Name: real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons
Uploaded: 2014-09-15T15:00:00.000+00:00
Duration: 10 min 53 s
Description: Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Gostaríamos de agradecer a Yue (Pauline) Hu, Rachel Sinit por sua assistência técnica. O estudo foi financiado pelo NIH concessão (PN2 EY016525) e pelo financiamento da Síndrome de Down Research Foundation e Tratamento eo Larry L. Fundação Hillblom.

Os procedimentos cirúrgicos e de animais são realizadas estritamente de acordo com o Guia do NIH para o Cuidado e Uso de Animais de Laboratório. Todos os experimentos que envolvem o uso de animais são aprovados por UCSD Animal Care Institucional e Comitê de Uso. 1. plasmídeo Clonagem, expressão e purificação do mono-biotinilado BDNF (mBtBDNF) NOTA: Construir pré-proBDNFavi e BirA cDNA em vetor pcDNA3.1 e co-expressar em células HEK293FT 10. Purificar mBtBDNF utilizando pérolas de Ni-NTA de acordo com o método previamente publicado de produzir factor de crescimento do nervo monobiotinylated madura e biologicamente activa (mBtNGF) 10. 2 Preparação de microfluídicos Chambers Microfluídicos dispositivo neurônio cultura torna possível isolar fluidicamente axônios de corpos celulares neurônio. Montar câmaras com lamelas recentemente pintadas direito antes de cada dissecção. Câmaras microfluídicos usadosneste protocolo são comercialmente adquirido (ver materiais e tabela do equipamento). Lave à mão e reutilizados câmaras adquiridas comercialmente até 5-6x. <ol><li> Somente lavagem à mão câmaras microfluídicos em 1% Alconox. Lavar três vezes com água Milli-Q durante 30 min cada. Câmaras de lavagem de mão novamente em etanol 70%. </li><li> Coloque para fora câmaras para secar em Parafilm na capela de fluxo laminar. Irradiar e esterilizar ambos os lados das câmaras, durante 20 min sob UV. Loja câmaras esterilizadas numa estéril 15 centímetros prato selado com Parafilm à temperatura ambiente. </li><li> Coloque 24 x 40 mm No. 1 lamínulas de vidro em um recipiente de vidro. Mergulhe as lamelas em ácido clorídrico a 35% durante a noite em um rotador. No dia seguinte, lavar as lamelas três vezes, durante 30 min cada, com água. </li><li> Esterilizar as lamelas, um por um, mergulhando cada lamela em etanol a 100% e ardor sobre um bico de Bunsen. Armazenar lamelas secas numa placa de Petri estéril e mantê-lo à temperatura ambiente até o revestimento. </li><li> Coloque UOt as lamelas em um 15 centímetros prato de cultura. Bata de cada lamela com 0,7 ml de 0,01% de poli-L-lisina (PLL) e incuba-se na capa, à temperatura ambiente. </li><li> Após 1 h, lavar as lamelas três vezes com água estéril. Lamelas secos com vácuo e coloque cada lamela em um prato de cultura 6 centímetros. </li><li> Para montar a câmara de microfluídica, coloque a câmara com o lado microgroove na parte inferior para a lamela PLL revestido com cuidado para não tocar nas estrias. Gentilmente pressionado a câmara com uma ponteira para garantir a câmara foi hermeticamente fechado. </li></ol> 3. Dissecção da Neuronal Cultura e Placa em Chambers <ol><li> Coloque dois hipocampos E17-E18 rato (um cérebro) dissecados em um tubo de 15 ml contendo 2 ml de tampão de dissecção (HBSS, sem cálcio, magnésio não, com 1% pen / strep e HEPES 10 mM. Lavar os tecidos 3x com 5 ml dissecção de tampão de cada vez. Remover o tampão de dissecção, tanto quanto possível, e adicionar 900 ul de di frescotampão ssection. </li><li> Para digerir o tecido, adicionar 100 ul de tripsina 10x (2,5%) no tampão de dissecção para tornar a concentração de trabalho 1x. Colocar o tubo cónico de 37 ° C Banho de água. Após 10 minutos de digestão, adicionar 100 ul de 10 mg / ml de ADNase I a uma concentração final de cerca de 1 mg / ml. </li><li> Usando uma pipeta Pasteur de vidro polido-fogo, triturar suavemente os tecidos pipetando para cima e para baixo 5-10x. Logo após a trituração, extinguir a tripsina com 2 ml de meio (chapeamento Neurobasal com 10% de FBS, 2 mM de GlutaMax, 2% B27). </li><li> Deixar a amostra na capa por 5 minutos para permitir que todos os restos dos tecidos para se estabelecer para o fundo. Remover cuidadosamente 2 mL de sobrenadante para um tubo de 15 ml estéril cónico limpo e centrifugar a 200 xg durante 5 min para sedimentar as células. Ressuspender o sedimento em 50 mídia ul chapeamento </li><li> Contar as células com hemocitômetro. Carga 15-20 ul de suspensão de células (~ 40.000 células) em um compartimento da câmara de microfluidos. Coloque a câmarana incubadora durante 10 minutos para permitir que as células se fixem à lamela. Após 10 min, adicionar mídia mais chapeamento para encher ambos os compartimentos da câmara. </li><li> No segundo dia da dissecção, substituir completamente os meios de chapeamento com meio de manutenção (Neurobasal com GlutaMax 2 mM, 2% B27), tanto no corpo da célula e o compartimento do axónio. Os axônios dos neurônios do hipocampo começa a atravessar as estrias no dia 3 e atingir o compartimento do axônio entre os dias 5-7. Durante este período de tempo, substitua metade da media cultura com meio de manutenção fresco todos os 24 ~ 48 horas. </li></ol> 4. axonal Transporte de QD-BDNF <ol><li> Antes da imagiologia de viver QD-BDNF transporte axonal, esgotar o BDNF, tanto do corpo da célula e os compartimentos de axónios da câmara de microfluidos por enxaguar ambos os compartimentos com o BDNF-livre, livre de soro mídia Neurobasal cada 30 min durante 2 horas. </li><li> Durante a depleção de BDNF, preparar os conjugados QD-BDNF. Misturar 50 nM de mono-biotinilado BDNF dímero com 50 nM QD655 conjugados com estreptavidina em meio Neurobasal e incubar em gelo durante 60 min. </li><li> Retirar mídia no compartimento axonal e adicionar 300 ul QD-BDNF com uma concentração final de 0,25 nM durante 4 horas a 37 ° C. Para minimizar a difusão de QD-BDNF no compartimento do corpo da célula, é muito importante para manter sempre um nível mais elevado de meios no compartimento do corpo de célula do que no compartimento do axónio. Lavar desvinculado QD-BDNF após incubação antes de imagem ao vivo. </li><li> Realizar imagens ao vivo de transporte QD-BDNF usando um microscópio invertido equipado com uma lente objectiva de óleo 100X. Aquecer o escopo e a câmara ambiental a ela ligada a uma temperatura constante (37 ° C) e de CO 2 (5%). Uso de um conjunto de cubos Texas excitação / emissão de vermelho para visualizar o sinal de QD655. </li><li> Adquirir e capturar imagens de lapso de tempo dentro dos axônios média na velocidade de um quadro / segundo para um total de 2 min usando uma câmera CCD. Use microgrooves sem axônios that não têm QD e, portanto, nenhum sinal de controlo para a infiltração. </li><li> Analisar transporte BDNF usando qualquer software de análise de imagem ou NIH ImageJ. </li></ol>

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Neste estudo, descrevemos o desenvolvimento de uma nova técnica para produzir biologicamente activo, monobiotinylated BDNF (mBtBDNF) que podem ser utilizados para rastrear o transporte axonal de BDNF. Conjugando a proteína a estreptavidina quantum dot, e usando uma câmara de microfluidos, o método permite detectar o transporte axonal de BDNF nos neurónios primários com sensibilidade única molécula, em tempo real e com resolução espacial e temporal. As ferramentas aqui utilizadas fornecer um meio pelo qual a es...

Os neurônios são as células cujos processos longos e muitas vezes altamente elaborados são fundamentais para estabelecer e manter a estrutura ea função dos circuitos neurais altamente polarizada. O axônio desempenha um papel vital no que transportam cargas de e para as sinapses. As proteínas e organelas sintetizados no soma célula necessita de ser transportado por axónios para chegar ao terminal pré-sináptico para suportar a função neuronal. Do mesmo modo, os sinais recebidos pelo axónios distais devem ser transduzidas e transportado para o soma. Estes processos são essenciais para a sobrevivência neuronal, diferenciação e manutenção. Em que o transporte axonal em neurónios algumas deve ser conduzida através de distâncias mais do que 1000 vezes o diâmetro do corpo da célula, a possibilidade é facilmente imaginado que mesmo pequenos défices pode impactar significativamente a função neuronal e o circuito. Derivado de factor neurotrófico cerebral (BDNF), um membro da família da neurotrofina de factores de crescimento, está presente na maregiões do cérebro, incluindo ny hipocampo, córtex cerebral, e do prosencéfalo basal. BDNF desempenha um papel crucial na formação da memória e cognição, através do apoio da sobrevivência, diferenciação e função de neurónios que participam em circuitos cognitivos. BDNF se liga ao seu receptor, a tirosina-cinase TrkB, no terminal axonal em que activa as vias de sinalização mediadas por TrkB, incluindo a proteína quinase activada por mitógeno / regulada por sinal extracelular da proteína cinase (MAPK / ERK), fosfatidilinositol-3-quinase (PI3K) e fosfolipase C-gama (PLCy). As proteínas que participam nestas vias de sinalização são empacotados em vesículas de endocitose para formar o BDNF / TrkB sinalização endosome 1-6 que são, então, transportado retrogradamente à soma neuronal. A câmara de cultura de microfluidos é uma plataforma muito útil para estudar a biologia axonal, em condições normais, bem como na definição de lesões e doenças 7,8. Por axônios isolandodos corpos celulares, o dispositivo tem permitido um para estudar o transporte especificamente em axônios 8-10. As plataformas microfluídicos baseados PDMS com 450 mM barreiras microgroove utilizados neste estudo foram adquiridos comercialmente (ver tabela de materiais). Para examinar o transporte BDNF, desenvolvemos uma nova tecnologia para produzir monobiotinylated BDNF (mBtBDNF). Tiramos proveito do peptídeo biotina aceitante, AP (também conhecido como AviTag). É uma sequência de 15 aminoácidos que contém um resíduo de lisina, que pode ser especificamente ligado a uma biotina pela Escherichia coli enzima biotina ligase, BirA. Nós fundido a AviTag para o C-terminal do rato pré-proBDNF cDNA por PCR (Figura 1A). A construção foi clonada no vector de expressão de mamífero, pcDNA3.1 Myc-His vector. Nós também clonado o DNA bacteriano BirA na pcDNA3.1 Myc-His vector. Os dois plasmídeos foram co-transfectadas transientemente em células HEK293FT para expressar ambas as proteínas. BirA catalisou a ligadura da biotaem que especificamente a lisina residir no AviTag no C-terminal do BDNF a uma razão de 1: 1 para produzir monobiotinylated monómero BDNF. Biotinilado, BDNF madura com uma massa molecular de ~ 18 kDa foi recuperada e purificada a partir dos meios utilizando Ni-resina (Figura 1C). A biotinilação de FNDC estava completa, conforme avaliado pela incapacidade para detectar o BDNF não modificado por imunotransferência (Figura 1E). Estreptavidina conjugada pontos quânticos, QD 655, foram utilizados para marcar mBtBDNF fazer QD-BDNF. A presença do AviTag não interferir com a actividade do BDNF como o mBtBDNF foi capaz de activar TrkB fosforilada (Figura 1E) e estimular o crescimento de neurites (Figura 1F) até ao ponto de BDNF recombinante humano (rhBDNF). Acms mostrado que QD-BDNF colocalized com TrkB em axónios do hipocampo, indicando que QD-BDNF é bioactivo (Figura 1G). Para estudar o transporte de BDNF, QD-BDNF foi adicionado ao compartimento do axónio distaiculturas de microfluidos contendo rato E18 neurónios do hipocampo (Figura 2A). QD-BDNF transporte retrógrado dentro de axônios foi capturado por em tempo real imagens ao vivo da etiqueta fluorescente vermelha (apoiando vídeos S1, S2). Ao analisar o quimógrafo gerado, QD-BDNF foi observada a ser transportados retrogradamente a uma velocidade de deslocamento de cerca de 1,06 pM / seg (Figura 3A). GFP ou mCherry-BDNF com etiquetas ter sido usado para controlar o movimento axonal de BDNF. As principais desvantagens são que eles não são brilhantes o suficiente para os estudos de moléculas individuais. Além disso, a presença de ambos os movimentos anterógrada e retrógrada BDNF torna difícil avaliar se ou não o BDNF transportado retrogradamente era um complexo de neurotrofina / receptor. Neste vídeo, demonstramos as técnicas utilizadas para examinar o tráfico ao vivo do QD-BDNF em câmaras microfluídicos usando neurônios primários. O ultrabrightness e excelente fotoestabilidade de pontos quânticos makes-se possível realizar de seguimento a longo prazo de transporte BDNF. Estas técnicas podem ser explorados para reforçar estudos de função axonal em AD, HD, e outras desordens neurodegenerativas.

<table><tbody><tr><td>Platinum pfx DNA polymerase&amp;nbsp;</td><td>Invitrogen</td><td>11708021</td><td></td></tr><tr><td>EcoRI&amp;nbsp;</td><td>Fermentas</td><td>FD0274</td><td></td></tr><tr><td>BamHI&amp;nbsp;</td><td>Fermentas</td><td>FD0054</td><td></td></tr><tr><td>HEK293FT cells</td><td>Invitrogen</td><td>R70007</td><td></td></tr><tr><td>DMEM-high glucose media</td><td>Mediatech</td><td>10-013-CV</td><td></td></tr><tr><td>D-biotin&amp;nbsp;</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>TurboFect&amp;nbsp;</td><td>Fermentas</td><td>R0531</td><td></td></tr><tr><td>PMSF&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A6279</td><td></td></tr><tr><td>Ni-NTA resins</td><td>Qiagen</td><td>30250</td><td></td></tr><tr><td>Protease inhibitors cocktail</td><td>Sigma</td><td>&amp;nbsp;S8820</td><td></td></tr><tr><td>Silver staining kit&amp;nbsp;</td><td>G-Biosciences</td><td>786-30</td><td></td></tr><tr><td>Human recombinant BDNF</td><td>Genentech</td><td></td><td></td></tr><tr><td>Microfluidic chambers</td><td>Xona</td><td>SND450</td><td></td></tr><tr><td>24 x 40 mm No. 1 glass coverslips&amp;nbsp;</td><td>VWR</td><td>48393-060</td><td></td></tr><tr><td>Poly-L-Lysine&amp;nbsp;</td><td>Cultrex</td><td>3438-100-01</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14185-052</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>GlutaMax&amp;nbsp;</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>B27&amp;nbsp;&amp;nbsp;</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>QD655-streptavidin conjugates</td><td>Invitrogen</td><td>&amp;nbsp;Q10121MP</td><td></td></tr><tr><td>anti-Avi tag antibody</td><td>GenScript</td><td>A00674</td><td></td></tr><tr><td>streptavidin-agarose beads&amp;nbsp;</td><td>Life Technology</td><td>&amp;nbsp;SA100-04</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma</td><td>T6399</td><td></td></tr><tr><td>HRP-streptavidin&amp;nbsp;</td><td>Thermo Scientific</td><td>N100</td><td></td></tr><tr><td>anti-pTrkB antibody</td><td>a generous gift from Dr M. Chao of NYU</td><td></td><td></td></tr><tr><td>anti-TrkB antibody</td><td>BD Science</td><td>610101</td><td></td></tr></tbody></table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF desempenha um papel importante em vários aspectos da sobrevivência neuronal, diferenciação e função. Défices funcionais e estruturais no axônios são cada vez mais vistos como uma característica precoce de doenças neurodegenerativas, incluindo a doença de Alzheimer (AD) e a doença de Huntington (DH). Como ainda é pouco claro o mecanismo (s) pelo qual é induzida lesão axonal. Nós relataram o desenvolvimento de uma nova técnica para produzir biologicamente activo, monobiotinylated BDNF (mBtBDNF) que pode ser utilizado para rastrear o transporte axonal de BDNF. Quantum dot marcado BDNF (QD-BDNF) foi produzido pela conjugação de ponto quântico 655 a mBtBDNF. Um dispositivo de microfluidos foi usada para isolar axónios de corpos celulares de neurónios. A adição de QD-BDNF ao compartimento axonal permitiu imagens ao vivo de transporte BDNF nos axônios. Nós demonstramos que QD-BDNF movido essencialmente exclusivamente retrogradamente, com muito poucas pausas, a uma velocidade de deslocamento de cerca de 1,06 pM / seg. Este sistema pode ser utilizado para me investigarcanismos de função axonal interrompido no AD ou HD, bem como outras doenças degenerativas.

Produção e Purificação de BDNF Mono-biotinilado Biologicamente Activo O vector de expressão de BDNF fundida com uma sequência de AviTag (GGGLNDIFEAQKIEWHE) foi criado de acordo com um protocolo previamente publicado 10. A massa molecular da proteína de fusão de comprimento completo foi previsto para ser ~ 32 kDa ( <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ) Monobiotinylated BDNF maduro com uma massa molecu...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

O transporte axonal de BDNF, um factor neurotrófico, é crítico para a sobrevivência e função de várias populações neuronais. Algumas desordens degenerativas são marcadas pela ruptura da estrutura e função axonal. Nós demonstramos as técnicas utilizadas para examinar o tráfico ao vivo do QD-BDNF em câmaras microfluídicos usando neurônios primários.

Imagens em tempo real de axonal Transporte de Quantum Dot marcado BDNF na Primária Neurônios

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Research

JoVE Journal

Neuroscience

15.1K visualizações.  University of California, San Diego.  O transporte axonal de BDNF, um factor neurotrófico, é crítico para a sobrevivência e função de várias populações neuronais. Algumas desordens degenerativas são marcadas pela ruptura da estrutura e função axonal. Nós demonstramos as técnicas utilizadas para examinar o tráfico ao vivo do QD-BDNF em câmaras microfluídicos usando neurônios primários. 

Vídeo: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagens ao vivo de vesículas com núcleo denso na Primária Cultivadas neurônios do hipocampo

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Biologia

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Tempo-lapso de imagem ao vivo e quantificação da dinâmica da filial dendrítica rápida no desenvolvimento de neurônios de Drosophila

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

Neurociência

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Um protocolo aprimorado para purificar e diretamente mono-biocombinado recombinante BDNF em um tubo para estudos de tráfico celular em neurônios

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imagem e Análise do Transporte de Neurofilamento em Nervo Tibial de Camundongo Excised

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Imagem de lapso de tempo da arborização neuronal usando rotulagem de vírus associado adeno esparsa de populações de células de retina geneticamente direcionadas

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Expansão do Kit de Ferramentas para Imagens In Vivo do Transporte Axonal

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient ...

<meta charset="utf8"></head><body>Visualização de proteínas em neurônios primários do hipocampo por imagens de células vivas

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>Autor em destaque: Compreendendo o impacto das proteínas patológicas no transporte axonal em doenças neurodegenerativas

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Imagens em tempo real de axonal Transporte de Quantum Dot marcado BDNF na Primária Neurônios

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