cálcio-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.