Agradecemos ao Dr. S. Stella e Helen Vuong para contribuições para o protocolo de imagem de cálcio. Agradecemos Sheets Dr. K. para filmar as cenas de entrevista. Agradecemos ao Dr. Arlene Hirano por seus comentários sobre o manuscrito. Este projeto de pesquisa e desenvolvimento foi realizado pelos autores na Geffen de Medicina David na UCLA e é possível graças a um acordo de contrato que foi concedido e administrado pela Research &amp; Material Médico Comando do Exército dos EUA (USAMRMC) ea Telemedicina e Tecnologia Avançada Centro de Pesquisa (TATRC), em Fort Detrick, Maryland sob Número do Contrato: W81XWH-10-2-0077. O suporte para esses estudos também vieram NIH EY04067 e uma revisão Mérito VA (NB). BCN é uma VA Carreira de Investigação Scientist.

Todos os experimentos foram realizados em conformidade com as orientações para o bem-estar dos animais experimentais de emissão da Política de Serviço de Saúde Pública dos EUA em Cuidado Humano e Uso de Animais de Laboratório e da Universidade da Califórnia-Los Angeles (UCLA) Comitê de Pesquisa Animal. Foram utilizados Macho e fêmea adultos ratos Sprague-Dawley (Charles River Lab, Wilmington, MA) entre a idade de 3-5 semanas. 1. Animais e Preparação de tecidos para Wholemount Retina e imunohistoquímica Protocolo <ol><li> Prepare os seguintes materiais e ferramentas: um microscópio de dissecação, 2 pinças com pontas muito finas, tesouras, papel de filtro de celulose, pipeta de plástico e uma lâmina de microscópio. </li><li> Eutanásia o rato numa câmara fechada por profundamente anestesiar os animais com 1-3% de isoflurano seguido por decapitação com uma guilhotina. Remover os olhos com um par de tesouras e de íris lugar em uma placa de Petri contendo a solução extracelular. Hibernate A, Ames-medium ou soluções fisiológicas são recomendados. </li><li> [Etapa opcional se aterro de CGR com Fluo-4 é desejado]. Realizar a rotulagem de Fluo-4, conforme descrito no 2,3-2,4 passos. </li><li> Utilizando o microscópio de dissecação (intensidade de luz: 1,6 x 10 8 fotões / mm 2 ⋅sec), remover a córnea cortando a parte dianteira do globo ocular. Retirar a lente e vítreo a partir da superfície interior da retina com uma pinça. Para remover o vítreo, estabilizar o globo ocular, mantendo a esclera firmemente com uma pinça. Descascar delicadamente a base vítrea para o centro da retina com a outra pinça. A preparação nesta fase é chamada a ocular, a qual é usada para criocortes. Mais detalhes sobre criocortes podem ser encontradas em referências 14,15. </li><li> Retire a retina do ocular. Em seguida, faça quatro cortes para permitir a retina para colocar o plano. Gentilmente montar a retina numa lâmina de microscópio com o fotorreceptor camada de cima. Adicionar uma gota de solução para a retina e colocar cepapel de filtro llulose na retina. Uma vez que a retina atribui ao papel de filtro, colocar a retina para trás em solução. Se necessário, achatar a retina com uma escova ou uma pinça. </li><li> Coloque as retinas wholemounted em PFA 4% por 10-15 min para fixação. </li><li> Lavam-se as retinas wholemounted três vezes durante 30 min em tampão fosfato 0,1 M (PB), pH 7,4. Bloquear as retinas com 5% de soro normal de cabra ou soro de burro em PB 0,1 contendo 0,3% de Triton X-100 a 0,1% e NaN 3 a 4 ° C durante a noite. Recomendamos um volume final de 500 mL para todas as etapas para salvar soluções de bloqueio e anticorpos. </li><li> No dia seguinte, incubar as wholemounts retinas com os anticorpos primários 5-7 dias a 4 ° C. As diluições ótimas deve ser determinado pelo usuário. </li><li> Lava-se a retina 3 x 30 min em 0,1 M PB e incubar durante a noite a 4 ° C, em que o anticorpo secundário conjugado com fluoróforo correspondente que é dirigido contra a espécie de anticorpo primário (concentração de 1: 1000). </li><li> Após três lavagens final de 30 min cada um com PB, montar as retinas em meio de montagem. Vedar as lamelas com unha polonês para o armazenamento prolongado. Armazene as amostras a 4 ° C e proteger da luz. </li></ol> 2 Rotulagem de células ganglionares e axônios Rat Wholemount Retinas com Cálcio Indicador Dye <ol><li> Preparar 1000 ml de Ringer de mamífero contendo (em mM) 125 de NaCl, 3 de KCl, 2 CaCl2, 1,25 NaH 2 PO 4, 1 de MgCl2, 25 de NaHCO3, 10 de glucose e borbulhado com 95% de O2 / 5% de CO &#173; 2. </li><li> Efectuar a dissecção como descrito no 1,2-1,4 passos. </li><li> Para carregar as células ganglionares da retina (RGCs) e seus axónios com um corante indicador de cálcio, injectar 0,5 ul de Fluo-4 sal pentapotássio (40 mM em H 2 O) para o coto do nervo óptico de cerca de 1 mm posterior ao globo ocular, utilizando uma seringa . Para medir um aumento dinâmico em células ganglionares da [Ca2 + </sup&gt;] i um indicador com elevada afinidade, tais como Fluo-4 com o seu K d de 345 nM, é óptima. Fluo-4 oferece a vantagem adicional de possuir uma elevada eficiência quântica, resultando num aumento das emissões de&gt; 100 vezes quando ligado ao cálcio. </li><li> Coloque a ocular no borbulhar com 95% de O2 / 5% de CO de Ringer de mamífero &#173; 2, durante 1 hora à temperatura ambiente no escuro. </li></ol> 3. Cálcio imagem Protocolo de células ganglionares da retina e axônios. <ol><li> Remover o olho, da lente e vítreo, tal como descrito nos passos 1,1-1,4. Isolar a retina do ocular e divida em quadrantes. Montagem lado de células ganglionares um quadrante se numa lâmina de vidro e usar uma grade fatia harpa para estabilizá-la. Mantenha as outras peças escuro adaptado em aeradas com 95% de O2 / 5% CO mamíferos de Ringer &#173; 2 em gelo para posterior utilização. </li><li> Superfuse a câmara grava com solução de Ringer e mamíferosmanter a solução borbulhar continuamente com 95% O2 / 5% de CO 2. </li><li> Imagem do tecido sob o microscópio. Realizar experiências à temperatura ambiente (~ 23 ° C), ou atingir temperaturas fisiológicas por aquecimento da fase, o aquecimento do banho de água por baixo da amostra, ou a aplicação de ar quente para a superfície do espécime. Nota: É importante notar que muitas funções celulares são regulados pela temperatura, que desempenha um papel fundamental na manutenção da homeostase intracelular 16. No entanto, a introdução de medidas de aquecimento pode aumentar a taxa de fotodegradação, a evaporação do desvio médio e foco causada pela expansão térmica 17. Utilização de temperatura ambiente diminui compartimentalização intracelular de Fluo-4 18. </li><li> Executar um controlo de dois de K + pulsos emparelhados elevados na ausência de drogas. Despolarizar RGCs e RGC axónios através do aumento da [K +] o de 3 mM a 60 mM durante 33 segundos, durante cada pulso para activarVGCCs com um sistema de superfusão impulsionado oito gravidade canal. Reduzir o Na + por 57 mM para manter isosmolarity na solução de elevado K +. </li><li> Avaliar a contribuição de diferentes VGCCs ao sinal de cálcio com o uso de bloqueadores dos canais de Ca gerais ou específicas. Por exemplo, a redução no sinal de cálcio após a administração de um bloqueador do canal de cálcio não-específica, o cobalto, fornecida uma medida semi-quantitativa da contribuição dos VGCCs ao sinal -evoked cálcio de K + elevado. </li><li> Adquirir os valores de intensidade de fluorescência através da colocação de uma região de interesse (ROI) em torno das CGR de interesse (Figura 2). </li><li> Normalizar a mudança na intensidade de fluorescência produzida pelo segundo pico de K + elevado para a alteração produzida por o primeiro pico de K + elevado. Em seguida, para o teste de bloqueadores dos canais de cálcio específicos, aplicar drogas apenas durante o segundo pulso de K + elevado de um par e normalizar este pico contrao primeiro valor de pico. Para medir o efeito do fármaco sobre a K + elevado pico, dividir a amplitude do segundo pico de K + pelo que a do primeiro. A análise deve ser executada em estes valores em relação aos seus controlos correspondentes derivados de K + elevado emparelhado picos medidos na ausência de fármaco. </li><li> Adquira imagens em intervalos de 5 seg. Para evitar a foto-branqueamento, manter o laser de excitação o mais baixo possível. Fornecer excitação pela linha 488 nm do laser de árgon, enquanto o tubo fotomultiplicador recolhe a emissão de fluorescência através de um filtro PB de 505 nm. </li></ol>

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Neste artigo, descrevemos duas técnicas diferentes: 1) Imunohistoquímica para mostrar Fluo-4 de localização para as células ganglionares da retina em wholemounts, e 2) Imagem de cálcio para analisar a dinâmica de cálcio em células ganglionares da retina e seus axônios. A imuno-histoquímica utilizando wholemounts foi usado para revelar a localização de proteínas no roedor retina 20-23. No entanto, existem algumas limitações. A qualidade da coloração depende muito ...

As células ganglionares da retina (RGCs) expressar L, N, P / Q e T-tipo VGCC como determinado por meio do bloqueio farmacológico desses componentes de toda a célula actual canal Ca 1,2. VGCC são proteínas transmembrana multimï¿½icos que estão envolvidos na liberação do transmissor, a transcrição do gene, regulação celular e sináptica 3,4,5 plasticidade. VGCCs funcionais são compostos de pelo menos três classes distintas de subunidades: grandes, transmembrana α 1 poro formando subunidades que estabelecem propriedades biofísicas e farmacológicas do canal, principalmente extracelular α auxiliar 2 δ subunidades e subunidades β intracelulares. Os dois últimos formam complexos heteromï¿½icas com diferentes α um subunidades e alterar a cinética de propagação e tráfico de canais para a membrana plasmática 6. Nas últimas décadas, várias técnicas têm sido empregadas para estudar expre proteínassion, tais como imuno-histoquímica, ligada ensaio imunoenzimático, análise de Western e citometria de fluxo. Estas técnicas requerem a utilização de anticorpos específicos para a detecção de uma determinada proteína de interesse e fornecem ferramentas poderosas para a localização e distribuição de proteínas específicas em diferentes tecidos. As técnicas usadas para detectar e quantificar níveis de uma proteína particular de expressão de ARNm, tais como a análise de manchas de Northern, RT-PCR, em tempo real quantitativa de RT-PCR, hibridação in situ, de microarrays e ensaio de protecção de ribonuclease proporcionam uma abordagem alternativa, quando os anticorpos não são prontamente os níveis de expressão ou se disponíveis de uma proteína particular são baixos 7. No entanto, uma limitação para a utilização de tais técnicas moleculares é necessária a identificação da sequência do gene. Para localizar as proteínas na retina, imuno-histoquímica pode ser realizada em retinas wholemount. Devido à acessibilidade das CGR, a wholemountpreparação fornece uma plataforma excelente para o estudo da localização de proteínas específicas para o RGC somata e seus axónios. Em adição à sua localização, algumas propriedades funcionais dos VGCCs em CGRs pode ser demonstrada através da utilização de técnicas de imagiologia de cálcio. Descreve-se um protocolo de imagens de cálcio para etiquetar selectivamente CGR com um corante indicador de cálcio para medir a dinâmica de cálcio intracelulares. A contribuição de diferentes VGCCs ao sinal de cálcio em diferentes compartimentos celulares podem ser isoladas com o uso de bloqueadores dos canais de Ca específicos do subtipo. Talvez um dos aspectos mais benéficos da técnica de imagiologia de cálcio descritos aqui é a capacidade de gravar, simultaneamente e de forma independente a partir de múltiplas CGR e seus axónios. Embora muitas técnicas fisiológicas, como a correção de células gravação braçadeira todo, proporcionar gravações de alta resolução temporal das correntes de membrana, o somático ou fonte axonal de these correntes gravados não podem ser discriminados e gravações só pode ser feita a partir de um único neurónio de cada vez. Matrizes multieletrodo (AMA) são capazes de gravar simultaneamente picos de muitas células, mas podem detectar e nem discriminar a ativação de, por exemplo, diferentes subtipos de canais de cálcio. Meas preferencialmente gravar a partir de células que estão localizadas na proximidade de um determinado eletrodo 8 e gravar a partir de células que geram grandes picos de 9. Métodos de imagem Optical fornecer uma estratégia alternativa para permitir que as gravações simultâneas e independentes de populações inteiras de células que podem ser integrados com as informações obtidas a partir de uma única célula de microeletrodos e remendo gravação de fixação e gravação de MEA. Embora tenham sido empregues as técnicas de imagiologia de cálcio descritos aqui para estudar a dinâmica de cálcio de CGRs, sujeição do emplastro e AAM também podem ser utilizados em paralelo para elucidar as correntes iónicas e propriedades spiking de CGRs. <pclass = &quot;jove_content&quot;&gt; Como as células amácrinas deslocadas compõem cerca de 60% ​​da população dos neurônios da camada de células ganglionares da retina do rato 10, o nosso objetivo era usar uma técnica de carga que rotula seletivamente CGR com um corante indicador de cálcio sintético em um preparação wholemount. Embora corantes indicadoras de cálcio sintéticos fornecem uma excelente plataforma para o estudo da dinâmica de cálcio intracelular, a sua utilização generalizada tem sido dificultado pela incapacidade para carregar de forma eficaz as populações específicas de neurónios no interior de uma determinada rede. Muitas técnicas, tais como volume de carga 11 e eletroporação 8,12 foram realizados para carregar populações inteiras de células, no entanto, essas técnicas não discriminar entre tipos específicos de células. Geneticamente codificado indicadores de cálcio fornecem a capacidade para identificar selectivamente populações específicas de células, no entanto, estes métodos requerem a geração de animais transgénicos 13. Nossa técnica descreve uma metod etiquetar seletivamente CGR na preparação wholemount via óptica injeção coto do nervo de um corante indicador de cálcio. Tomadas em conjunto, as técnicas estruturais e fisiológicas descritas neste artigo fornecer uma plataforma para o estudo da localização e da contribuição dos VGCCs para o sinal de cálcio no CGR e seus axônios.

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			<td>Invitrogen</td>
			<td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=A1247501">A12475-01</a></td>
			<td>Media</td>
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			<td><a href="https://www.google.com/url?sa=t&amp;rct=j&amp;q=&amp;esrc=s&amp;source=web&amp;cd=1&amp;cad=rja&amp;sqi=2&amp;ved=0CC0QFjAA&amp;url=http%3A%2F%2Fwww.vectorlabs.com%2Fcatalog.aspx%3FcatID%3D279&amp;ei=VAHbUb_fBceeiQLK2IHIDw&amp;usg=AFQjCNHijFTTB8p19FMPQ5hg_qTawXU9mA&amp;sig2=FJCfB9fzKR_TmjxoGvhLcg&amp;bvm=bv.48705608,d.cGE">Mounting Media for Fluorescence</a></td>
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			<td height="21" style="height:21px;width:216px;">Microscope slide</td>
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			<td height="21" style="height:21px;width:216px;">Zeiss LSM 5 Pascal microscope</td>
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			<td height="42" style="height:42px;width:216px;">Axioplan 40x (NA 0.8) objective lens</td>
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			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:112px;">Molecular Probes</td>
			<td style="width:119px;">A-11034</td>
			<td>Secondary antibody</td>
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			<td height="42" style="height:42px;width:216px;">RBPMS</td>
			<td style="width:112px;">ProSci, Poway, CA</td>
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			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
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immunohistochemical and calcium imaging methods in wholemount rat retina

Neste artigo descrevemos as ferramentas, os reagentes e os passos práticos que são necessários para: 1) preparação bem-sucedida de retinas wholemount para imunohistoquímica e, 2) imagens de cálcio para o estudo da tensão fechado de canais de cálcio (VGCC) mediada sinalização de cálcio na retina células ganglionares. O método de imagem de cálcio que descrevem problemas contorna relativas à carga não-específica de células amácrinas deslocadas na camada de células ganglionares.

Imunomarcação com anticorpos específicos proporciona uma plataforma para o estudo da localização de proteínas de interesse particular na retina e na técnica de imagiologia de cálcio permite o estudo da contribuição dos VGCCs para a dinâmica de cálcio nas células ganglionares da retina e seus axónios. Ao utilizar um anticorpo contra as células ganglionares RBPMS proteína (proteína de ligação de ARN com splicing múltipla) que rotula selectivamente CGRs 19, fomos ...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Imunohistoquímica protocolos são usados ​​para estudar a localização de uma proteína específica na retina. Técnicas de imagem de cálcio são utilizados para estudar a dinâmica de cálcio nas células ganglionares da retina e seus axônios.

Os métodos de imagem de imuno-histoquímica e cálcio em Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

immunohistochemical-calcium-imaging-methods-wholemount-rat

Research

JoVE Journal

Neuroscience

14.4K visualizações.  University of California, Los Angeles.  Imunohistoquímica protocolos são usados ​​para estudar a localização de uma proteína específica na retina. Técnicas de imagem de cálcio são utilizados para estudar a dinâmica de cálcio nas células ganglionares da retina e seus axônios. 

Vídeo: Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Imagem de cálcio funcional em desenvolvimento de redes Cortical

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Neurociência

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Imuno-histoquímica para Topografia Wholemount cerebrais, Complexo

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

In vivo Neuronal Calcium Imaging in C. elegans

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

In vivo Neuronal Calcium Imaging in C. elegans

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

Na imagem cálcio vivo Neuronal em C. elegans

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

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Análise imuno-histoquímica no sistema nervoso central e periférico Rat linfonodo Tissue Secções

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Parênquima não-invasivo, Vascular e Metabólica alta freqüência ultra-som e Fotoacústica Rat Cerebral Profunda Imagem

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imagem Subcelular da Manipulação Neuronal de Cálcio In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Imagem de cálcio de dois fótons e campo amplo no colículo superior

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Autor em destaque: Revelando a codificação neural e os mecanismos de processamento visual no colículo superior 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Imagem de cálcio no colículo superior de camundongos

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...