El trabajo fue apoyado por DK065872 (PEV), una familia Smith Premio de la Fundación de Excelencia en Investigación Biomédica (PEV), F31 DA023760 (BMG, PEV) y P30 NS047243 (Tufts Center for Neuroscience Research).

<ol>
	<li>Geiger, B. M., Behr, G. G., Frank, L., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+defective+mesolimbic+dopamine+exocytosis+in+obesity-prone+rats.">Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats.</a> FASEB Journal. 8, 2740-2746 (2008).</li><li>Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dopamine+quantal+size+in+midbrain+and+hippocampal+neurons.">Regulation of dopamine quantal size in midbrain and hippocampal neurons.</a> Behavioural Brain Research. 130, 203-207 (2002).</li><li>Pothos, E. N., Larsen, K. E., Setlik, W., Gershon, M. D., Krantz, D., Liu, Y. -. J., Edwards, R. H., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+transporter+expression+regulates+vesicle+phenotype+and+quantal+size.">Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.</a> The Journal of Neuroscience. 20, 7297-7306 (2000).</li><li>Pothos, E. N., Davila, V., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+recording+of+quanta+from+midbrain+dopamine+neurons+and+modulation+of+the+quantal+size.">Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size.</a> The Journal of Neuroscience. 18, 4106-4118 (1998).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+mechanisms+that+regulate+quantal+size.">Presynaptic mechanisms that regulate quantal size.</a> Reviews in the Neurosciences. 11, 159-212 (2000).</li></ol>

Los métodos descritos aquí permiten la resolución de multa de características morfológicas y neuroquímicas de las neuronas dopaminérgicas centrales que se que no esta disponible en un enfoque sistémico o en vivo.

Los materiales están incluidos en el texto del protocolo.

primary dissociated midbrain dopamine cell cultures from rodent neonates

La capacidad de crear cultivos celulares primarios de neuronas de la dopamina permite el estudio de las características pre-sináptica de las neuronas de dopamina en el aislamiento de la entrada general a partir de otras partes del cerebro. En nuestro laboratorio, usamos estas neuronas para evaluar la cinética de liberación de la dopamina con amperometría de fibra de carbono, así como los niveles de expresión de genes relacionados con la dopamina y las proteínas utilizando la PCR cuantitativa y la inmunocitoquímica. En este video, te mostramos cómo generar estas culturas de los recién nacidos de roedores. El proceso consiste en varios pasos, incluyendo el revestimiento de los astrocitos corticales gliales, el condicionamiento de medios de cultivo celular neuronal por el sustrato gliales, la disección del cerebro medio, en los recién nacidos, la digestión, extracción y revestimiento de las neuronas de dopamina y la adición de factores neurotróficos a garantizar la supervivencia celular. Las aplicaciones adecuadas para esta preparación incluye la electrofisiología, la inmunocitoquímica, PCR cuantitativa, la microscopía de vídeo (es decir, de la fusión vesicular en tiempo real con la membrana plasmática), los ensayos de viabilidad celular y otras pantallas toxicológicos.

Watch this Scientific Journal Video about Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates at JoVE.com

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

Primaria disociada del cerebro medio de cultivos celulares permiten la dopamina para el estudio de las características pre-sináptica de las neuronas dopaminérgicas. Pueden ser utilizados para monitorear en tiempo real la cinética de la liberación de dopamina y de la proteína / niveles de ARNm de los reguladores de la exocitosis de dopamina. Aquí te mostramos cómo generar estas culturas de los recién nacidos de roedores.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

primary-dissociated-midbrain-dopamine-cell-cultures-from-rodent

Research

JoVE Journal

Biology

14.8K vistas.  Tufts University. Primaria disociada del cerebro medio de cultivos celulares permiten la dopamina para el estudio de las características pre-sináptica de las neuronas dopaminérgicas. Pueden ser utilizados para monitorear en tiempo real la cinética de la liberación de dopamina y de la proteína / niveles de ARNm de los reguladores de la exocitosis de dopamina. Aquí te mostramos cómo generar estas culturas de los recién nacidos de roedores.

Video: Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES células derivadas de células de las Culturas neuroepiteliales

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Investigación

Biología

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.

Cultivos neuronales primarios del cerebro de Drosophila pupas etapa tardía

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

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.

Preparación de cultivos neuronales a partir de embriones de Drosophila Midgastrula Etapa

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

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparación de los cultivos disociados ratón neuronas corticales

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

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

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 into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Las culturas de las células nerviosas de Aplysia para imágenes de alta resolución de los Conos de Crecimiento

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 ...

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.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Modelo murino de enfermedad de Parkinson: de la lesión 6-OH dopamina a prueba de comportamiento

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

El establecimiento de cultivos primarios de fibroblastos de adultos de los roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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.

Cultivos de células primarias de Drosophila Gástrula embriones

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

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

La eliminación rápida de fibroblastos de alta densidad Humanos culturas de células madre embrionarias

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...