A maior parte do trabalho e do pessoal envolvido foram apoiados pelo NIH conceder NS060677 e em parte pelo NIH concede MH099559 e MH104069 (a BSK). Alguns dos trabalhos também foi apoiado pela Fundação CHDI.

Todos os protocolos de animais estavam de acordo com os Institutos Nacionais de Saúde Guia para o Cuidado e Uso de Animais de Laboratório e foram aprovados pelo Comitê de Cuidados e Uso de Animais Institucional na UCLA. 1.1) Prepare Micropipeta e AAV2 / 5 Vírus Carregando <ol><li> Use micropipetas de vidro de borosilicato finas para a injecção do vírus. Puxe a micropipeta usando um programa puxando de duas etapas com um puxador vertical. Bisel da pipeta com um ângulo de 40 °, utilizando um moinho de pipeta. A pipeta que é ideal para uso terão um diâmetro de ponta de 20 - 40 mm e um comprimento da haste de 6-7 mm. Autoclave a pipeta e instrumentos cirúrgicos. A broca é esterilizada por limpeza com etanol a 70%. </li><li> Armazenar o vírus AAV2 / 5-GfaABC 1 D.cyto-GCaMP3.SV40 (1,5 x 10 13 gc / ml), a -80 ° C em alíquotas de 10 ul. Pouco antes da cirurgia, tire as alíquotas do congelador e guarde no gelo até ser usado para o preenchimento de the micropipeta. </li><li> Encher a micropipeta com vírus, utilizando uma bomba de seringa. Firmemente ligar uma extremidade de um pedaço de tubo, através de uma tubagem de compressão de tamanho adequado encaixe, para uma seringa de vidro com um êmbolo reforçado. Encaixe a micropipeta autoclavado para a outra extremidade do pedaço de tubo através de uma agulha de tamanho apropriado compressão encaixe removível. </li><li> Antes de carregar o vector viral, remover bolhas de ar do sistema de bombagem, incluindo tubos, seringas e micropipeta enchendo o sistema com óleo mineral colorida com Sudan IV vermelha (~ 1 mg / 50 ml) numa seringa de 1 ml com uma agulha (27 L ). </li><li> Coloque um pedaço de parafilm limpo sob a micropipeta de vidro, e dispensar 5-10 ul de o vector viral para o parafilme. </li><li> Sugam o vector viral, movendo o êmbolo da seringa para trás a 0,5 ul / min, e marcam o limite entre o vector e o óleo sobre a pipeta de vidro. </li></ol> 1.2) A anestesia, corte de cabelo, Head Fixação e Preparação para Craniotomia <ol><li> Coloque um rato (P49-63, C57BL / 6) para a câmara cheia com N 2 O e O 2 e 5% de isoflurano. Avaliar a profundidade da anestesia, pela perda de movimento intencional e uma taxa respiratória lento (~ 60-90 / min). Cortar cabelo na cabeça quando o mouse é anestesiado. </li><li> Montar o mouse no quadro estereotáxico, com a cabeça protegida por barras de ouvido sem corte e seu nariz colocados em um sistema de anestesia e ventilação. Manter contínua isoflurano a 2 - 3%. Aplicar pomada lágrimas artificiais para ambos os olhos desde que os ratos perdem o reflexo de piscar sob anestesia. </li><li> Administrar 0,05 ml de buprenorfina (0,1 mg / ml) por injecção subcutânea para o alívio da dor. </li><li> Limpo área cirúrgica com 10% de iodo de povidona e 70% de álcool três vezes, alternando entre as duas soluções e começando com povidona iodo. Comece pela frente e limpe para trás para remover todos os cabelos já cortados. </li><li> Faça uma incisão na pele em cima of a cabeça, que se inicia entre os olhos e termina entre as orelhas. </li></ol> 1.3) Craniotomia e Microinjeções <ol><li> Retire o periósteo passando com um cotonete e depois secar a superfície do crânio com bolas de algodão dental. Fazer uma marca em torno do local da injecção, e perfurar em torno da borda da marca usando uma pequena rebarba de aço alimentado por uma broca de alta velocidade. </li><li> Remover o osso e limpar a superfície com solução salina. </li><li> Coloque a pipeta no dorsal e lateral esquerda da striatum com as seguintes coordenadas (mm): 0,8 ântero-posterior, médio-lateral: 2,0, dorsal-ventral da superfície pial: -2,4 13. Note-se que estas coordenadas significa que a ponta da agulha de microinjecção fica logo acima do estriado dorsolateral (Figura 1A, B), o que significa que só penetra ligeiramente dentro deste núcleo. Evite vasos sanguíneos prejudiciais. Se a pipeta passa a ser direito em um vaso sanguíneo, um pequeno ajuste nacoordenadas por ~ 0,1-0,2 mm. </li><li> Iniciar a injecção com uma taxa de injecção ajustado a 0,2 ul / min, e injectar tipicamente de 1 a 1,5 ul de vírus. </li><li> Deixar a pipeta no local após a injecção durante 10 min, e em seguida, retirar a pipeta lentamente. </li><li> Finalize fechando a ferida cirúrgica com sutura contínua de nylon externo sutura. </li></ol> 1.4) Recuperação <ol><li> Coloque o mouse em uma gaiola limpa em uma almofada de aquecimento, durante 24 horas. Não devolva um rato que foi submetido a cirurgia para a companhia de outros animais até que esteja totalmente recuperado. Não deixe o mouse sem vigilância até que tenha recuperado a consciência suficiente para manter decúbito esternal. </li><li> Adicionar Sulfamethoxiazole Trimetoprim (5 ml por 500 ml de água, contendo 40 mg de trimetoprim e 200 mg sulfamethoxiazole) na água durante uma semana. Administrar Buprenorfina duas vezes por dia durante 3 dias após a cirurgia. </li><li> Manter o mouse em um ciclo claro-escuro de 12 horas, alimentados e regada diariamente, umnd é ponderada uma vez por dia até 3 dias após a cirurgia. Nestes três dias, todo o rato com uma perda de mais de 10% do peso corporal é considerado como comprometida, e serão sacrificados em vez de ser submetido a experimentação. </li><li> Separadamente, mudar a gaiola do mouse duas vezes por semana, e monitor para a saúde em geral, pelo menos uma vez por dia. Normalmente, o mouse é usado dentro de 2-3 semanas após as microinjeções estereotáxica. </li></ol> 2. aguda do cérebro Slice Preparação para confocal Ca 2+ imagem 2.1) Preparação de Corte e Soluções de gravação. <ol><li> Preparar solução compreendendo (mM) de corte: 194 de sacarose, 30 NaCl, 4,5 de KCl, 10 D-glucose, 1 MgCl2, 1.2 NaH 2 PO 4, 26 e NaHCO 3, saturado com 95% O2 e 5% de CO 2. </li><li> Preparar a solução de gravação compreendendo (mM): NaCl 124, KCl 4,5, MgCl 2 1, 10 D-glicose, 2 de CaCl2, 1,2 de NaH 2 PO 4 </sub&gt;, e 26 de NaHCO3; pH 7,3 - 7,4 290-295 mOsm, saturado com 95% O2 e 5% de CO 2. Encha o copo fatia do cérebro segurando com solução de gravação, e mantê-lo a 34 ° C. </li></ol> 2.2) O fatiamento <ol><li> Duas a três semanas após microinjeções estereotáxica, profunda e terminal anestesiar o mouse, e depois decapitá-lo. </li><li> Extraia o cérebro, e usar uma lâmina para remover o hemisfério direito não injectada, e montar a esquerda na bandeja vibratome usando super-cola. Encher o tabuleiro de vibratome com tampão de corte a frio de gelo, e manter saturando-a com 95% de O2 e 5% de CO 2. </li><li> Cortar fatias de cérebro do estriado coronais ou sagitais a 300 mm de espessura. Normalmente, 4-5 coronal, ou 6-7 fatias estriatais sagital podem ser coletadas. </li><li> Transfira as fatias para o copo fatia segurando aquecido a 34 ° C, e mantê-los lá por 30 min antes de armazená-los em temperatura ambiente por recordi posteriorng. </li><li> Incubar as fatias de cérebro de gravação em tampão oxigenado à temperatura ambiente durante pelo menos 30 min antes de [Ca 2+] i imagiologia. </li></ol> 3. Imuno-histoquímica (IHQ) <ol><li> Duas semanas após a injecção de vírus, perfundir o rato transcardialmente com PBS, seguido por 10% de formalina em PBS. </li><li> Retirar, pós-fixar durante a noite, e cryoprotect o cérebro em tamponada 30% de sacarose por pelo menos dois dias. </li><li> Prepare 40 mm seções usando um micrótomo criostato. </li><li> Enxágüe seções 3 vezes com PBS a 10 min de intervalo. </li><li> Incubar secções durante 1 h em PBS com 10% de soro de cabra normal e com 0,5% de Triton X-100. </li><li> Incubar secções durante a noite a 4 ° C em anticorpo primário preparadas em PBS com 0,5% de Triton X-100. Os anticorpos primários utilizados foram como se segue: galinha anti-GFP 1: 1.000, de ratinho anti-S100β 1: 400, de ratinho anti-NeuN 1: 600, de rato anti-glutamina sintetase 1: 300. </li><li> Enxágüe seções 3 vezes com PBS a 10 mem intervalos. </li><li> Incubar as secções com anticorpos secundários preparadas em PBS com 10% de soro de cabra normal durante 2 horas à temperatura ambiente. Os anticorpos secundários foram os seguintes: anticorpos de cabra anti-murganho Alexa546 1: 500, de cabra anti-galinha Alexa488 1: 500. </li><li> Monte as secções em lâminas de vidro, seco e aplique algumas gotas de antifade meio de montagem, em seguida, cubra lentamente as seções com uma lamela de vidro. Armazenar as lâminas a 4 ° C. </li><li> Tome imagens num microscópio confocal com uma lente de imersão em óleo de 40X com uma abertura numérica de 1,3. </li></ol> 4. confocal [Ca 2+] i Imagem NOTA: [Ca 2+] i de imagem foi feita usando um microscópio confocal com uma lente objetiva de 40X de imersão em água com uma abertura numérica de 0,8. <ol><li> Entre 1 e 5 h após o corte, fatia lugar na câmara de gravação e gravação superfuse com tampão oxigenado à temperatura ambiente com uma taxa de fluxo de 1-2 ml pormin. Em seguida, coloque uma harpa de platina com cordas de nylon em cima da fatia para minimizar o movimento durante o experimento. </li><li> Visualizar e localizar o striatum dorsal com brilhante luminescência campo sob uma objectiva de imersão em água 10X. </li><li> Alterne para a lente objetiva de 40X de imersão em água, e ligue a linha 488 nm do laser de argônio. Ao alterar o plano focal, encontrar células localizadas cerca de 20 mm abaixo da superfície da fatia, exibindo fluorescência basal em Soma, ramos e raminhos. De nota, as células comprometidas normalmente apresentar nível de fluorescência acima da média, o que deve ser evitado. </li><li> Use o modo &quot;quadro varredura &#39;para iniciar a digitalização. Normalmente, a intensidade do laser ajustado para 0,5 - 5% da potência máxima é suficiente para a imagem [Ca 2+] i com o cito-GCaMP3. Para monitorar [Ca 2+] i dinâmica em todo o território dos astrócitos, a &quot;varredura frame &#39;com uma velocidade de varredura de 1 segundo por quadro ou mais rápido é recomeNDED, mas isso tem de ser decidido de acordo com o experimento em questão. </li><li> Se necessário, a imagem [Ca2 +] i em processos, utilize ampliação digital de 2-3 vezes para monitorar 1-2 astrócitos em todo o campo de visão. </li><li> Para evitar pixels saturados durante a gravação, utilize o modo de pesquisa &quot;HiLo&quot; a pseudo as células. Sempre comece com uma &quot;gravação piloto&quot; por cerca de 5 minutos, para testar se a cor vermelha aparece como indicação de saturação. Se for assim, diminuir a potência do laser, ou diminuir a PMT / Gain / compensar os valores e realizar uma outra gravação piloto para testar os valores de pixel estão na faixa não-saturada. Tipicamente, os parâmetros combinados utilizados para imagiologia de astrócitos [Ca 2+] i no corpo estriado são as seguintes: potência de laser de saída 0,5-5% (de 10 mW), PMT 550-650 volts, Ganho de 2,0 - 3,5 vezes maior, e deslocamento 0 - 5%. </li></ol>

<ol>
	<li>Agulhon, C., 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=What+is+the+role+of+astrocyte+calcium+in+neurophysiology.">What is the role of astrocyte calcium in neurophysiology.</a> Neuron. 59, 932-946 (2008).</li><li>Khakh, B. S., North, 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=Neuromodulation+by+extracellular+ATP+and+P2X+receptors+in+the+CNS.">Neuromodulation by extracellular ATP and P2X receptors in the CNS.</a> Neuron. 76, 51-69 (2012).</li><li>Gourine, A. V., 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=Astrocytes+control+breathing+through+pH-dependent+release+of+ATP.">Astrocytes control breathing through pH-dependent release of ATP.</a> Science. 329, 571-575 (2010).</li><li>Florian, C., Vecsey, C. G., Halassa, M. M., Haydon, P. G., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-derived+adenosine+and+A1+receptor+activity+contribute+to+sleep+loss-induced+deficits+in+hippocampal+synaptic+plasticity+and+memory+in+mice.">Astrocyte-derived adenosine and A1 receptor activity contribute to sleep loss-induced deficits in hippocampal synaptic plasticity and memory in mice.</a> J Neurosci. 31, 6956-6962 (2011).</li><li>Shigetomi, E., Jackson-Weaver, O., Huckstepp, R. T., O'Dell, T. J., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPA1+channels+are+regulators+of+astrocyte+basal+calcium+levels+and+long-term+potentiation+via+constitutive+D-serine+release.">TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive D-serine release.</a> J Neurosci. 33, 10143-10153 (2013).</li><li>Tong, X., Shigetomi, E., Looger, L. L., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+calcium+indicators+and+astrocyte+calcium+microdomains.">Genetically encoded calcium indicators and astrocyte calcium microdomains.</a> Neuroscientist. 19, 274-291 (2013).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat Neurosci. 13, 759-766 (2010).</li><li>Shigetomi, E., 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=Imaging+calcium+microdomains+within+entire+astrocyte+territories+and+endfeet+with+GCaMPs+expressed+using+adeno-associated+viruses.">Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.</a> J Gen Physiol. 141, 633-647 (2013).</li><li>Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+near+plasma+membrane+and+global+intracellular+calcium+dynamics+in+astrocytes.">Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes.</a> J Vis Exp. 26, (2009).</li><li>Reeves, A. M., Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+loading+of+calcium+indicator+dyes+to+study+astrocyte+physiology:+key+limitations+and+improvements+using+morphological+maps.">Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps.</a> J Neurosci. 31, 9353-9358 (2011).</li><li>Li, D. D., Agulhon, C., Schmidt, E., Oheim, M., Ropert, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+investigating+astrocyte-to-neuron+communication.">New tools for investigating astrocyte-to-neuron communication.</a> Frontiers in Cellular Neuroscience. 7, (2013).</li><li>Davila, D., Thibault, K., Fiacco, T. A., Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+molecular+approaches+to+understanding+astrocyte+function+in+vivo.">Recent molecular approaches to understanding astrocyte function in vivo.</a> Front Cell Neurosci. 7, 272 (2013).</li><li>Paxinos, G., Franklin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2012).</li><li>Perea, G., Yang, A., Boyden, E. S., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+astrocyte+activation+modulates+response+selectivity+of+visual+cortex+neurons+in+vivo.">Optogenetic astrocyte activation modulates response selectivity of visual cortex neurons in vivo.</a> Nat Commun. 5, 3262 (2014).</li><li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119, 7-35 (2010).</li><li>Eid, 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=Loss+of+glutamine+synthetase+in+the+human+epileptogenic+hippocampus:+possible+mechanism+for+raised+extracellular+glutamate+in+mesial+temporal+lobe+epilepsy.">Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.</a> Lancet. 363, 28-37 (2004).</li><li>Eid, T., Williamson, A., Lee, T. S., Petroff, O. A., de Lanerolle, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+astrocytes--key+players+in+human+mesial+temporal+lobe+epilepsy.">Glutamate and astrocytes--key players in human mesial temporal lobe epilepsy.</a> Epilepsia. 49 Suppl 2, 42-52 (2008).</li><li>Tong, X., 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=Astrocyte+Kir4.1+ion+channel+deficits+contribute+to+neuronal+dysfunction+in+Huntington's+disease+model+mice.">Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.</a> Nat Neurosci. 17, 694-703 (2014).</li><li>Ortinski, P. I., 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=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nat Neurosci. 13, 584-591 (2010).</li><li>Zhang, Y., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr Opin Neurobiol. 20, 588-594 (2010).</li><li>Reimsnider, S., Manfredsson, F. P., Muzyczka, N., Mandel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+transgene+expression+after+intrastriatal+pseudotyped+rAAV2/1,+rAAV2/2,+rAAV2/5,+and+rAAV2/8+transduction+in+the+rat.">Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat.</a> Mol Ther. 15, 1504-1511 (2007).</li></ol>

Os autores não têm nada a revelar.

Os métodos aqui descritos nos permitiram expressar cito-GCaMP3 em astrócitos do estriado in vivo para posterior [Ca 2+] i imaging in situ. Este método apresenta vantagens em relação ao uso transgénica ou knock-em ratinhos, incluindo expressão robusta da proteína, rapidez e flexibilidade orientada de aplicação experimental e especificidade anatómica. A expressão de GCaMP3 usando AAV2 / 5, foi encontrada para ser mais específico e robusto. A combinação de GFAP GfaABC ...

Os astrócitos são células gliais ubíquas e abundantes do cérebro. Está bem estabelecido que os astrócitos servir de suporte vital e papéis homeostáticos incluindo buffer de K + concentração no espaço extracelular, a captação de neurotransmissores, bem como o fornecimento de nutrientes. No entanto, estudos recentes mostram que eles também exibem [Ca 2+] i sinais, que ocorrem espontaneamente e são aumentados pela atividade neuronal 1. A existência de astrócitos [Ca 2+] i sinalização tem sido cada vez mais pensado para provocar a sua comunicação com os neurônios, e, como tal, tem sido interpretada como uma forma de &quot;Ca 2+ excitabilidade&quot; dentro de astrócitos. Os dados disponíveis sobre as últimas duas décadas sugerem duas configurações em que os astrócitos e neurônios podem se comunicar, talvez, de forma bidirecional. Em primeiro lugar, os astrócitos, muitas vezes respondem com um aumento na [Ca 2+] i, quando activado pelos neurotransmissores eneuromoduladores liberados de neurônios 2. Em segundo lugar, [Ca 2+] i aumenta em astrócitos causam a liberação de moléculas de sinalização de astrócitos, que por sua vez podem afetar os neurônios e vasos sanguíneos. As evidências sugerem que as moléculas liberadas a partir de astrócitos levar a alterações nas funções de sinapses, circuitos e, em última instância comportamento 3-5 via de sinalização astrócitos-a-neurônio. No entanto, esta continua a ser uma área de pesquisa em rápido desenvolvimento, e tem-se argumentado que uma compreensão melhor e detalhada de astrócitos [Ca 2+] i é necessário para resolver algumas das incertezas atuais 6. Em trabalhos anteriores, foi demonstrado que o carregamento em massa de orgânicos Ca 2+ corantes indicador em astrócitos não consegue detectar com fiabilidade [Ca 2+] i sinais dentro astrócitos inteiras em cultura e in situ 10/07. Estes resultados foram discutidos por nós e outros 6,11,12. A Emerging imagem é que [Ca 2+] i sinais dentro dos processos de astrócitos (por exemplo, ramos e raminhos), que são os principais sítios de interação com neurônios e vasos sanguíneos, raramente têm sido explorados em detalhe. Recentemente, a utilização de indicadores de cálcio geneticamente codificados (Gecis), tais como GCaMP3 citosólica, GCaMP5G e GCaMP6 e membrana de plasma (por exemplo, versões tethered, Lck-GCaMP3) permitiu o estudo da [Ca 2+] i sinais em pequenos compartimentos de astrócitos tais processos como finas, perto da membrana do plasma e dentro territórios inteiros 7,8. No entanto, Gecis têm uma desvantagem sobre orgânicos Ca 2+ corantes indicadores e que é a exigência de métodos genéticos para entregar os genes que codificam para selectivamente astrócitos in vivo por períodos de semanas para a Gecis a ser apropriadamente expressa. Expressão in vivo é tipicamente alcançado utilizando camundongos transgênicos, bater-nos ratos ou com app entrega vírus baseadosbaratas. No presente artigo, relatamos JoVE métodos e procedimentos empregados para entregar Gecis para astrócitos do estriado usando vírus adeno-associados. Nós concentrar em cito-GCaMP3 como um exemplo, mas o mesmo procedimento básico funciona para qualquer outro GECI ou fluorescente repórter base de proteína.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>704506</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Capillaries</td>
			<td >World Precision Instruments</td>
			<td >1B100-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Narishige</td>
			<td >PC-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette grinder</td>
			<td >Narishige</td>
			<td >EG-40</td>
			<td></td>
		</tr>
		<tr >
			<td >pZac2.1 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I mL syringe</td>
			<td >BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe needle</td>
			<td >BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr >
			<td >AAV2/5 virus</td>
			<td >UPenn vector core</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan red IV</td>
			<td >Sigma-Aldrich</td>
			<td >67386</td>
			<td></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >CVS Pharmacy</td>
			<td>152355</td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat</td>
			<td >Leica</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
			<td >900LS</td>
			<td></td>
		</tr>
		<tr >
			<td >High Speed Rotary Micromotor Kit</td>
			<td >FOREDOM</td>
			<td >K.1070</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Santa cruz biotechnology</td>
			<td >sc-281692</td>
			<td></td>
		</tr>
		<tr >
			<td >Super Glue</td>
			<td>Krazy&#174;Glue</td>
			<td>KG925</td>
			<td></td>
		</tr>
		<tr >
			<td >Microslicer</td>
			<td >Ted Pella</td>
			<td >DTK-Zero 1</td>
			<td></td>
		</tr>
		<tr >
			<td >Confocal microscopes</td>
			<td >Olympus</td>
			<td >FV300 and FV1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal goat serum</td>
			<td >Vector</td>
			<td >S-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >chicken anti-GFP</td>
			<td >Abcam</td>
			<td >ab13970</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-s100&#946;</td>
			<td >Sigma-Aldrich</td>
			<td >S2532</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-NeuN</td>
			<td >Millipore</td>
			<td >MAB377</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-glutamine synthetase</td>
			<td >Millipore</td>
			<td >MAB302</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-mouse-Alexa546</td>
			<td >Invitrogen</td>
			<td >A11003</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-chicken-Alexa488</td>
			<td >Invitrogen</td>
			<td >A11039</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover Glass</td>
			<td >Fisher Scientific</td>
			<td >12-548-5J</td>
			<td></td>
		</tr>
		<tr >
			<td >Mounting Medium</td>
			<td >Vector</td>
			<td >H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Para a expressão especifica de astrócitos de cito-GCaMP3 no striatum, utilizou-se vírus adeno-associado (AAV), do serotipo 5, e a PGFA GfaABC 1 D promotor (Figura 1A), que tem sido mostrado previamente para dirigir GCaMP3 robusta e o gene repórter expressão no hipocampo e astrócitos corticais 8,14. Duas semanas após a micro-injecção de vírus para o corpo estriado do rato, o rato (~ 10 semanas de idade) foi perfundido e IHC foi realizada em secções de cérebro finas para...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

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 ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Research

JoVE Journal

Neuroscience

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

14.7K visualizações.  University of California Los Angeles. 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. 

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

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.

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 ...

Neurociência

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

De gravação simultânea de sinais de cálcio dos neurônios identificados e comportamento alimentar de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelagem astrocitoma Patogênese<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Usando Cortical astrócitos ou células-tronco neurais de condicional, Geneticamente Modificadas Ratos

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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 ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Dissecação de locais de Ca2 + sinais em pilhas cultivadas pelos indicadores de membrana-alvo Ca2 +

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Indicadores únicos de sinapse de liberação de glutamato e absorção em fatias cerebrais agudas de ratos normais e huntington

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualizando e analisando o transporte intracelular de organelas e outras cargas em astrócitos

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Minimizando a hipóxia em fatias hipocampais de camundongos adultos e envelhecidos

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.