Este trabalho foi financiado por uma bolsa NIH NS27501 para DKOD. Suporte adicional para este trabalho foi fornecida por um Grant Programa HHMI Professor para DKOD.

I. Dissecção de cérebros da mosca adulta <ol><li> Coloque uma pequena gota (~ mL 100) de dissecar solução no centro de uma placa de Petri 35 mm. </li><li> Pegar fêmea adulta utilizando aspirador. Sob microscópio de dissecação, use duas agulhas de seringa para decapitar a voar. </li><li> Cabeça no lugar uma pequena gota de solução salina de dissecação (com papaína adicionados) numa placa de Petri. </li><li> Posição com cabeça com tromba de frente para fundo do prato. </li><li> Segure a cutícula que cobre o olho composto esquerdo com agulha 1. Faça um corte diagonal que se estende desde o dorsal para a superfície ventral com agulha 2, apenas medial a agulha 1. </li><li> Segure o aparelho bucal com agulha e usar uma agulha de 2 para fazer um corte horizontal que se estende desde a superfície ventral do olho esquerdo para o olho direito, ao nível da base da tromba. </li><li> Segure a cutícula que cobre o olho composto direito, com agulha 1. Faça um corte diagonal que se estende desde o dorsal para a superfície ventral com agulha 2, apenas medial da agulha 1. </li><li> Gire a cabeça para que o lado rostral está na superfície placa de Petri. Insira a agulha 1 entre a cutícula rostral e cérebro, e usar agulhas de 2 a seguir o mesmo caminho como a primeira agulha. Idealmente, a cápsula será se desfazer do cérebro com ambos os lobos óptica anexada uma vez que estes são importantes na estabilização do cérebro para a gravação. </li><li> Traquéia, sacos de ar, e outros tecidos conectivos são cuidadosamente removidos com duas pinças de ponta fina. </li><li> Dissecção todo deve tomar 30-10 minutos </li></ol> II. Montagem do CNS <ol><li> O cérebro é transferido para a câmara de gravação usando uma pipeta de ponta amarela. </li><li> Na câmara de gravação, o cérebro é estabilizado pela colocação do quadro de platina de forma que dois mira bem fazer contato com o tecido na junção entre os lobos ópticos e região do cérebro central. </li><li> Cada cérebro é permitido descansar na câmara de gravação com perfusão contínua com solução salina oxigenada (95% de oxigênio e dióxido de carbono de 5%) durante pelo menos 10 minutos. Perfusão da câmara com solução salina oxigenada é continuado durante todo o período de gravação. Preparação é visualizada utilizando um microscópio vertical (Axioskop 2FS; Zeiss, Oberkochen, Alemanha), com um palco fixo e uma objetiva de imersão 40x de água (Achroplan; abertura numérica, 0,8; Zeiss) e Nomarski óptica. GFP foi visto com um 505-530 BP de filtros de fluorescência. </li></ol> III. Eletrofisiologia <ol><li> Pipetas de 14/08 Mohms são usados ​​para gravações de células inteiras </li><li> Gravações braçadeira de tensão e corrente-clamp são realizadas usando uma Lista de EPC7 ou um amplificador Axopatch 200B, a Digidata 1322A DA conversor (dispositivos Molecular, Foster City, CA), um Dell Dimension 8200 computador (Dell Computer, Round Rock, TX), e pClamp 9 software (dispositivos Molecular). </li></ol>

<ol>
	<li>Gu, H., O Dowd, D. K., K, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+Adult+Drosophila+Kenyon+cells+in+situ.">Cholinergic synaptic transmission in Adult Drosophila Kenyon cells in situ.</a> Journal of Neuroscience. 26, 265-272 (2006).</li><li>Wilson, R. I., Turner, G. C., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+olfactory+representations+in+the+Drosophila+antennal+lobe.">Transformation of olfactory representations in the Drosophila antennal lobe.</a> Science. 303, 366-370 (2004).</li></ol>

A preparação do cérebro isolado todo ilustramos neste vídeo permite a avaliação dos mecanismos celulares subjacentes a excitabilidade ea transmissão sináptica em neurônios identificados, incluindo aqueles em vias de processamento olfativo, no adulto Drosophila cérebro (Gu e O Dowd, 2006). Esta abordagem é complementar ao estudo da atividade neuronal no cérebro de adultos intactos Drosophila (Wilson et all, 2004), em grande parte as gravações mesma forma dos neurônios em uma fatia do cérebro de mamíferos são complementares ...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>#5 Foceps</td>
<td>Tool</td>
<td>FST by DUMONT</td>
<td>No. 11251-10 </td>
<td>Be careful, never damage the fine tips of forceps</td>
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<tr>
<td>Papain</td>
<td>Reagent</td>
<td>Worthington</td>
<td>3126</td>
<td>20 U/ml activated by 1 mM L-cysteine</td>
</tr>
<tr>
<td>Dissecting microscope</td>
<td>SMZ-2B Model:C-PS</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Equipped with gooseneck fiber optic lights positioned a low angle</td>
</tr>
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<td>Needle & Syringe </td>
<td>&nbsp;</td>
<td>PrecisionGlide&#174;?, Becton Dickinson & Co </td>
<td>305175 & 309602</td>
<td>Cheap and durable</td>
</tr>
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<td>Upright microscope</td>
<td>Axioskop2 FS plus</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Equipped with a fixed stage, 40X water immersion objective, Nomarski optics, and fluorescent lamp</td>
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<td>Patch clamp amplifier</td>
<td>200 B</td>
<td>Molecular device</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Digidata D-A converter </td>
<td>1322A</td>
<td>Molecular device</td>
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whole cell recordings from brain of adult drosophila

Neste vídeo, demonstramos o procedimento para isolar toda cérebros de adultos Drosophila em preparação para gravar a partir de neurônios individuais. Começamos por descrever a solução de dissecação e captura das fêmeas adultas usadas em nossos estudos. O procedimento para a remoção de todo o cérebro intacto, incluindo ambos os lobos óptica, é ilustrado. Dissecção da traquéia sobrejacente também é mostrada. O cérebro isolado não é apenas pequena, mas necessita de cuidados especiais no manuseio, nesta fase, para evitar danos aos neurônios, muitos dos quais estão perto da superfície externa do tecido. Mostramos como um suporte especial que desenvolvemos é usado para estabilizar o cérebro na câmara de gravação. A eletrofisiologia padrão criado é usado para gravar a partir de neurônios individuais ou pares de neurônios. Uma imagem fluorescente, vistos através do microscópio de gravação, de uma linha de condução GAL4 expressão GFP (GH146) ilustra como os neurônios de projeção (PNs) são identificados no cérebro vivo. A imagem de alta potência Nomarski mostra uma visão de um único neurônio que está sendo alvo para gravação de células inteiras. Quando o cérebro é removido com sucesso sem danos, a maioria dos neurônios são espontaneamente ativos, disparando potenciais de ação e / ou exibindo entrada sináptica espontânea. Esta em preparação local, em que a gravação de células inteiras de neurônios identificados em todo o cérebro pode ser combinado com manipulações genéticas e farmacológicas, é um modelo útil para explorar fisiologia celular e plasticidade no SNC adulto.

Watch this Scientific Journal Video about Whole Cell Recordings from Brain of Adult Drosophila at JoVE.com

Whole Cell Recordings from Brain of Adult Drosophila

Este vídeo demonstra o procedimento para isolar toda cérebros de adultos Drosophila em preparação para gravar a partir de neurônios individuais usando a tecnologia de célula padrão todo. Inclui imagens de células GFP rotulados e neurônios visto durante a gravação.

Todo Recordings celular do cérebro de adultos Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

whole-cell-recordings-from-brain-of-adult-drosophila

Research

JoVE Journal

Biology

16.4K visualizações.  University of California, Irvine (UCI). Este vídeo demonstra o procedimento para isolar toda cérebros de adultos Drosophila em preparação para gravar a partir de neurônios individuais usando a tecnologia de célula padrão todo. Inclui imagens de células GFP rotulados e neurônios visto durante a gravação.

Vídeo: Whole Cell Recordings from Brain of Adult Drosophila

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissecação de discos imaginais de 3 larvas Drosophila Instar

Biologia

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Transplante de Medula Renal inteiro em Zebrafish Adulto

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

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 ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

De célula inteira Recordings of Light Evocados Excitatórios Correntes Synaptic na Slice Retinal

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissecção de Órgãos do Zebrafish Adulto

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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 ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissecção da Oenocytes de Adultos Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e Purificação de cinesina partir Drosophila Os embriões

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificação de Transcrições e metabolitos de<em&gt; Drosophila</em&gt; Cabeças

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...