Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Placa de 20.000 células ES de ratinho diferenciadas (MES) / poço em placas de 96 lisina revestidos assim Poli-D e manter no neurónio motor meios de diferenciação terminal por 5-7 dias. Administração 1. Composto e Intoxicação com BoNT / A Realizar todos os seguintes trabalhos em um gabinete BSL2 para manter a conformidade com as diretrizes do CDC / NIH. <ol><li> Preparar soluções de estoque 10 mM de cada composto da biblioteca em 100% de DMSO em placas de 96 poços de polipropileno. Usar o estoque 10 mM para preparar uma placa de diluição intermédia que é 10 vezes maior do que a concentração de triagem desejado. Prepare 100 uM placa intermediária dispensando estoque 10 mM em placa de 96 poços e diluir a 100 uM utilizando meios de cultura. Dispensar 10 ul dos compostos sobre a 90 ul de meio sobre as células 11. Teste os compostos em 10 uM concentração final e garantir a porcentagem final de DMSO não exceda 0,5%. </li><li> Realize todos garanhãos que utilizam células vivas e toxina ativa sob nível de biossegurança 2 condições. Substituir as velhas mídias nas placas de 96 poços com 80 mL de fresco mídia diferenciação terminal usando um manual de 12 canais pipeta. Realizar aspiração e dispensar passos por linhas para evitar a exposição das células sem mídia para o ar ambiente. </li><li> Pretreat células com compostos antes da aplicação da toxina através da adição de 10 ul de compostos de 10x a placa de fonte utilizando uma pipeta de 12 canais, seguido por mistura por aspiração (duas vezes). Para cada placa de 96 poços, tratamento de duas colunas de 6 poços com 10x DMSO diluído nos meios de comunicação para servir como sinal de alta e controles de sinal baixo. A coluna sem tratamento BoNT exibe o nível mais elevado de todo o comprimento SNAP-25 (controlo de sinal alta). Trate a outra coluna com BoNT sozinho (sem quaisquer compostos) como o baixo controle de sinal. Use baixo controle para observar a eficácia de BoNT clivagem de SNAP-25 e sua detecção com o anticorpo BACS (Figura 2). </li><li> Incubar as células com os compostos durante 30 min a 37 ° C e 6% de CO 2 na incubadora de cultura de células. </li><li> Prepare a neurotoxina botulínica A partir de um 1 mg / ml fonte comercial em solução salina tamponada com fosfato (PBS) para uma final 10x estoque de trabalho por diluição com meios de diferenciação terminal. </li><li> Iniciado intoxicação por adição de 10 ul de 10x de BoNT / A estoque em poços designados para tratamento e baixo sinal controlos utilizando uma pipeta de canais múltiplos (Figura 2). Tratar as cavidades designadas como de controlo elevado com meio isolado. </li><li> Descontaminar todos os utensílios de plástico e outros materiais descartáveis ​​que entra em contacto com BoNT / A durante o estudo por imersão em solução de hipoclorito de 0,825% em água durante pelo menos 20 min. Realize todos os procedimentos de biossegurança em armários e descontaminar áreas de trabalho, antes e depois de experimentos com 5% de detergente desinfetante limpador como solução MICROCHEM. </li><li> Rotular as placas claramente para denotar a utilização de toxina e incubcomeram placas tratadas com 1 nM / L de BoNT / A durante 4 horas a 37 ° C e 6% de CO 2 num incubador de cultura de células. Mostrar sinalização adequada para informar os colegas que a toxina está em uso no laboratório. </li><li> Parar BONT / A clivagem proteolítica pela fixação metanol. Remover meios contendo toxina e compostos de cada poço com uma pipeta multicanal e descartar em solução de lixívia a 10%. Adicionar metanol arrefecido em gelo (100 ul) directamente a cada poço. </li><li> Incubar as placas durante 15 minutos à temperatura ambiente (TA) para permitir a fixação de ocorrer. </li><li> Após a fixação, lidar com segurança com os reagentes descartados (considerado neutralizados) com protocolos de nível de biossegurança 1 e descartar adequadamente. </li><li> Descartar metanol e 100 ul de usar PBS para lavar as células e hidratar-los antes da imunocoloração. Realizar duas lavagens com PBS adicionais. </li><li> Após a lavagem final, deixe PBS nos poços, placas de vedação com película adesiva de microplacas e armazenar a 4 ° C até a análise. Armazene as placas a 4 ° C for vários dias. </li></ol> 2. Immunostaining O procedimento immunostaining é, uma operação de várias etapas de trabalho intensivo que inclui reagentes repetitivos ciclos de distribuição / aspiração e placa de lavagem extensa que pode levar à introdução potencial de variabilidade intraplaca e Interplate significativo. Uma abordagem semi-automática é aplicada a economizar o tempo do pessoal de laboratório, aumentar a taxa de transferência de ensaio, e minimizar imunomarcação variabilidade. <ol><li> Use uma estação de trabalho automatizado equipado com uma cabeça de 96 poços capaz de transferir volumes de até 250 mL e integrado com um braço robótico garra para mover o material de laboratório em locais diferentes, na plataforma modular (ver Materiais e Equipamentos) para este protocolo. <ol><li> O deck modular foi concebido para acolher diferentes tipos de material de laboratório e pode suportar até 11 itens de uma só vez. O manipulador de microplacas automático é equipado com um compartimento duplo microplaca empilhador, a fim de manter emanipular até 50 placas por ciclo. </li><li> A estação de trabalho automatizado está localizado dentro de uma cabine de segurança biológica Classe II projetado para laboratório equipamentos de automação para proporcionar esterilidade e segurança. Para imunocoloração automatizado, o convés está disposta como mostrado na Figura 3, para permitir o acesso do reagente conforme necessário e sem intervenção humana. </li></ol></li><li> Placas pré-carga contendo células fixas na revista placa e transferência para o convés como necessário para operações de movimentação de líquidos. Use a cabeça 96 pipeta equipado com 250 ul dicas para aspirar PBS dos poços até 10 jul. Permeabilizar as membranas celulares para facilitar a ligação do anticorpo a alvos intracelulares, com tampão de permeabilização (0,1% de Triton X-100). Dispensar tampão de permeabilização (100 ul) em cada poço antes de retornar as placas para o segundo compartimento de armazenamento do empilhador para uma microplaca de 15 minutos de incubação à temperatura ambiente (RT). </li><li> Remover tampão de permeabilização e eplavagem da placa com PBS rform (duas vezes) como descrito anteriormente no passo 1.13. </li><li> Após o último passo de lavagem, remover o tampão PBS e dispensar 100 ul de tampão de bloqueio contendo 1% de soro de cavalo e 0,1% de Tween 20 em PBS em todos microplacas antes devolvê-los ao compartimento placa durante 1 hora de incubação à temperatura ambiente. </li><li> Usar dois anticorpos primários para imunocoloração: um anticorpo policlonal de coelho anti-rato de anticorpo BACS, para a detecção de comprimento completo SNAP-25 e um anticorpo monoclonal de rato anti-tubulina III anticorpo para a detecção de neurónios (ver Materiais e Equipamento). Após 60 min de incubação, remover o tampão de bloqueio e dispensar 50 ul de 4ug / ml de anticorpo BACS e 0,5 ug / ml de III-tubulina em tampão de bloqueio em todas as placas. </li><li> Incubar durante 1 h à TA remover os anticorpos primários e lavar 3 vezes com 100 ul de PBS contendo 0,05% de Tween (PBST). </li><li> Usar uma IgG anti-ratinho marcada com Alexa Fluor 647 para visualizar a -III tubulina e anti-IgG de coelho marcado com Alexa Fluor 568 para visualizar o anticorpo BACS. Prepare ambos os anticorpos como uma diluição de 2 ug / ml em tampão de bloqueio e dispensar 50 ul da mistura em cada poço. NOTA: Os anticorpos secundários seleccionados para imunocoloração são marcados com diferentes fluoróforos para permitir a detecção da sua luz fluorescente emitida nos comprimentos de onda distintos, utilizando diferentes canais dentro do detector de o conteúdo de alta Imager. </li><li> Incubar durante 1 h à TA. Aspirar os anticorpos secundários e lavar 3 vezes com 100 uL de PBST. </li><li> Após a lavagem final, adicionar 100 ul de PBS contendo corante Hoechst (32 uM) para corar o ADN para a detecção de núcleos. </li><li> Selar as placas com uma película impermeável luz para proteger os fluoróforos de fotodegradação. As placas podem ser armazenadas a 4 &#160; ° C durante semanas sem perda significativa de sinal. </li></ol> 3. Imagem NOTA: Realizar a aquisição de imagens utilizando alta Conteúdo de imagem SysTEM (ver Materiais e Equipamentos). <ol><li> Especificação de Alta Definição Imager conteúdo: <ol><li> Selecione o tipo de placa, layout, número de campos, objetivo: Greiner u claro, formato de 96 poços, 16, 20X de água, respectivamente. </li><li> Selecione os canais: núcleo de excitação EX: 405 nm como canal 4; citoplasma EX: 644 nm como o canal 2; antígeno específico EX canal anticorpo: 561 nm como canal 3 e Configure potência de excitação de cada laser, experimento Instalação como duas exposições, uma exposição com uma excitação a 644 nm, Exposição 2 com duas excitações em 405 nm e 561 nm. Selecione binning como BIN2. </li><li> Use poços de controlo elevados para a otimização da exposição com intensidade máxima como canal SNAP-25 e baixa de poços de controlo de intensidade mínima. </li><li> Ajustar a exposição, de modo que a intensidade de cada canal é de cerca de metade do nível de saturação (2000-2500 unidades relativas). </li><li> Identificar o plano Z ideal no canal da máscara de célula usando o script de correção delta. Veja Figura 5 para resultados após immunostaining e imagem. Exportar dados para o servidor onde o software de análise de imagem está residindo. </li></ol></li></ol> 4. Análise de Imagem (Figura 6) NOTA: Os passos seguintes descrevem aplicação dos algoritmos de software Columbus. <ol><li> Selecione um algoritmo apropriado para o segmento dos objetos primários (núcleos). Se necessário, ajustar o limiar de fundo e de parâmetros de contraste. O software Columbus oferece quatro métodos de detecção de núcleos. Selecione o método que os núcleos do segmento com precisão por inspeção visual núcleos segmentados (em F igura método 6a M é selecionado). </li><li> Selecione o algoritmo de neurite (CSIRO Neurite Análise 2) para o segmento de neurites Se necessário, ajustar os parâmetros de limite de fundo e contraste para remover o fundo. Veja a Figura 6b para os parâmetros usados ​​para detectar neurites. </li><li> Identificar as regiões neurite (segmentos neurite) based em objectos secundários (neurites), utilizando uma ampliação de pixel -3 do canal β-tubulina III (Alexa Flour 640) como máscara (Figura 6c). </li><li> Calcule a intensidade dos canais de sinal (SNAP-25, (Alexa Farinha 568) para a região de neurites e canal tubulina β-III (Figura 6d e 6e). </li><li> Selecione os parâmetros desejados para a exportação, como comprimento neurite, intensidades médias de SNAP-25, β-tubulina III, número de núcleos, número do segmento neurite, etc. (Figura 6f). </li><li> Placas de transferência de tabuleiros usando o robô e carga para o Imager Conteúdo alta para aquisição de imagem. </li></ol> 5. Análise de Dados: Para avaliar a robustez do ensaio desenhado, calcular os seguintes parâmetros a partir do experimento à base de chapa. <ol><li> Signal (S) a fundo (B): S / B = intensidade do sinal de controle de alta média) / média intensidade de baixo controle de sinal </li> &lt;li&gt; Coeficiente de Variação (CV) de sinal e o fundo (para medir a uniformidade do sinal específico): CV = (Desvio Padrão (DP) / média de amostra) * 100 (%), para determinar a variabilidade no manuseamento de líquidos, reagentes, etc. </li><li> Signal to Noise: S = (intensidade de sinal significa - intensidade de baixo controle de sinal média) / SD de baixo controle <ol><li> Calcular o factor de Z &#39;com intoxicação de uma metade de uma placa de 96 poços e uma intoxicação simulada da outra metade. Z &#39;= 1-3 * (SD de alto controle de sinal + SD de baixo controle de sinal) / (média de controle de alta sinal - média de baixo controle do sinal). Para a análise posterior dos dados de rastreio, normalizar alguns dos parâmetros de matérias primárias sobre uma placa de base para permitir a comparação entre as placas e entre os diferentes dias de experiências. </li></ol></li><li> Percentagem de clivagem, reflectindo a redução do sinal associado com o comprimento total de SNAP-25 detectada por um anticorpo específico (BACS): Clivagem% = 100% * (meaintensidade n de controle sinal alto - intensidade da amostra) intensidade de controle de alta sinal / média. </li><li> Porcentagem de inibição da clivagem:% inibição = 100% * (intensidade da amostra - intensidade de baixo controle de sinal média) / (intensidade de controle de alta sinal significa - intensidade de baixo controle de sinal média). </li></ol>

<ol>
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Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

A alta potência de neurotoxinas botulínicas e relativa facilidade de seu armamento resultou em sua classificação como Categoria A (prioridade mais alta) agentes biothreat pelos Centros dos EUA para Controle e Prevenção de Doenças. Infelizmente, não há terapias aprovadas pela FDA para contrariar BoNT intoxicação após a toxina tenha sido internalizados pelos neurónios motores. Qualquer mecanismo druggable que promove a recuperação neuronal de BoNT intoxicação pode levar para o desenvolvimento de uma terap...

A bactéria Clostridium botulinum produz neurotoxina botulínica, um dos mais potentes toxinas biológicas conhecidas para o homem 1. Existem 7 sorotipos BoNT distintas (TB / AG). BoNT / A-Gall induzir paralisia na junção neuromuscular, devido à proteólise complexo SNARE 2,3. SNARE proteólise impede-neurotransmissor vesícula de membrana de fusão e, por conseguinte, bloqueia a exocitose do neurotransmissor 4. O alvo SNARE específica depende do serotipo particular, BoNT envolvidos no processo de intoxicação. BoNT / A e BoNT / E se apegam SNAP-25 enquanto que cliva BoNT / C ambos SNAP-25 e syntaxin 5. O restantes serotipos sinaptobrevina clivam (também chamado de proteína de membrana de vesícula-associado (VAMP). BoNT / A foi escolhida para o desenvolvimento do ensaio, uma vez que é responsável por uma proporção elevada de ocorrência natural botulismo e tem a maior duração de acção 6. Desenvolvimento de pequena molécula terapêutica contra BoNT / A é uma meta importante parao nosso programa de descoberta de drogas e utilizou métodos tradicionais à base de alvo para identificar inibidores de sítio proteolíticas activas 7, 8-10. No entanto, a criação de inibidores de sítio activo com actividade de largo espectro contra vários serotipos e eficácia pós-exposição irá provavelmente ser um desafio. Temos, portanto, implementado uma abordagem inovadora, fenotípicas descoberta droga que usa BoNT SNAP-25 clivagem como um desfecho funcional para identificar pequenas moléculas que podem bloquear BoNT mediada por intoxicação do neurônio motor. SNAP-25 é necessária para a libertação de neurotransmissores, como a degradação de SNAP-25 é preditivo da paralisia e letalidade in vivo. Por exemplo, o rastreio baseado em células poderia levar à descoberta de novos moduladores de factores celulares responsáveis ​​pela inactivação da toxina ou a inibição das vias de toxina no interior das células-alvo. Um fator importante no desenvolvimento de ensaio fenotípico é a seleção de modelos biológicos fisiologicamente relevantes. We e outros descreveram rato estaminais embrionárias (ES) derivadas de células de neurónios motores que recapitulam o carácter imunológico de neurónios motores principais, incluindo a expressão de SNAP-25, 11- 13. É importante notar que estes sistemas celulares são altamente sensíveis a BoNT / A intoxicação e demonstrar a clivagem dependente da dose de SNAP-25 em resposta a concentrações crescentes de toxina 11,12. Os neurónios motores diferenciadas também são produzidos em quantidades que são suficientes para uma análise baseada placa de alto rendimento e permitiram a construção de uma variedade de ensaios celulares. O ensaio fenotípico é um método de imunofluorescência utilizando dois anticorpos diferentes para quantificar a clivagem de comprimento total endogenamente expressa SNAP-25 durante BoNT / A intoxicação da cultura do rato do neurônio motor. Um terminal carboxilo BoNT / A (BACS) anticorpo sensível à clivagem que reconhece apenas o comprimento total SNAP-25 permite a avaliação de BoNT / A proteólise mediada da SNAP-25expressão no motor de rato neurônios 10. Um diagrama esquemático do ensaio IHC está representado na Figura 1.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Botulinum neurotoxin A&#160;</td>
			<td style="width:180px;">Metabiologics</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtitre plates</td>
			<td style="width:180px;">Greiner&#160;</td>
			<td style="width:119px;">655946</td>
			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BACs antibody</td>
			<td style="width:180px;">Lampire Biological</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microchem</td>
			<td style="width:180px;">National&#160;</td>
			<td style="width:119px;">0255</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Methanol</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">A412-20</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formaldehyde</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Horse serum</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">16050</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">PE JANUS MDT Mini Automated Workstation</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">AJMDT01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opera</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Triton X 100</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BIII tubulin antibody</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">BAM1195</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342dye&#160;</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">3570</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A21236</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A10042</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 2.4</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PlateStack</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
		</tr>
	</tbody>
</table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Os dados dos controlos altos e baixos criadas duas populações distintas com a diferença de duas medianas superiores a 3 desvios padrão (Figura 7A). O objectivo do processo de triagem é para encontrar os compostos dentro da população da amostra com valores próximos das populações de controlo positivo, assumindo uma distribuição normal dentro da população da amostra (Figura 7B, (i)). Os pontos de dados que são três desvios padrão além da média são considerados estatist...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

A alta Ensaio imagem Conteúdo de Identificação da neurotoxina botulínica Inibidores

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

8.5K visualizações.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Vídeo: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

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 ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Imagem Funcional do Cortex Auditivo em gatos adultos usando de alta campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Isolamento e Quantificação da neurotoxina botulínica a partir de matrizes complexas usando os BoTest Matrix Ensaios

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

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 ...

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 ...

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-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Análise do elevado-throughput de comportamento Locomotor no ensaio de ilha de Drosophila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.