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>

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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><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 GfaABC&lt;sub&gt;1&lt;/sub&gt;D.cyto-GCaMP3</td><td>Addgene</td><td>44331</td><td>a plasmid sent to UPenn Vector Core for virus packaging</td></tr><tr><td>1 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&amp;reg;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&amp;beta;</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

Universidad de Cartagena UDEC

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

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Medição Perto membrana plasmática e global Dynamics cálcio intracelular em astrócitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biologia

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induzir Plasticidade de astrocítica Receptores de Manipulação de Neuronal queima de moeda

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Neurociência

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Cultivo In Vivo-gosto murino astrócitos usando o protocolo AWESAM rápida, simples e barato

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivo Imagem de célula específica cálcio sinalização em sinapse tripartida do diafragma de rato

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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.

In vivo Imagem de cálcio em rato de azeitona inferior

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 ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Imagem pericínica cerebral de cálcio e hemodinâmica em camundongos transgênicos em Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Imagem Mitocondrial Ca2+ Absorção em Astrócitos e Neurônios usando Indicadores Ca2+ Codificados Geneticamente (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

In vivo Imagem de cálcio de campo amplo e de dois fótons de um rato usando uma grande janela craniana

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In vivo Imagem com cálcio de conjuntos neuronais em redes de neurônios sensoriais primários em gânglios intactos da raiz dorsal

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Imagens em tempo real para atividade de neurônios em células do giro dentado de camundongos 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Autor em destaque: Investigando a atividade neural de células granulares do giro dentado com microscópio em miniatura

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Imagem de cálcio in vivo de células granulares no giro dentado do hipocampo em camundongos 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

Imagens intracelular Ca

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