Este trabalho foi financiado pelo NIH concede DA15169 e DA035224 a HEM

Todo o manuseio de animais e colheita de tecidos foi efetuada de acordo com as diretrizes da Universidade do Institutional Animal Care Massachusetts Medical School Comitê de Uso (IACUC), seguindo o protocolo de número A1506 aprovado (Melikian, PI). Soluções necessárias Artificial líquido cefalorraquidiano (ACSF) - Faça fresco diariamente NaCl 125 mM, KCl 2,5 mM, 1,2 mM de NaH 2 PO 4, 1,2 mM de MgCl2, 2,4 mM de CaCl2, 26 mM de NaHCO 3, e 11 mM de glicose Nota: Preparar ACSF como uma solução estoque de 10x, com exclusão de NaHCO3 e glicose. Faça soluções de trabalho 1x diariamente a partir do estoque de 10x, completando com fresco glicose NaHCO3 e. Suplementados com sacarose ACSF (SACSF) - Faça fresco diariamente 250 mM sucessoaumentou, KCl 2,5 mM, 1,2 mM de NaH 2 PO 4, 1,2 mM de MgCl2, 2,4 mM de CaCl2, 26 mM de NaHCO 3, e 11 mM de glucose Nota: Preparar SACSF como uma solução estoque de 10x, com exclusão de NaHCO3 e glicose. Faça soluções de trabalho 1x diariamente a partir do estoque de 10x, completando com fresco glicose NaHCO3 e. Sulfo-N-hydroxysuccinyl-SS-Biotina (sulfo-NHS-SS-biotina, Pierce Chemical Company) As soluções de reserva deve ser de 200 mg / ml em DMSO e são resistentes a múltiplos ciclos de congelação / descongelação. Alíquotas são armazenadas a -20 ° C. O éster succinilo é rapidamente hidrolisado em solução aquosa, pelo que as soluções de trabalho deve ser preparado imediatamente antes de aplicar a fatias. Fatia Solução Quench ACSF suplementado com 100 mM de glicina A lise RIPA Buffer 10 mM Tris, pH 7,4, NaCl 150 mM, EDTA 1,0 mM, 1% Triton-X-100, 0,1% SDS, 1% de desoxicolato de Na RIPA com Inibidores da Protease (RIPA / PI) - Faça fresco diariamente RIPA suplementado com 1 fiM de leupeptina, 1 jiM de pepstatina, 1 uM de aprotinina, e 1 mM de fluoreto de fenilmetil sulfonilo. 1. Prepare fatias cerebrais <ol><li> Faça SACSF 1x fresco e 1x ACSF </li><li> Refrigerar SACSF em gelo num copo pequeno. Isto irá ser utilizado para segurar o cérebro do rato recém-colhido. </li><li> Saturar o ACSF e SACSF com oxigénio fazendo borbulhar com 95% / 5% de O 2 / CO 2, 20 min em gelo. </li><li> P30-38 ratinhos devem ser usadas para a viabilidade do tecido óptima. Sacrifício de animais por deslocamento cervical e decapitação, e remover rapidamente os cérebros em pré-arrefecido, SACSF oxigenado. </li><li> Usando um micrótomo vibrando, fazer 300 seções mM cerebrais na região de interesse. </li><li> Se desejar, fatias pode ser ainda mais dissecado anterior à recuperação de enriquecer para regiões cerebrais particulares ou direito distinto e hemisférios esquerdo para usar como controle e fatias experimentais, respectivamente. </li><li> Usando uma pipeta Pasteur polida-fogo, transfira as fatias de malha de fundo câmaras definidas em placas de 24 poços. </li><li> Permitir fatias a recuperar durante 40 min, 31 ° C em ACSF oxigenado, com contínua, borbulhamento suave. </li></ol> 2. Tratamento de Drogas (se for o caso) e Slice Biotinilação <ol><li> Após a recuperação, lavagem 3x em fatias pré-aquecido (37 ° C), oxigenado ACSF borbulhar constantemente com 95% / 5% de O 2 / CO 2. </li><li> Adicionar os compostos de teste e incuba-se com oxigenação constante. <ol><li> Por conveniência, adicionar um 1/10 de volume de 10x droga concentrada e misturar invertendo suavemente. </li><li> Agitar suavemente as placas num banho de água à temperatura desejada. </li></ol></li><li> Após o tratamento de drogas, rapidamentefatias frio por lavar 3x no gelo ACSF frio. </li></ol> 3. Proteínas de superfície biotinilar <ol><li> Prepare a 1,0 mg / ml de sulfo-NHS-SS-biotina em gelo ACSF frio imediatamente antes da marcação. </li><li> Adicionar 0,75 ml sulfo-NHS-SS-biotina para fatias e fatias incubar no gelo, 45 min. </li><li> Lave as fatias três vezes rapidamente com o frio do gelo ACSF, em seguida, incubar por 10 min no gelo ACSF frio no gelo. </li><li> Lave as fatias três vezes com tampão de fatia de têmpera gelada e incubar com 0,75 ml de tampão de têmpera fatia duas vezes, 25 min, no gelo para saciar livre sulfo-NHS-SS-biotina. </li></ol> 4. Prepare Lysates Tecido <ol><li> Lavar as fatias de três vezes em gelo ACSF gelado e transferir cada fatia para um tubo de microcentrífuga utilizando uma pipeta de Pasteur polidas ao fogo. </li><li> Gentilmente pelota fatia por centrifugação de 200 xg, 1 min e ACSF cuidadosamente aspirado restante. </li><li> Adicionar 400 mL de gelo frio RIPA / PI e romper o tecido pipetando cima e para baixo uma vez através de um pip P200ette. </li><li> Transferência dissociado fatia / RIPA para um novo tubo e incubar 30 min, 4 ° C, em rotação, para completar a lise. </li><li> Sedimentar os detritos celulares por centrifugação, 18.000 xg, 15 min, 4 ° C. </li><li> Determinar a concentração de proteína de lisado usando o ensaio de proteína BCA, com albumina de soro bovino (BSA) como padrão. </li></ol> 5. Isolar Biotinylated Proteínas <ol><li> Otimizar relação proteína do grânulo / total. Nota: os níveis de expressão de proteínas individuais podem variar muito entre diferentes regiões cerebrais. A não ser que toda a proteína biotinilada de uma dada quantidade de tecido ligado é capturado, não é possível detectar com precisão todas as mudanças potenciais na expressão à superfície. Portanto, é imperativo para determinar empiricamente a relação ideal talão / total de proteína para uma determinada região de proteína / cérebro antes de embarcar em um novo estudo fatia biotinilaï. <ol><li> Incubar 25 ul estreptavidina-agarose com uma quantidade cada vez maiors de lisado de tecido (intervalo aproximado de 25-200 mg), e em seguida, proceder conforme descrito a seguir para a ligação, lavagem e eluição passos. </li><li> Quantificar as bandas imunorreactivas resultantes e escolher um rácio de grânulo / lisado na gama linear de ligação que irá permitir a quantificação exacta de qualquer aumento ou diminuição da expressão da proteína de superfície. </li></ol></li><li> Preparar contas de estreptavidina-agarose <ol><li> Determinar o volume total talão necessário para todas as amostras. Prepare um volume talão suficiente para que a quantidade mais um extra (ou seja, 4 amostras de 25 mL / amostra = 100 contas ul + um extra = 125 contas mL. </li><li> Vortex Streptavidin estoque talão e pipeta fora desejado volume. Se for utilizada uma pipeta de P200, cortar a extremidade da ponta para impedir o bloqueio ou grânulo danos. </li><li> Lave contas 3 vezes em 0,5-1,0 ml RIPA / PI para remover conservantes, vórtex após cada adição RIPA e coleta de contas entre lavagens por centrifugação 18.000 xg, 1 min, na sala de temperamentoature. </li><li> Aspirar fora tampão entre lavagens utilizando uma pipeta de Pasteur de vidro ligada a um frasco de vácuo. Uma dica P200 plástico preso à extremidade da pipeta Pasteur de vidro proporciona um controle mais preciso quando aspiração. Não é necessário remover completamente todo o tampão para as duas primeiras lavagens, uma vez que os riscos de aspiração grânulos para a pipeta. Após a lavagem final, remover tanto excesso de tampão quanto possível sem aspirar os grânulos. </li><li> Adicionar RIPA / PI para trazer contas de volta ao seu volume original, pipetando para cima e para baixo para voltar a suspender. Evitar vórtice nesse ponto, como grânulos vão ficar à parede do tubo. </li><li> Contas Alíquota em microtubos usando um P200 com a ponta cortada. Pipeta cima e para baixo várias vezes entre a amostragem para garantir contas permanecem uniformemente suspenso e disperso entre os tubos. </li></ol></li><li> Proteínas biotiniladas Bind para estreptavidina contas <ol><li> Distribuir os lisados ​​celulares para os tubos contendo as pérolas de agarose. Para experiências whe<html> re várias amostras estão sendo comparados, certifique-se de usar a mesma quantidade de proteína para cada amostra para comparações precisas. </li><li> Adicionar RIPA / PI adicional para levar a amostras de um volume mínimo de 200 ul. Isto assegura que as amostras se misturar adequadamente durante a incubação e também unifica as concentrações de proteína através de amostras. </li><li> Colocar os tubos num rotor tubo e misturar durante a noite a 4 ° C. </li><li> Em tubos separados, dispensar um equivalente do volume de lisado total utilizado para cada amostra. Alternativamente, se os volumes de amostra são elevados, uma fracção do volume total de lisado pode ser reservado em vez disso, para acomodar volumes de carga máxima em géis de SDS-PAGE. Estas serão usadas para normalizar a expressão de superfície para a quantidade total de proteína. </li><li> Adicionar ou um volume igual de 2x ou 1/5 do volume de tampão de amostra de SDS-PAGE de 6x e ou incubar a 4 ° C, em paralelo com as amostras de grânulo, ou armazenar a -20 ° C até serem analisadas amostras. </li></ol></li></ol>ove_title &quot;&gt; 6. Elute e analisar amostras <ol><li> Granulado de grânulos por centrifugação de 18.000 x g, 2 minutos, à temperatura ambiente. </li><li> Sobrenadante Aspirar e lavar contas três vezes com 0,75 ml RIPA. Para minimizar a perda de talão, deixe um pequeno volume de tampão cabeça acima contas entre lavagens. Após a lavagem final, retirar o máximo RIPA quanto possível, sem perturbar o sedimento talão. </li><li> Eluir proteínas a partir de contas de estreptavidina biotinilados através da redução da ligação dissulfureto. Adicionam-se 25 ul de SDS-PAGE 2x tampão de amostra redutora, de vórtice e assim rodar as amostras 30 min, temperatura ambiente. Nota: Muitas proteínas da membrana têm uma elevada tendência para se agregar quando fervidos em tampão de amostra de SDS-PAGE, prejudicando gravemente a sua mobilidade electroforética. Se este é o caso para a proteína a ser investigado, evitar aquecimento das amostras e, em vez disso, eluir-se lentamente à temperatura ambiente. Se ferver é absolutamente necessário (por exemplo, se uma outra proteína avaliada em paralelo requer ebulição), sample tampão pode ser suplementado com ureia 2 M (concentração final) para minimizar a agregação. Embora eficaz na redução da agregação, em alguns casos, ureia frequentemente compromete a aparência da banda. </li><li> Analisar amostras por immunoblot. </li><li> Descongele as amostras totais lisado e rodar em paralelo com amostras de talão, 30 min, a temperatura ambiente. </li><li> Proteínas separadas em géis de SDS-PAGE. </li><li> Identificar proteína (s) de interesse por immunoblotting. <ol><li> Esteja certo de que as bandas são detectados na faixa linear de detecção para a quantificação adequada. </li><li> Para garantir que o reagente biotinilaï não teve acesso a proteínas intracelulares via células danificadas / comprometidos. Isto é melhor realizado por imunotransferência em paralelo para uma proteína intracelular específica para o tipo de célula a ser investigado. </li><li> Quantificar densidades banda e calcular relativa densidade de superfície da proteína como um por cento do nível total de expressão da proteína. </li></ol></li></ol>

<ol>
	<li>Conner, S. D., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+portals+of+entry+into+the+cell.">Regulated portals of entry into the cell.</a> Nature. 422, 37-44 (2003).</li><li>Zastrow, M., Williams, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+neuromodulation+by+receptor+membrane+traffic+in+the+endocytic+pathway.">Modulating neuromodulation by receptor membrane traffic in the endocytic pathway.</a> Neuron. 76, 22-32 (2012).</li><li>Leto, D., Saltiel, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+transport+by+insulin:+traffic+control+of+GLUT4.">Regulation of glucose transport by insulin: traffic control of GLUT4.</a> Nat. Rev. Mol. Cell Biol. 13, 383-396 (2012).</li><li>Liu, Y. W., Lukiyanchuk, V., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+membrane+trafficking+defects+of+disease-associated+dynamin+2+mutations.">Common membrane trafficking defects of disease-associated dynamin 2 mutations.</a> Traffic. 12, 1620-1633 (2011).</li><li>Li, X., DiFiglia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recycling+endosome+and+its+role+in+neurological+disorders.">The recycling endosome and its role in neurological disorders.</a> Prog. Neurobiol. , 127-141 (2012).</li><li>Sandvig, K., Pust, S., Skotland, T., van Deurs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-independent+endocytosis:+mechanisms+and+function.">Clathrin-independent endocytosis: mechanisms and function.</a> Curr. Opin. Cell Biol. 23, 413-420 (2011).</li><li>Kumari, S., Mg, S., Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+unplugged:+multiple+ways+to+enter+the+cell.">Endocytosis unplugged: multiple ways to enter the cell.</a> Cell Res. 20, 256-275 (2010).</li><li>Barry, M. F., Ziff, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+trafficking+and+the+plasticity+of+excitatory+synapses.">Receptor trafficking and the plasticity of excitatory synapses.</a> Curr. Opin. Neurobiol. 12, 279-286 (2002).</li><li>Bredt, D. S., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+at+excitatory+synapses.">AMPA receptor trafficking at excitatory synapses.</a> Neuron. 40, 361-379 (2003).</li><li>Kerchner, G. A., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+synapses+and+the+emergence+of+a+postsynaptic+mechanism+for+LTP.">Silent synapses and the emergence of a postsynaptic mechanism for LTP.</a> Nat. Rev. Neurosci. 9, 813-825 (2008).</li><li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+and+synaptic+plasticity.">AMPA receptor trafficking and synaptic plasticity.</a> Annu. Rev. Neurosci. 25, 103-126 (2002).</li><li>Borgland, S. L., Malenka, R. C., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+cocaine-induced+potentiation+of+synaptic+strength+in+the+ventral+tegmental+area:+electrophysiological+and+behavioral+correlates+in+individual+rats.">Acute and chronic cocaine-induced potentiation of synaptic strength in the ventral tegmental area: electrophysiological and behavioral correlates in individual rats.</a> J. Neurosci. 24, 7482-7490 (2004).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine-induced+potentiation+of+synaptic+strength+in+dopamine+neurons:+Behavioral+correlates+in+GluRA(-/-)+mice.">Cocaine-induced potentiation of synaptic strength in dopamine neurons: Behavioral correlates in GluRA(-/-) mice.</a> PNAS. 101, 14282-14287 (2004).</li><li>Hyman, S. E., Malenka, R. C., Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+addiction:+the+role+of+reward-related+learning+and+memory.">Neural mechanisms of addiction: the role of reward-related learning and memory.</a> Annu. Rev. Neurosci. 29, 565-598 (2006).</li><li>Thomas, M. J., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+mesolimbic+dopamine+system.">Synaptic plasticity in the mesolimbic dopamine system.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358, 815-819 (2003).</li><li>Sorkina, T., Hoover, B. R., Zahniser, N. R., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+and+protein+kinase+C-induced+internalization+of+the+dopamine+transporter+is+mediated+by+a+clathrin-dependent+mechanism.">Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.</a> Traffic. 6, 157-170 (2005).</li><li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical,+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat. Neurosci. 8, 881-888 (2005).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J. Biol. Chem. 278, 22168-22174 (2003).</li><li>Melikian, H. E., Buckley, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+trafficking+regulates+the+activity+of+the+human+dopamine+transporter.">Membrane trafficking regulates the activity of the human dopamine transporter.</a> J. Neurosci. 19, 7699-7710 (1999).</li><li>Daniels, G. M., Amara, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+trafficking+of+the+human+dopamine+transporter.+Clathrin-mediated+internalization+and+lysosomal+degradation+in+response+to+phorbol+esters.">Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters.</a> J. Biol. Chem. 274, 35794-35801 (1999).</li><li>Sorkina, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screen+reveals+an+essential+role+of+Nedd4-2+in+dopamine+transporter+ubiquitination+and+endocytosis.">RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.</a> J. Neurosci. 26, 8195-8205 (2006).</li><li>Eriksen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+dopamine+transporter+trafficking+in+live+neurons+by+use+of+fluorescent+cocaine+analogs.">Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs.</a> J. Neurosci. 29, 6794-6808 (2009).</li><li>Rao, A., Simmons, D., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+subcellular+distribution+of+endosomal+compartments+and+the+dopamine+transporter+in+dopaminergic+neurons.">Differential subcellular distribution of endosomal compartments and the dopamine transporter in dopaminergic neurons.</a> Mol. Cell Neurosci. 46, 148-158 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-752 (2011).</li></ol>

Os autores não têm interesses financeiros que estão em conflito com este trabalho.

Apesar do conhecimento de longa data que o tráfico endocítico criticamente impactos sinalização sináptica no cérebro, tem-se revelado difícil de medir quantitativamente mudanças na expressão da proteína de superfície em neurônios adultos. Neste trabalho, relatamos uma abordagem confiável para rotular proteína de superfície ex vivo em fatias cerebrais agudas. Preparações fatia do cérebro têm uma longa história de utilidade para registros eletrofisiológicos, que mantenham conexões sináptica...

Tráfico endocítico é um mecanismo celular ubíquo que ajusta a apresentação da membrana plasmática de uma variedade de proteínas de membrana integrais. Endocitose fornece nutrientes vitais para o ambiente intracelular e um dessensibiliza a sinalização do receptor em resposta à activação do receptor 2. Endocítica reciclagem de volta para a membrana plasmática pode adicionalmente melhorar a sinalização celular, aumentando os níveis de expressão de proteínas na superfície da célula 3. Além disso, as perturbações de tráfico de membrana estão implicados em inúmeras doenças e condições patológicas 4,5, salientando a necessidade de investigar os mecanismos moleculares que governam proteína tráfico endocítica. Embora muitas proteínas utilizar mecanismos de internalização clássicos dependente de clatrina, a evidência de montagem ao longo dos últimos anos demonstra que vários mecanismos de endocitose independente de clatrina governar o potencial endocítica de uma crescente variedade deproteínas de 6,7. Assim, a necessidade de investigar os mecanismos de endocitose que facilitam o tráfico de sistemas relevantes fisiológicas tem crescido consideravelmente. No cérebro, o tráfico de endocitose de receptores, canais iônicos e transportadores de neurotransmissores tem um papel primordial no estabelecimento de plasticidade sináptica 8-11 e resposta a drogas de abuso 12-15, em última análise, que impactam a excitabilidade neuronal e respostas sinápticas. Até à data, a maioria dos estudos de tráfico neuronal dependem tanto sistemas de expressão heterólogos ou neurônios primários em cultura, nenhum dos quais pode refletir de forma confiável mecanismos em jogo em neurônios adultos. Aqui, apresentamos uma abordagem que usa biotinilaï superfície para medir quantitativamente os níveis da proteína de superfície em fatias cerebrais agudas derivadas de roedores adultos. Usando essa abordagem, apresentamos dados que demonstram que o mouse do transportador de dopamina no estriado internaliza rapidamente em reposta de forbol de éster mediada por proteína-quinase C (PKC) a activação.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

Tráfico endocítico Regulamentado é o mecanismo central facilitar uma variedade de eventos neuromoduladores, controlando dinamicamente receptor, canal iônico, e transportador de apresentação da superfície celular em uma escala minutos tempo. Há uma grande diversidade de mecanismos que controlam o tráfico endocítica de proteínas individuais. Estudos que investigam as bases moleculares do tráfico têm dependido principalmente sobre biotinilaï superfície para medir quantitativamente mudanças na proteína da membrana expressão de superfície em resposta a estímulos exógenos e manipulação genética. No entanto, esta abordagem tem sido limitado principalmente para cultura de células, o que pode não refletir fielmente os mecanismos fisiologicamente relevantes em jogo em neurônios adultos. Além disso, as abordagens de células cultivadas podem subestimar as diferenças específicas da região em mecanismos de tráfico. Aqui, descrevemos uma abordagem que se estende biotinilaï superfície celular para a preparação fatia cerebral aguda. Nósdemonstrar que este método fornece uma abordagem de alta fidelidade para medir rápidas mudanças nos níveis de superfície de proteínas de membrana em neurônios adultos. Esta abordagem é provável que tenha ampla utilidade no domínio do tráfico de endocítico neuronal.

O transportador de dopamina neuronal é internalizado em resposta à activação de PKC nas linhas celulares 16-20. Apesar de muitos relatórios que provam induzidas PKC perdas superficiais DAT em ​​uma variedade de linhas celulares e sistemas de expressão, tem sido um desafio para confirmar esse achado em neurônios dopaminérgicos cultivadas 21-23. Usamos fatias estriado do rato para testar diretamente se DAT internaliza em resposta à ativação PKC em neurônios dopaminérgicos adultos. Ap...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Tráfico de membrana neuronal controla dinamicamente membrana disponibilidade de proteína plasmática e significativamente impactos neurotransmissão. Até à data, tem sido um desafio para medir tráfico endocítico neuronal em neurônios adultos. Aqui, descrevemos um método altamente eficaz, quantitativo para medir as mudanças rápidas na expressão de proteínas de superfície ex vivo em fatias cerebrais agudas.

Cérebro Fatia Biotinilação: Uma<em&gt; Ex Vivo</em&gt; Abordagem medir Região específicas do tráfego de proteínas de membrana plasmática em adultos Neurônios

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.8K visualizações.  University of Massachusetts Medical School.  Tráfico de membrana neuronal controla dinamicamente membrana disponibilidade de proteína plasmática e significativamente impactos neurotransmissão. Até à data, tem sido um desafio para medir tráfico endocítico neuronal em neurônios adultos. Aqui, descrevemos um método altamente eficaz, quantitativo para medir as mudanças rápidas na expressão de proteínas de superfície ex vivo em fatias cerebrais agudas. 

Vídeo: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Neurociência

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imagiologia pHluorin-tagged Inserção receptor para a membrana plasmática em primários de rato cultivadas Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Usando um α-Bungarotoxina Binding Tag Site para Estudar GABA um receptor de membrana Localização e Tráfico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Agudo Adulto Cérebro Cortando na temperatura fisiológica

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenética Dissection de Circuitos medo no cérebro Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imagens ao vivo da proteína GLUT4 tráfico de Mouse de neurônios hipotálamo primário usando microscopia de deconvolução

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Imagem de cálcio para visualizar as respostas do cérebro a sinalização endócrina em Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

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

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

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

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

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