Este trabalho foi financiado pelo apoio através da Fundação McNair, NARSAD, e conceder NINDS R00NS064171-03.

1. Na fixação do cérebro situ * Prepare uma seringa de 10 ml (28 agulha) preenchido com tampão fosfato salino (PBS). * Prepare uma seringa de 10 ml (28 agulha) preenchido com paraformaldeído a 4% (PFA) em PBS. Reserve um adicional de 50-10 ml de PFA / PBS para fixação post. <ol><li> Injetar por via intraperitoneal rato experimental com uma dose letal de Avertin ou Nembutal. </li><li> Uma vez que o mouse está sedado, molhar o abdômen com etanol e segura o mouse para a parte inferior de uma bandeja em camadas com a cortiça, a cera, ou elastômero de silicone utilizando os pinos de dissecação. Com abdômen virado para cima, segura as quatro patas para a superfície - espalhando-os o mais amplo possível. </li><li> Agarrar a pele com uma pinça ao nível do esterno, e cortado transversalmente para expor o fígado. Corte lateral e depois para cima através das costelas e diafragma. Remover o retalho de tecido costela e continue cortando até que o coração está acessível. </li><li> Furar ou cortar o átrio direito para drenar o sangue circular. </li><li> Lave sangue residual do sistema circulatório por perfusão de PBS através do ventrículo esquerdo. </li><li> Acessando o buraco mesma agulha, corrigir rato inteiro por perfusão subsequente de PBS / PFA através do ventrículo esquerdo. </li></ol> 2. Dissecção e extração de cérebro <ol><li> Retire a pele da cabeça, pescoço e crânio, reduzindo em torno de canal auditivo e as órbitas dos olhos, seguida puxando a pele anteriormente para expor o crânio. * Veja a Figura 2 para uma ilustração de passos 2.2) a 2.5) </li><li> Usando uma tesoura osso, cortado transversalmente do olho do soquete através do corneto nasal ossos. Faça isso para cada lado. O crosscut deve ser anterior aos olhos e bulbos olfatórios. </li><li> Corte longitudinal de cada canal de orelha a cavidade ocular. </li><li> Usando uma tesoura pequena dissecção, corte a placa de osso occipital que cobre o cerebelo de um canal do ouvido para o outro. </li><li> Com a tesoura mesmo dissecção, corte anteriormente ao longo da linha média ao nível dos bulbos olfativos. </li><li> Usando pinça, retire cada placa óssea a partir do cérebro, tendo o cuidado de remover qualquer tecido conjuntivo para que o cérebro permanece intacto ao levantar fora do osso. </li><li> Nervos cranianos e Snip óptica, corte as vértebras cervical superior e remova o cérebro em fixador. </li><li> Pós-fix por 1-2 horas a 4 ° C. Lavar 3 vezes 15 min cada em PBS. </li></ol> 3. Incorporação de agarose <ol><li> Derreta 2% de alta resistência, com baixo ponto de gel de agarose tipo IB (Sigma número de catálogo: A0576) em PBS salina com forno de microondas. </li><li> Transferência de agarose fundida a um tubo de ensaio. Coloque o tubo de ensaio em um banho de água morna ou incubadora (40 ° -41 ° C) até a temperatura de equilíbrio. </li><li> Cola tecido cerebral fixa para o êmbolo da seringa amostra com cola de cianoacrilato (Figura 3A). Pincel uma fina camada de solução de sacarose-PBS saturada na superfície do tecido cerebral para facilitar a liberação posterior do anel de agarose as fatias de cérebro. </li><li> Sacar o êmbolo com o tecido do cérebro dentro do alojamento de êmbolo (Figura 3B). </li><li> Pipeta ou derramar agarose quente dentro da caixa do êmbolo, cobrindo o tecido cerebral. Evite bolhas de interceptação no agarose. Bolhas de ar no agarose irá comprometer a qualidade fatia. </li><li> Grampo da seringa com uma amostra de frio (° C a -25 ° C 0) bloco de refrigeração por 1 minuto para definir a agarose (Figura 3C). </li></ol> 4. Seccionamento fatia do cérebro com o Compresstome <ol><li> Monte a seringa contendo a amostra de tecido cerebral no Compresstome (Figura 3D). </li><li> Alinhe a extremidade da lâmina de barbear em estreita colaboração com a saída da seringa de amostra (Figura 3E). </li><li> Encher o tanque com a solução tampão PBS. </li><li> Gire o micrômetro de espessura uma fatia para a frente a extrusão do bloco de tecido cerebral agarose-out da seringa espécime. </li><li> Pressione o botão &quot;iniciar&quot; na unidade de controle do Compresstome para iniciar o processo de corte. </li><li> Repita os passos 4.4) a 4.5) até fatias desejados são obtidos em determinar a espessura. </li><li> Colheita fatias de cérebro com pincel ou pipeta para frascos ou poços coloração preenchido com PBS (Figura 3F). </li></ol> 5. Clearing óptica <ol><li> Prepare um 75% vol: vol solução de glicerol: PBS. </li><li> Pour (ou pipeta) off metade do volume de PBS de lavagem dos fatias de cérebro coletados. </li><li> Adicionar novamente o volume removido com glicerol / PBS. </li><li> Deixar equilibrar com a mistura suave a 4 ° C até a pia fatias. </li><li> Repita os passos 6.2) a 6,4) até uma concentração de glicerol final atinja os 75%. </li><li> Substituir solução com 90% de glicerol em PBS e deixar equilibrar. * Nota: Tome cuidado para não introduzir bolhas de ar, enquanto a adição de soluções de glicerol. Despeje lentamente soluções e misture delicadamente, uma vez que as bolhas de ar irá interferir com uniforme equilibração e será transferido para montagem de carros. </li></ol> 6. Fatia de montagem para imagens <ol><li> Corte de dupla face adesiva de vedação (SA-S-1L, Grace Bio-Labs) em um retângulo aberto para se ajustar à área visível de um slide micro (Superfrost Plus, 25 X 75 X 1 gato mm,. # 48311-703, VWR ) com lados 2-3 mm de largura. </li><li> Coloque fatias de cérebro dentro do centro do retângulo de fita adesiva, utilizando uma pipeta ou pincel. Remover o glicerol em excesso com a aspiração suave. </li><li> Arrume as fatias de cérebro em slide na ordem desejada com um pincel. </li><li> Tampa de vidro delicadamente mais baixo (24 X 60 mm, gato. # 48383-139, VWR) sobre a fita adesiva, tomando cuidado para não introduzir bolhas de ar. Aplique uma suave pressão sobre tampa de vidro para garantir o contato com fita adesiva. </li><li> Aspirar glicerol em excesso das bordas entre lâmina e lamínula. </li><li> Selo de slides e lamela utilizando unha polonês claro. </li><li> Deixar secar antes de imagem, ou armazenamento de longo prazo em caixas de slides, a 4 ° C. </li></ol> 7. Resultados representativos: Processamento, de imagem, e analisar o tecido cerebral fluorescente etiquetado tornaram-se indispensáveis ​​para o estudo da neurobiologia. Muitas dessas investigações requerem sofisticados manipulações genéticas para obter expressão repórter em subconjuntos direcionados neuronal, seguido por ambas as análises de imagem de baixa e alta resolução. Muitas vezes as limitações técnicas experimentais e torná-la impossível obter estes tipos de dados a partir do mesmo animal, ou de uma forma rápida. Preparação de opticamente-desmarcada, seções semi-grossa cérebro para lâmpadas fluorescentes imagem aids neste desafio. Um exemplo de uma fatia do cérebro intacto espessura rotulados com um vírus geneticamente modificados para expressar raiva engenharia EGFP, e fotografada por microscopia de epifluorescência e confocal é mostrado na Figura 4 e Figura 5, respectivamente. Para geração de imagens de epifluorescência usamos uma Leica M205 FA, e para aquisição de imagem confocal foi utilizada uma Zeiss LSM 510. Devido à relativa simplicidade do protocolo anexo, este método é capaz de produzir dados de imagem úteis a partir do tecido expressar repórteres fluorescente com um tempo de retorno de menos de um dia, e é compatível tanto com a microscopia de luz baixa e alta resolução. Se o pesquisador escolhe para incorporar métodos postmortem adicionais rotulagem, coloração imuno-histoquímica, ou fazer cortes mais finos, o protocolo alonga em conformidade. No entanto, o método descrito acima representa um método simples e relativamente high-throughput screening para os padrões de expressão repórter vital no cérebro intacto. <img src="/files/ftp_upload/2807/2807fig1.jpg" alt="Figura 1"/> Figura 1. Fluxograma diagramação a fixação, a dissecção, corte, limpeza e procedimento de montagem de fatias de espessura semi-cérebro. <img src="/files/ftp_upload/2807/2807fig2.jpg" alt="Figura 2"/> Figura 2. Diagrama de passos seqüenciais de corte para extrair o cérebro de camundongos intactos. <img src="/files/ftp_upload/2807/2807fig3.jpg" alt="Figura 3"/> Figura 3. Passos para montar e cortar o tecido cerebral usando o Compresstome. A) Colocação de tecido do cérebro em corte êmbolo com supercola. B) Desenho baixo do tecido cerebral montada no êmbolo para a incorporação de agarose. C) Solidificando uma ficha cérebro agarose usando um bloco de compressão refrigerados. D) Inserção de agarose ficha cérebro e êmbolo em Compresstome câmara de corte. E) O alinhamento da lâmina de barbear para o dispositivo de êmbolo. F) Corte e coleta de fatias de cérebro em Compresstome câmara de buffer. <img src="/files/ftp_upload/2807/2807fig4.jpg" alt="Figura 4"/> Figura 4. Imagens de microscopia de luz de uma grossa fatia de glicerol limpo tecido cerebral através do córtex frontal expressando a proteína verde fluorescente melhorada (EGFP). Uma fatia) Coronal através de um cérebro de camundongo (200 um de espessura) marcada com um vetor viral EGFP expressar e fotografada em baixa resolução usando um estereoscópio de epifluorescência. Escala de bar, 2 mm. <img src="/files/ftp_upload/2807/2807fig5.jpg" alt="Figura 5"/> Figura 5. Ampliação de imagem de alta da camada fluorescente etiquetado 06/05 neurônios corticais em uma fatia do cérebro limpo de espessura. A) Imagem de alta resolução confocal de uma projeção máxima Z-stack (150 hm de espessura) através da região em destaque na (Figura 4). Barra de escala, 25 um.

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Jian-Qiang Kong é um empregado de Precisionary Instruments, Inc., que fabrica um produto utilizado neste artigo.

Dada a aplicação generalizada do uso de proteínas fluorescentes para atingir subconjuntos neuronal para investigação por microscopia óptica, a necessidade de rapidamente de tela, imagem, e analisar as redes neurais dentro do tecido cerebral intacto tem valor inestimável. Avanços tecnológicos no desenvolvimento de user-friendly vetores virais, em técnicas de eletroporação in vivo, e linhagens de camundongos geneticamente modificados agora fornece uma fon...

<table border="1"&gt;<tbody&gt;<tr&gt;<th&gt; Nome do item</th&gt;<thEmpresa&gt;</th&gt;<th&gt; Número de catálogo</th&gt;<th&gt; Comments (opcional)</th&gt;</tr&gt;<tr&gt;<td&gt; Osso tesoura</td&gt;<td&gt; FST</td&gt;<td&gt; 16044-10</td&gt;<td&gt; Ou equivalente</td&gt;</tr&gt;<tr&gt;<td&gt; Tesouras de dissecação</td&gt;<td&gt; FST</td&gt;<td&gt; 14084-08</td&gt;<td&gt; Ou equivalente</td&gt;</tr&gt;<tr&gt;<td&gt; Tipo IB agarose</td&gt;<td&gt; Sigma</td&gt;<td&gt; A0576</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Compresstome</td&gt;<td&gt; Instrumentos Precisionary</td&gt;<td&gt; VF-200</td&gt;<td&gt;-Outros vibratomes são compatíveis</td&gt;</tr&gt;<tr&gt;<td&gt; Adesiva dupla face</td&gt;<td&gt; Grace Bio-Labs</td&gt;<td&gt; SA-S-1L</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Além disso Superfrost slides</td&gt;<td&gt; VWR</td&gt;<td&gt; 48311-703</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Tampa de vidro</td&gt;<td&gt; VWR</td&gt;<td&gt; 48383-139</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Glicerol</td&gt;<td&gt; EMD Chemicals Inc.</td&gt;<td&gt; GX0185-6</td&gt;<td&gt; Ou equivalente</td&gt;</tr&gt;</tbody&gt;</table&gt;

a rapid approach to high-resolution fluorescence imaging in semi-thick brain slices

Um objetivo fundamental tanto para a neurociência básica e clínica é compreender melhor a identidade, composição molecular e padrões de conectividade que são característicos para os neurônios do cérebro em ambos os normais e doentes. Para isso, um grande esforço foi colocado na construção de mapas de alta resolução neuroanatômicas 1-3. Com a expansão da genética molecular e os avanços em microscopia de luz veio a capacidade de consulta não só morfologias neuronal, mas também a composição molecular e celular dos neurônios individuais e suas redes associadas 4. Grandes avanços na capacidade de marcar e manipular neurônios através gene transgênico e tecnologias de direcionamento no roedor agora permitir que os pesquisadores subconjuntos neuronal &quot;programa&quot; à vontade 5-6. Indiscutivelmente, uma das contribuições mais influentes para a neurociência contemporânea foi a descoberta e clonagem de genes que codificam proteínas fluorescentes (PQ) em invertebrados marinhos 7-8, ao lado de seus subseqüentes de engenharia para produzir uma caixa de ferramentas cada vez maior de repórteres vital 9. Exploração de atividade do promotor de células de tipo específico para conduzir direcionados expressão FP em discretos populações neuronais agora oferece investigações neuroanatômicas com precisão genética. Engenharia expressão FP em neurônios melhorou muito nossa compreensão da estrutura e função cerebral. No entanto, os neurônios individuais de imagem e suas redes associadas em tecidos cerebral profunda, ou em três dimensões, manteve-se um desafio. Devido ao alto teor lipídico, o tecido nervoso é bastante opaco e apresenta fluorescência auto. Estas propriedades inerentes biofísicos dificultam a visualização e imagem neurônios fluorescente etiquetado em alta resolução através de microscopia confocal de epifluorescência ou padrão profundidades além de dezenas de microns. Para contornar este desafio investigadores muitas vezes empregam imagens seção fina de série e métodos de reconstrução 10, ou 2-fótons de microscopia de varredura a laser 11. Inconvenientes atuais para essas abordagens são a preparação do tecido associado de trabalho intensivo, ou de custo proibitivo de instrumentação, respectivamente. Aqui, apresentamos um método relativamente rápido e simples de visualizar células fluorescente etiquetado na fixa semi-grossas fatias de cérebro de rato, limpando óptica e de imagem. No protocolo anexo descrevemos os métodos de: 1) o tecido cerebral fixação in situ, através de perfusão intracardíaca, 2) dissecção e remoção do cérebro inteiro, 3) a incorporação do cérebro estacionário em agarose, 4) elaboração de precisão fatia semi-grossa usando instrumentação vibratome nova , 5) o tecido cerebral de compensação através de um gradiente de glicerol, e 6) de montagem em lâminas de vidro para microscopia de luz e z-stack reconstrução (Figura 1). Para preparar fatias de cérebro implementamos um pedaço relativamente novo de instrumentação chamado de &quot;Compresstome&quot; VF-200 (http://www.precisionary.com/products_vf200.html). Este instrumento é um micrótomo semi-automatizado, equipado com um avanço motorizados e sistema de vibração da lâmina com características semelhantes em função de vibratomes outros. Ao contrário de outras vibratomes, o tecido a ser cortado é montado em um plugue de agarose dentro de um cilindro de aço inoxidável. O tecido é extrudado em espessuras desejadas do cilindro, e corte pela lâmina para a frente avançando vibrando. A ficha de agarose / sistema de cilindro permite tecido reprodutíveis de montagem, alinhamento e corte de precisão. Em nossas mãos, o &quot;Compresstome &#39;rende fatias de alta qualidade de tecidos para eletrofisiologia, imuno-histoquímica, e dirigir-tecido fixo de montagem e de imagem. Combinados com a óptica, aqui demonstrar a preparação de semi-grossas fatias de cérebro fixa de alta resolução de imagem fluorescente.

Watch this Scientific Journal Video about A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices at JoVE.com

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

Aqui nós descrevemos um método rápido e simples de células de imagem fluorescente etiquetado em fatias grossas semi-cérebro. Através da fixação, corte, limpeza e opticamente tecido cerebral descrevemos como imagens de epifluorescência ou confocal padrão pode ser usado para visualizar as células individuais e redes neuronal no tecido nervoso intacto.

Uma abordagem rápida para alta resolução de imagem de fluorescência em fatias grossas Semi-Cérebro

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

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Research

JoVE Journal

Neuroscience

16.2K visualizações.  Baylor College of Medicine (BCM). Aqui nós descrevemos um método rápido e simples de células de imagem fluorescente etiquetado em fatias grossas semi-cérebro. Através da fixação, corte, limpeza e opticamente tecido cerebral descrevemos como imagens de epifluorescência ou confocal padrão pode ser usado para visualizar as células individuais e redes neuronal no tecido nervoso intacto.

Vídeo: A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Um Ensaio Slice organotípicas para High-Resolution Imaging Time Lapse de migração neuronal no cérebro pós-natal

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Neurociência

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

De alta resolução funcionais Métodos ressonância magnética para Midbrain Humanos

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta resolução<em&gt; In Vivo</em&gt; Manual Protocolo Segmentação para hipocampo humano subcampos Utilizando Ressonância Magnética 3T

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

De alta resolução de ressonância magnética estrutural do Subcórtex Humano<I&gt; In Vivo</I&gt; E Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

In Vivo funcional do cérebro Imagiologia Abordagem Baseada em cálcio Bioluminescent Indicador GFP-aequorina

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

De alta resolução de imagem latente Confocal da barreira sangue - cérebro: geração de imagens, reconstrução 3D e quantificação de Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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

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

Imagem de dois fotões cálcio em dendritos neuronais no cérebro fatias

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

De alta resolução Volume Imaging de neurônios pela exclusão da utilização da fluorescência método e dedicado Microfluidic dispositivos

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

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

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

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

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

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

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

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Imagem latente 3D de alta resolução da infecção do vírus da raiva no tecido solvente-cancelado do cérebro

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...