Gostaríamos de agradecer Ryan Maneri (Oystercatcher Productions, LLC) para filmar o nosso procedimento e Rodney McPhail (Departamento de Ciências Biológicas, Purdue University) para a assistência com a edição do vídeo de dissecação. Pesquisa no laboratório Suter é suportado por concessões do NIH (NS049233 R01) e do Centro de Biociências Bindley da Universidade Purdue, a DMS

Soluções <ul><li> L15-ASW meio de cultura celular (1l) <ul><li> 1 saco de pó L15 </li><li> adicionar 800 ml de H 2 O ultrapura </li><li> NaCl 400 mM </li><li> MgSO 4 27 mM </li><li> MgCl 2 28 mM </li><li> L-Glutamina 4mM </li><li> Gentamicina 50 mg / ml </li><li> HEPES 5 mM </li><li> Ajustar o pH 7,9 </li><li> Adicione gota a gota, CaCl 2 9,3 mM (parar se precipita) </li><li> Adicionar H 2 O ultrapura a 1 l </li><li> Verifique osmolaridade (950-1000 mmol / kg) com osmômetro (pressão de vapor Wescor, # 5520) </li><li> Filtro de 0,22 mM usando uma unidade de filtragem de pressão positiva (Filtros: SVGV010RS Millipore) </li><li> Armazenar a 4 ° C por até 1 mês </li></ul></li><li> Poli-L-lisina (70-150 kD) 200 mg / ml de solução estéril em H 2 O ultrapura mantida a -20 º C </li><li> 0,5 M MgCl 2 solução </li></ul> Dia 1: Dissecção da Aplysia gânglio abdominal Preparação da solução de dissociação enzimática <ol><li> Pesar 90-10 mg de Dispase enzima (Worthington, Cat: LS02111) em um tubo Eppendorf </li><li> Adicionar 900 mL de L15-ASW meio de cultura, e 100 l de H 2 O estéril ultrapura </li><li> Misture e coloque em temperatura ambiente (RT) </li></ol> Dissecção do gânglio abdominal <ol><li> Encha uma seringa de 50 ml com 0,5 M MgCl 2 solução. </li><li> Tome um animal do tanque, lidar com cuidado para evitar resposta animal de defesa de tinta. </li><li> Coloque o Aplysia do seu lado em uma placa de dissecção (isopor board), lado rostral para o lado, certo caudal para a esquerda. </li><li> Inserir a agulha no animal logo atrás da cabeça e injectar toda a solução MgCl 2 na cavidade do corpo. </li><li> Suavemente esfregue o animal para dispersar a solução de MgCl 2 em toda cavidade do corpo, aguarde alguns minutos para que o animal ser completamente anestesiado. </li><li> Pin a cabeça ea cauda do animal para o conselho de dissecação com duas agulhas. </li><li> Use uma pinça para tirar a pele, corte através da pele e da camada muscular com tesoura grande para a cabeça ea cauda para fazer uma grande abertura na lateral. </li><li> Encontrar o gânglio abdominal, sob o ovário escova-like. Use uma pinça para segurar os nervos conectando cerca de 1 cm à frente do gânglio abdominal, cortar os nervos com tesouras de dissecação em frente à posição de fórceps segurando e atrás do gânglio abdominal. </li><li> Gânglio lugar na solução Dispase e incubar por 15-16 horas a 22 ° C em banho-maria com temperatura controlada. </li></ol> Dia 2: Chapeamento de células saco Aplysia Preparando poli-lisina revestido lamínulas <ol><li> Em um banco de fluxo, preparar três pratos de petri 35 milímetros como trabalhar pratos para dissecção do gânglio, enchendo-os com 4 ml de meio L15-ASW cada. Transferir o gânglio abdominal a partir da solução dispase a um dos pratos úteis após 15,5 horas de digestão. </li><li> Utilizando uma pinça tomar ácido-limpos # 1.5 lamínulas (22x22 mm) armazenados em etanol, remover o álcool e esterilizar lamínulas por flaming-los brevemente em bico de Bunsen. Deixe-os esfriar antes de colocar brevemente as lamelas na placas de cultura. </li><li> Prepare uma ponta plating curvas, dobrando uma ponta amarela sobre o bico de Bunsen (chama apenas brevemente para evitar derretimento do plástico!). </li><li> Preparar a quantidade adequada de 20 mg / solução de poli-lisina ml, diluindo 200 mg / ml de solução estéril com H 2 O ultrapura. </li><li> Cobrir cada lamela com 500 mL de 20 mg / ml poli-lisina e incubar por 20 minutos em temperatura ambiente. </li><li> Lave cada lamela 3-5 vezes com 500 mL estéreis H 2 O ultrapura cada. </li><li> Retirar a água de lamínulas; preencher cada prato com 4 ml de L15-ASW meio de cultura. </li></ol> Dissociação de células saco <ol><li> Sob um microscópio de dissecação, corte o gânglio abdominal em meia tesoura e pinça de dissecção utilizando previamente limpas com etanol a 95% (linha 1 Figura 1). </li><li> Em uma metade do gânglio, corte entre clusters saco célula e as células hemiganglion (linha 1 Figura 2), e cortar extensões nervosas remanescentes do outro lado do saco células (linha 1 Figura 3). Trabalhe em uma metade do gânglio de cada vez, deixe a outra metade no prato, enquanto as células do primeiro cluster estão sendo folheado. </li><li> Utilizando duas pinças (ou uma pinça e tesouras de dissecção), a liberação aglomerado de células saco, empurrando de lado a bainha de tecido conjuntivo ao redor do aglomerado de células saco. Cortar tecido conjuntivo com tesouras de dissecação, se necessário. Cuidado para não apertar e tocar o cluster de células saco muito durante este processo. </li><li> Quando a maioria do tecido conjuntivo é removido, a transferência do bag aglomerado de células em um prato novo a trabalhar com a ponta curvada amarelo preparado anteriormente. Tenha cuidado para não deixar que o cluster de células saco de ficar preso dentro da ponta amarela ou preso na interface ar / médio. </li><li> Triturar o cluster com ponta amarela para remover as células saco individual. Faça isso por pipetagem do cluster dentro e fora da ponta. Comece com baixa forças de cisalhamento e aumentar a força quando necessário. Sempre usar a menor força necessária para remover neurônios sem matar células demais. </li><li> Coletar 3-5 células saco saudável e transferi-los para um prato com uma lamela poli-lisina-revestido. Evitar a captação de células mortas (eles são preto / transparente no centro) e restos de tecido conjuntivo. </li><li> Repita a etapa 6. e coloque cerca de 15-25 neurônios vivem no centro de cada lamela. Se as células mortas ou detritos são transferidos, bem como, retirar o material indesejado por pegá-la ou assoar-lo longe das células saudáveis. Este processo leva cerca de 2-3 horas em ambos os clusters. </li><li> Uma vez terminado manter os pratos da cultura no banco de fluxo para pelo menos 2 horas em temperatura ambiente para permitir que as células attach (manter banco de fluxo blower off para evitar vibrações indesejadas). </li><li> Coloque os pratos em uma incubadora a 14 ° C durante a noite, ou até à sua utilização. Cones de crescimento pode se desenvolver em 10/04 horas. </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="Figura 1. Esquemática de Aplysia californica gânglio abdominal. Linhas de cor indicam onde cortar para obter grupos de células do saco." /> Figura 1. Esquemática de Aplysia californica gânglio abdominal. Linhas de cor indicam onde cortar para obter grupos de células do saco. Dia 3: Configurando câmara para imagens ao vivo de células saco Aplysia Montagem de imagens da câmara <ol><li> Antes de utilizar células saco para experimentos com imagens de células vivas, inspecionar as células em um microscópio de baixa potência. Se as células não se desenvolveram cones crescimento saudável, fonte de meio fresco e deixe em temperatura ambiente por 1-2 horas. </li><li> Para fornecer placas com o meio-L15 ASW fresco, remover metade (~ 2ml) de meio de idade, com uma linha de vácuo, e adicionar 2 ml de novos L15-ASW média por placa de Petri. É importante não remover completamente todos os suportes do prato de uma só vez, pois isso irá remover as células da lamela. </li><li> Para imagens de células saco, montamos duas lamínulas e dois espaçadores de plástico em uma câmara de imagem sanduíche-like. A lamela superior no sanduíche é cortar mais curto para permitir a troca fácil de médio e reagentes. Durante a montagem, lamínulas com células saco anexado deve ser submerso em L15-ASW médio em todos os momentos para impedir as células de desalojar. </li><li> Para preparar a câmara, cortado ~ 2 milímetros do lado direito de um milímetro lamínulas # 1 22x22 com diamante caneta (cortador de vidro) e régua. </li><li> Usando uma seringa de 1 ml, aplique graxa de alto vácuo para os dois lados da lamela mais curta, e anexar espaçadores de plástico para cada lado. Aplique graxa de vácuo para a parte superior de ambos os espaçadores. </li><li> Inverter a lamela e montagem espaçador, e coloque em placa de Petri contendo células saco em uma lamela. Alinhar e pressione suavemente para baixo para furar lamela cima para baixo com lamínula na extremidade oposta de dois Q-dicas. </li><li> Utilizando uma pinça, levantar delicadamente montagem sanduíche fora da placa de Petri sem o meio vazando. Alinhe as duas lamelas, se necessário, e retire o excesso com uma média Kimwipe. Coloque o sanduíche para a parte inferior do titular lâmina de microscópio e coloque a tampa com cuidado usando clipes. </li><li> Limpar o centro da parte superior do sanduíche com Q-dicas dabbed no limpador de vidros (Sparkle é usado como um limpador de vidro). Rapidamente secar o limpador de vidros com um segundo, seca Q-dica para evitar filas raia. Faça o mesmo para a lamela inferior. Lamínulas limpa livre de linhas de raia são importantes para geração de imagens de alta qualidade. </li></ol> Células bolsa no microscópio Uma vez que as culturas de células neuronais saco Aplysia estão em baixa densidade, é melhor se a sua configuração microscópio tem funções estágio posicionamento para facilitar a mudança entre as células. Células saco pode ser facilmente mantidos em temperatura ambiente por várias horas no palco durante as experiências de imagem. Troca do meio periodicamente para evitar a evaporação.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Neurônios de células Aplysia fornecer um saco de soro livre de sistema de cultura de células neuronais, com muito poucas células não-neuronais. Estes neurônios formam cones de crescimento muito grande, adequado para tratar uma série de questões importantes células biológicas. Neurônios de células saco pode ser facilmente manipulado e fotografada em temperatura ambiente durante várias horas. Usando Microscopia fluorescente Speckle (FSM) pode-se analisar quantitativamente os vários parâmetros de F-actina e polimerização dos microtúbulos ea dinâmica de translocação. Estas ferramentas de imagem, juntamente com o recentemente lançado informações sobre o genoma Aplysia, bem como técnicas de expressão melhor fazer esses neurônios de um sistema poderoso modelo para estudar os mecanismos moleculares e celulares da motilidade crescimento neuronal cone e orientação.

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<tr>
<td>#1 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>48366067</td>
<td>&nbsp;</td>
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<td>#1.5 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>Corning</td>
<td>2870-22</td>
<td>&nbsp;</td>
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<td>35 mm Petri dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
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<td>Filters for medium filtration</td>
<td>Tool</td>
<td>Millipore</td>
<td>SVGV010RS</td>
<td>&nbsp;</td>
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<td>High vacuum grease</td>
<td>Tool</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>&nbsp;</td>
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<td>Osmometer</td>
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<td>Wescor</td>
<td>5520</td>
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neuronal cell cultures from aplysia for high-resolution imaging of growth cones

Cones de crescimento neuronal são as estruturas altamente móveis na ponta dos axônios que podem detectar sinais de orientação no meio ambiente e transdução esta informação em movimento direcional para a célula de destino. Para entender completamente como a informação é transmitida a orientação da superfície da célula para a dinâmica subjacente redes do citoesqueleto, um precisa de um sistema modelo adequado para imagens de células vivas da dinâmica de proteínas na resolução temporal e espacial de alta. Típicos cones de crescimento vertebrados são pequenos demais para analisar quantitativamente F-actina e dinâmica de microtúbulos. Neurônios da lebre do mar Aplysia californica são 5-10 vezes maior do que os neurônios dos vertebrados, podem ser facilmente mantidos em temperatura ambiente e são células muito robusta para micromanipulação e medidas biofísicas. Cones seu crescimento tem muito definido regiões do citoplasma e um sistema bem-descrito do citoesqueleto. Os corpos celulares neuronais podem ser microinjeção com uma variedade de sondas para estudar a motilidade cone de crescimento e orientação. No presente protocolo que demonstrar um procedimento para a dissecção do gânglio abdominal, a cultura de neurônios de células saco e criação de uma câmara de imagem para imagens de células vivas de cones de crescimento.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neurônios Aplysia californica desenvolver cones grande crescimento na cultura que são excelentes para imagens de alta resolução da motilidade cone de crescimento e orientação. Aqui, apresentamos um protocolo para a dissecção e chapeamento de neurônios células Aplysia saco, bem como para a criação de uma câmara para imagens de células vivas.

Culturas de células neuronais de Aplysia para imagens de alta resolução de Cones de Crescimento

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Nanyang Technological University

Research

JoVE Journal

Biology

11.3K visualizações.  Purdue University. Neurônios Aplysia californica desenvolver cones grande crescimento na cultura que são excelentes para imagens de alta resolução da motilidade cone de crescimento e orientação. Aqui, apresentamos um protocolo para a dissecção e chapeamento de neurônios células Aplysia saco, bem como para a criação de uma câmara para imagens de células vivas.

Vídeo: Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Biologia

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Preparando-se Fator de Crescimento de Células T esplenócitos Rat

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

De alta resolução de vídeo de Rastreamento Locomotion em Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparação de Aplysia Sensório-motor Neuronal culturas de células

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Culturas de células primárias de Drosophila Os embriões Gástrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Geração de Disperso Presomitic mesoderme culturas de células for Imaging da Segmentação Relógio Zebrafish em células individuais

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

O método ex vivo para High Resolution Imaging de Motilidade Cilia em Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Usando plusTipTracker software para medir Microtubule Dynamics em<em&gt; Xenopus laevis</em&gt; Cones de Crescimento

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...