Este trabalho foi financiado pelo NEI e NIMH.

1. VideoSwitcher 1,1. Ligue VideoSwitcher a um computador O VideoSwitcher (Figura 1) recebe de vídeo analógico (VGA) entradas e é usada para acionar tubo de raios catódicos (CRT) monitores coloridos. Antes de usar o VideoSwitcher, verifique se o computador tem placa de vídeo ou uma porta VGA (Figura 2A) ou Digital Visual Interface (DVI-I) porta (Figura 2B), que transmite tanto sinais de vídeo digitais e analógicas. Um adaptador DVI-to-VGA (Figura 2C) é necessário para conectar a porta DVI-I para o VideoSwitcher. Desligue o computador eo monitor e desligue o monitor da placa gráfica do computador. Conecte o cabo VGA ou porta DVI-I da placa gráfica do computador à porta de entrada do VideoSwitcher usando o cabo fornecido VGA. Use uma chave de fenda pequena para proteger as conexões. Conecte o cabo VGA do monitor à porta de saída da uma VideoSwitchernd proteger as conexões. Conecte o VideoSwitcher a uma fonte de alimentação usando o adaptador de alimentação fornecido. Ligue o computador eo monitor. 1,2. Dois modos de exibição do comutador de vídeo O botão interruptor na VideoSwitcher permite ao usuário voltar-e-vem em dois modos de exibição. No modo de cor, o VideoSwitcher não altera o sinal de vídeo a partir da placa gráfica do computador. No modo de escala de cinzentos, o visor torna-se monocromática, com a sua luminância determinada principalmente pelo sinal de vídeo a partir do canal de azul da placa gráfica, ligeiramente afectado por o sinal do canal de vermelho, e nenhuma contribuição do canal de verde. Isto é porque a rede resistente dentro do VideoSwitcher atenua o sinal a partir do canal de vermelho por um factor de cerca de 128, eo sinal do canal verde não contribui de todo. A razão entre as contribuições da azul umcanais D vermelhas para a saída do VideoSwitcher é chamado azul-de-vermelho (razão BRratio). O BRratio é propriedade de um comutador de vídeo, independente da configuração da tela. Ele pode ser determinada experimentalmente para cada VideoSwitcher (ver 1.3). O modo de exibição também pode ser ligado através do envio de um sinal especial pelo canal verde da placa de vídeo usando uma chamada de função em Matlab: PsychVideoSwitcher (&#39;Switchmode&#39;, whichScreen, ToGrayMode); onde ToGrayMode de 1 ou 0 meio para mudar para tons de cinza ou modo de cor. A Figura 3 mostra o diagrama esquemático de um VideoSwitcher. 1,3. Meça BRratio O BRratio pode ser medido para cada VideoSwitcher usando uma onda quadrada tarefa de detecção de grade. Os dois níveis de onda quadrada são atribuídos valores RGB, tais como (0, 0, 40) e (x, 0, 39). Pode-se ajustar o valor de x até que a grade de onda quadradadesaparece, ponto em que o valor x é a BRratio --- a magnitude do sinal no R-canal que conduz à intensidade produzido por uma unidade de sinal no canal B. Embora se possa medir a BRratio electronicamente, por exemplo, para medir a razão entre as saídas de tensão de um VideoSwitcher com valores de RGB de (0, 0, 255) e (255, 0, 0), o método psicofísico aqui descrito é muito simples e preciso. 1,4. Mostrar Calibração Para a maioria dos monitores, iluminação de exibição não aumenta linearmente com o nível de pixel cinza especificado pelo cartão de computação gráfica. A relação pode ser descrito como: <img src="/files/ftp_upload/3312/3312eq1.jpg" alt="Equação 1" /> onde L max, L min e γ são determinados com um processo de correcção gama, ou usando fotómetros ou por uma combinação de procedimentos psicofísicos e medições fotométricas 7,8. Calibração Gamma é important para produzir contrastes precisos em uma tela, que deve ser realizado para cada dispositivo de exibição. Com um fotómetro, pode-se simplesmente exibir um quadrado grande uniforme em graylevels pixel diferentes (por exemplo, de 0 a 255), medir a luminância do centro do quadrado, e se encaixam os resultados com a Equação 1 para se obter γ. O procedimento envolve a criação de psicofísico, em uma área da superfície de visualização, tão uniforme quanto possível uma mistura (no espaço e no tempo) de quantidades iguais de zero e de intensidade de pixels completo e determinação do valor graylevel de pixels de uma área adjacente homogénea ( em que todos os pixels têm a mesma intensidade) que produz uma correspondência psicofísico para a luminância da área misturado-pixel. A primeira partida determina o valor graylevel durante 1/2 de luminância máxima. Uma mistura de pixels com intensidades de luminância de 1/2 e 1 é usado para determinar o valor 3/4, e este procedimento é repetido até que sete valores from 1/8 a 7/8 foram determinados. Estes jogos são repetidos, e várias verificações de consistência são feitas, tais como a verificação de que uma mistura de 3/4 e 1/4 partidas 1/2. Neste procedimento, tanto o B e os níveis de P da zona homogénea são ajustadas, e U = B + R / BRratio. Os resultados podem ser encaixar com a Equação 1 para se obter γ. Depois de obter os valores de L min, max L e γ, podemos definir a luminância de fundo L 0 = (L + L max min) / 2, A relação entre pixels de contraste c (U) e pixel cinzento nível U é: <img src="/files/ftp_upload/3312/3312eq2.jpg" alt="Equação 2" /> Podemos reescrever a equação para resolver o nível exigido de cinza para um contraste dado: <img src="/files/ftp_upload/3312/3312eq3.jpg" alt="Equação 3" /> em que U é normalmente um número não inteiro. O valor RGB do pixel correspondente é: R = BRratio * [U-int (U)], G = 0, B =int (U), onde int () leva o valor inteiro da sua entrada. 1,5. Demonstração de uma grade de onda senoidal O efeito da escala de cinzentos resolução aumentada pode ser mostrado por comparação de duas grelhas de onda de seno. A grade do lado esquerdo usa a capacidade total do VideoSwitcher com cada intensidade de pixel especificado pela R apropriado e os valores de B. A grade do lado direito é o mesmo que o da esquerda, excepto que apenas o U arredondada é usado como valor B de cada pixel, mimetizando a saída de um dispositivo de exibição de 8-bit. 2. RTbox 2,1. Ligue o RTbox a um computador Ligue o RTbox (Figura 4) para uma porta USB de um computador com o cabo fornecido. Se necessário, baixar o driver do dispositivo de <a href="http://www.ftdichip.com/Drivers/VCP.htm">http://www.ftdichip.com/Drivers/VCP.htm</a> . Instale o driver seguindo as instruções no site. 2,2. Use o RTcaixa para medir o tempo de resposta O RTbox pode ser usado em duas formas diferentes. Se o programa do usuário pode retornar vezes estímulo confiáveis ​​início, como o tempo retornado pela Screen (&#39;Flip&#39;) e PsychPortAudio em Psychtoolbox [8], o RTbox pode ser usado como um teclado. Porque os tempos de estímulo início e os horários botão RTbox baseiam-se os relógios do computador host eo RTbox, respectivamente, é preciso sincronizar os dois relógios. Isto é feito com um RTBox chamada de função (&#39;clear&#39;) antes de cada início do estímulo para apagar os eventos indesejados no buffer. O tempo de resposta pode ser simplesmente medido como t resp = t - t início, onde tonset é o estímulo retornado do programa do usuário, e t é o tempo botão retornado pela chamada de função [t, botão] = RTBox (timeout). Isto é porque o condutor RTBox converte automaticamente o tempo de botão a partir do momento RTbox para acolher o tempo de computador. Se o programa do usuário não podevoltar vezes início de confiança de estímulo, indicando sinais de disparo inícios de estímulo devem ser fornecidas ao RTbox. Uma solução é a utilização de um sensor de luz para detectar o aparecimento de um estímulo, ou um fragmento de luz que está sincronizado com o estímulo. A saída do sensor de luz é ligado à porta de luz do RTbox. Uma segunda solução consiste em utilizar a saída de disparo do VideoSwitcher. O gatilho pode ser conectado diretamente à porta de pulso da RTbox. A terceira solução é ligar o sinal de áudio do computador host para a porta de som do RTbox. Para gerar um gatilho início do estímulo da VideoSwitcher, nós &quot;ligar&quot; o canal verde dos pixels na porção central do primeiro quadro do estímulo visual. Porque não contribui para a rede de resistências, o sinal no canal verde é invisível para o sujeito. Uma chamada de função [t, botão] = RTBox (&quot;pulso&quot;, timeout) retorna o tempo de resposta, onde &quot;pulso&quot; instrui o RTbox para calcular t<html> ele resposta relativa de tempo para o gatilho. A chamada de função é utilizado o mesmo para o gatilho de som. Para acionadores do sensor de luminosidade, a chamada de função é [t, botão] = RTBox (&quot;luz&quot;, timeout). 3. Integrar VideoSwitcher e RTbox Temos programado um programa de demonstração que usa o VideoSwitcher para controlar a exibição, o RTbox para coletar o tempo de resposta precisa, eo RTbox para enviar código do evento e as respostas para um sistema de ERP. O programa pode ser descarregado a partir <a href="http://lobes.usc.edu/videoswitcher/VideoSwitcherRTBoxERP_demo.m">http://lobes.usc.edu/videoswitcher/VideoSwitcherRTBoxERP_demo.m</a> . 4. Os resultados representativos <img src="/files/ftp_upload/3312/3312fig1.jpg" alt="A Figura 1" /> Figura 1. Imagem de um comutador de vídeo. Tem entrada VGA e portas de saída (tanto do sexo feminino), um botão para alternar entre dois modos de exibição, e uma porta de saída de disparo. ONTEÚDO &quot;&gt; <img src="/files/ftp_upload/3312/3312fig2.jpg" alt="A Figura 2" /> Figura 2. Fotos de VGA (A) e DVI-I (b) portas e uma DVI-I para VGA (C). <img src="/files/ftp_upload/3312/3312fig3.jpg" alt="A Figura 3" /> Figura 3. O esquemática de um comutador de vídeo. O multiplexador tem dois modos de entrada, controlados por um nível de tensão. Quando se está no modo mostrado no esquema, os canais RGB de um monitor de receber o sinal mesmo, de modo que o mostrador é em tons de cinzento modo. A tensão de comutação é gerado por um chip CPLD, controlado por sinais do G-canal eo botão interruptor. O gatilho está também controlado pela entrada no G-canal. <img src="/files/ftp_upload/3312/3312fig4.jpg" alt="A Figura 4" /> Figura 4. Imagem de um RTbox. Ele tem uma porta USB-B, uma porta de entrada de luz, uma porta de entrada de pulso / som, uma porta de entrada externa botão e uma porta de saída TTL.

<ol>
	<li>Li, X., Lu, Z. -. L., Xu, P., Jin, J., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+high+gray-level+resolution+monochrome+displays+with+conventional+computer+graphics+cards+and+color+monitors.">Generating high gray-level resolution monochrome displays with conventional computer graphics cards and color monitors.</a> Journal of Neuroscience Methods. 130 (1), 9-18 (2003).</li><li>Pelli, D. G., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+control+of+contrast+on+microcomputer+displays.">Accurate control of contrast on microcomputer displays.</a> Vision Research. 31, 1337-1350 (1991).</li><li>Li, X., Liang, Z., Kleiner, M., Lu, Z. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTbox:+A+device+for+highly+accurate+response+time+measurements.">RTbox: A device for highly accurate response time measurements.</a> Behavior Research Methods. 42 (1), 212-225 (2010).</li><li>Lu, Z. L., Sperling, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-order+reversed+phi.">Second-order reversed phi.</a> Perception Psychophysics. 61, 1075-1088 (1999).</li><li>Colombo, E., Derrington, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+calibration+of+CRT+monitors.">Visual calibration of CRT monitors.</a> Displays. 22, 87-95 (2001).</li></ol>

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O comutador de vídeo fornece um método compacto, barato e fácil de usar para gerar resolução alta luminância exibe usando computação gráfica convencionais cartões e monitores de cor analógicas. Ele é conectado entre as saídas analógicas da placa de vídeo eo monitor. Também pode alternar entre os modos de exibição monocromáticas e cromáticas, proporcionando maior comodidade. Seu canal de disparo permite que os pesquisadores para sincronizar outros equipamentos para apresentações visuais. <p clas...

enabling high grayscale resolution displays and accurate response time measurements on conventional computers

Sistemas de visualização baseados em placas gráficas de computador convencionais são capazes de gerar imagens com resolução de 8-bit dos níveis de cinza. No entanto, a maioria das experiências em investigação visão requerem exibe com mais de 12 bits de resolução de luminância. Várias soluções estão disponíveis. Bit + + 1 e 2 DataPixx usar o Digital Visual Interface de saída (DVI) de placas gráficas e de alta resolução (14 ou 16-bit) digital-analógico conversores para conduzir dispositivos de vídeo analógicos. O 3 VideoSwitcher descrito aqui combina sinais de vídeo analógicos dos canais vermelho e azul de placas gráficas com pesos diferentes, utilizando uma rede resistente passivo 4 e um circuito ativo para entregar os sinais de vídeo idênticas para os três canais de monitores coloridos. O método fornece uma maneira barata de permitir displays monocromáticos de alta resolução usando cartões de gráficas convencionais e monitores analógicos. Ele também pode fornecer sinais de disparo que podem ser usados ​​para marcarlatências de estímulo, tornando mais fácil para sincronizar apresentações visuais com gravações fisiológicas ou medições de tempo de resposta. Apesar de teclados e mouses de computador são freqüentemente usados ​​para medir tempos de resposta (TR), a precisão dessas medidas é bastante baixa. O RTbox é um hardware especializado e solução de software para medições precisas RT. Conectado ao computador host através de uma conexão USB, o motorista do RTbox é compatível com todos os sistemas operacionais convencionais. Ele usa um relógio e microprocessador de alta resolução para registrar as identidades e de tempo de eventos de botão, que estão em buffer até que o computador host recupera-los. Os eventos de botão gravados não são afetados pelas incertezas de tempo ou potenciais vieses associados com a transmissão de dados e processamento no computador anfitrião. O armazenamento assíncrona simplifica o desenho de programas do usuário. Vários métodos estão disponíveis para sincronizar os relógios do RTbox ea Comput anfitriãoer. O RTbox pode também receber accionadores externas e ser utilizado para medir RT com respeito aos eventos externos. Ambos VideoSwitcher e RTbox estão disponíveis para os usuários comprarem. A informação relevante e de demonstração pode ser encontrada em <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a> .

Watch this Scientific Journal Video about Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers at JoVE.com

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Hardware convencional não pode gerar estímulos visuais com suficientemente alto em tons de cinza tempos de resposta e de resolução de medida com uma precisão suficiente. Nós descrevemos como usar o VideoSwitcher para produzir displays de alta resolução monocromática, eo RTbox para medir tempos de resposta com alta precisão em hardware convencional.

Ativando alta escala de cinza monitores de resolução e Medidas resposta precisa de tempo em computadores convencionais

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

enabling-high-grayscale-resolution-displays-accurate-response-time

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9.3K visualizações.  The Ohio State University. Hardware convencional não pode gerar estímulos visuais com suficientemente alto em tons de cinza tempos de resposta e de resolução de medida com uma precisão suficiente. Nós descrevemos como usar o VideoSwitcher para produzir displays de alta resolução monocromática, eo RTbox para medir tempos de resposta com alta precisão em hardware convencional.

Vídeo: Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

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

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

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

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 ...

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 Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

De alta resolução de imagem ao vivo de comportamento de células no desenvolvimento neuroepitélio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Em Medidas Vivo eletrofisiológicos nos nervos ciático Rato

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Detecção de Axonally localizada mRNAs em seções do cérebro Usando de alta resolução<em&gt; In Situ</em&gt; Hibridização

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

High-Resolution Análise Quantitativa immunogold de Receptores de Membrana na retina fita Sinapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Provocando estresse celular e morte utilizando convencional UV laser microscopia confocal

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Aumento Time-dependente na resposta da rede à estimulação de culturas de pilha neuronais em matrizes do micro-elétrodo

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...