Name: isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains
Uploaded: 2015-02-19T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

NOTA: Gli animali sono stati mantenuti e gestiti in conformità con le linee guida istituzionali e tutte le procedure di animali sono stati approvati dal benessere degli animali del Imperial College e Ethical Review Body (AWERB) e il Ministero degli Interni e la Harvard University Istituzionale Animal Care and Use Committee (IACUC), in conformità alle normative federali e statali. 1. reagente e installazione attrezzature <ol><li> Thaw, aliquota e conservare 1 mg / ml soluzione laminina a -80 ° C. Sciogliere 2 microlitri in 1 ml DMEM / F12, ottenendo una concentrazione di rivestimento di 1-2 mg / cm 2. NOTA: Thaw laminina lentamente sul ghiaccio e si dissolvono in freddo DMEM / F12 e aggiungerlo ai piatti / coprioggetti immediatamente. </li><li> Diluire 75 ml di 0,01% Poly-L-ornitina in 425 ml di PBS e conservare a 4 ° C. NOTA: La durata della soluzione di lavoro è fino a un mese. </li><li> Sciogliere 25% (peso / volume) BSA in PBS, pH 7.4, e conservare a 4 ° C per un massimo di un year. </li><li> Preparare 50% FBS (in HBSS) mezzi di disattivazione. </li><li> Preparare terreno completo aggiungendo 50 U / ml di penicillina e streptomicina, 1x N2 supplemento, 5% (vol / vol) FBS, 0,36% D - (+) - Glucosio (peso / volume), 0,25% di BSA (peso / volume) di DMEM / F12 e conservare a 4 ° C. NOTA: La durata è fino a 10 giorni. </li><li> Luogo coprioggetto in ebollizione il 70% (vol / vol) di etanolo per almeno 30 minuti per rimuovere eventuali tracce di grasso e di autoclave. </li><li> Luogo coprioggetto in piastre da 24 pozzetti e aggiungere 500 microlitri soluzione Poli-L-ornitina in ciascun pozzetto. Incubare 1 ora sotto il cofano di coltura tissutale a temperatura ambiente e poi, lavare 3 volte con 500 microlitri acqua. Aggiungere la soluzione laminina 500 microlitri per ogni piatto e incubare O / N in umidificata coltura tissutale incubatore a 37 ° C. NOTA: Anche se non sono necessari lavaggi dopo il trattamento laminina, lavare i poli-L-ornitina meno di tre volte provoca la morte delle cellule 24 ore dopo coltura. </li><li> Lucidare le punte delle pipette di vetro oltre a gas naturale o torcia fzoppo. NOTA: Dopo questo passo il diametro della punta dovrebbe essere circa la metà della sua dimensione normale. </li><li> Tagliare i tappi di provette da 1,5 ml microcentrifuga, utilizzando una forbice autoclave e autoclave. </li></ol> 2. dissezione embrionali ventrale Mesencefalo <ol><li> Narcotizzare gravide temporizzato (E12.5) dighe di CO 2 inalazione e quindi eutanasia da dislocazione cervicale. </li><li> Spruzzare l&#39;addome con il 70% di etanolo e fare un&#39;incisione addominale fino a quando le sacche uterine sono tutti esposti. Uso pinze tagliare l&#39;attaccamento vaginale e rimuovere l&#39;utero. Inserire l&#39;utero in ghiacciata HBSS, in una piastra di Petri 100 mm. </li><li> Rimuovere embrioni dall&#39;utero e sacco amniotico, usando pinze (Figura 1C). Luogo embrioni in HBSS ghiacciata fresca. NOTA: Il rettangolo in figura 1D indica la posizione del mesencefalo ventrale embrionale. </li><li> Mettere gli embrioni al microscopio dissezione (10x) e sezionare il mesencefalicaarch tagliando il cervello al istmo e mesencefalica-diencephalic regione di confine, con il bioscissor e pinza (Figura 1E, si prega di fare riferimento alla Figura 1A per le linee di incisione). </li><li> Dopo aver preso l&#39;intera mesencefalo, fare un taglio nel mesencefalo mediodorsale (Figura 1F). </li><li> Rimuovere meningi, utilizzando due pinze (Figura 1H). NOTA: PUNTO CRITICO: Questo passaggio è fondamentale per la resa ottimale neuronale e la sopravvivenza. Poiché le condizioni di coltura sono ottimali per le cellule cerebrali, la maggior parte delle cellule del tessuto circostante muoiono dopo placcatura e il segnale apoptotico da questo tessuto può danneggiare l&#39;integrità delle culture. </li><li> Appiattire il tessuto mesencefalo sul piatto Petri per osservare la forma a farfalla e tagliare circa la metà di ogni ala, utilizzando una lama da barba sterilizzato (Figura 1H, I). NOTA: Figura 1J descrive isolato mesencefalo ventrale. </li><li> Trasferire ilpezzo di mesencefalo ventrale in ghiaccio-freddo HBSS in un tubo da 15 ml e procedere con il successivo dell&#39;embrione. NOTA: i passaggi 2,1-2,8 non dovrebbe prendere più di 1 ora. Mantenere gli embrioni di là di questo lasso di tempo sul ghiaccio diminuisce vitalità cellulare in modo significativo. </li></ol> 3. La dissociazione delle cellule ventrale Midbrain <ol><li> Trasferire i pezzi di mesencefalo ventrale sotto una cappa a flusso laminare. Rimuovere il HBSS e aggiungere 1 ml di pre-riscaldato (37 ° C) 0,05% tripsina-EDTA nel tubo conico (volumi sufficienti per un massimo di 12 pezzi di mesencefalo ventrale). Incubare il tessuto a 37 ° C per 5-10 minuti. </li><li> Rimuovere tripsina-EDTA sotto il cofano e aggiungere 1 ml di mezzo di disattivazione (siero disattiverà tripsina) al tessuto. </li><li> Rimuovere il mezzo disattivazione e lavare il tessuto in 1 ml di mezzo completo due volte. NOTA: evitare di rimuovere tutto il supporto dal tubo e pipettando il tessuto, per evitare che scartando le cellule dissociate. </li><li> Aggiungere 1 ml terreno completo e triturato il tessuto con un bicchiere pipetta fuoco lucido. Evitare la formazione di bolle e continuare triturazione fino al raggiungimento sospensione di cellule singole. NOTA: Di solito, 8-10 passa attraverso la punta ristretta sono necessari per la completa dissociazione. </li><li> Centrifugare le cellule a 400 xg per 5 minuti a RT e rimuovere il supporto. </li><li> Risospendere il pellet in 1 ml di media completo lentamente pipettando su e giù per un massimo di quattro volte. </li></ol> 4. Cellule placcatura ventrale Midbrain <ol><li> Miscelare 10 ml di sospensione cellulare con 90 microlitri Trypan blue soluzione e contare il numero di cellule con un emocitometro. NOTA: la vitalità delle cellule può essere controllato in questa fase. </li><li> Mettere tappi provetta sterilizzati in 100 millimetri piastre di Petri e trasferire i coprioggetti Poly-L-ornitina / laminina rivestite, utilizzando pinze, una alla volta sulla parte superiore dei tappi. NOTA: Non lavare i coprioggetti ed evitare l&#39;essiccazione della laminina, poiché questo può causare uneveculture n e diminuzione sopravvivenza delle cellule. </li><li> Regolare il volume a 1500 cellule per 1 ml aggiungendo mezzo completo e aggiungere 100 microlitri (totale di 150.000 cellule) della sospensione cellulare sopra l&#39;coprioggetto (il volume è ottimale per coprire la superficie del vetrino senza perdite). Chiudere le piastre di Petri e incubare per 1 ora in un incubatore umidificato coltura tissutale (37 ° C, 5% CO 2). NOTA: PUNTO CRITICO: Questo passo è necessario per migliorare il collegamento e la vitalità delle cellule e per aumentare il numero di lamelle per dell&#39;embrione. In alternativa, le cellule possono essere trasferiti direttamente nei pozzetti (contenenti coprioggetto), ma il numero di celle deve essere aumentata a 350.000 per pozzetto (invece di 150.000). In altre parole, con questo metodo, 8-10 coprioggetto possono essere acquisiti, mentre il trasferimento diretto produce circa 3-4 vetrini a causa delle cellule, che si attaccano alle piastre (accanto o sotto i coprioggetti). La viabili mediaty delle culture, con questo metodo è stato di circa il 90%. </li></ol> 5. Cultura crescita e manutenzione <ol><li> Dopo 1 ora di incubazione, trasferire accuratamente i coprioggetti compreso il terreno in piastre da 24 pozzetti, contenente 400 ml di pre-riscaldato (a 37 ° C) terreno completo (volume totale: 500 ml). Incubare le cellule O / N a 37 ° C. </li><li> Entro 24 ore dopo incubazione dei pozzetti, aggiungere delicatamente 500 microlitri terreno completo in ogni pozzetto. NOTA: Le prime ore è la fase più critica per la sopravvivenza dei neuroni dopaminergici e il numero di cellule sopravvissute non cambia significativamente oltre questo punto. </li><li> Non aggiungere supporti supplementare alle colture per le prime due settimane o finché il terreno diventa gialla. Se coltura per più di due settimane o cambiamenti di colore medio al giallo, cambio la metà dei mezzi di comunicazione (500 ml) con mezzi freschi completi pre-riscaldato (tipicamente ogni due settimane). NOTA: i neuroni dopaminergici nelculture sarebbero sopravvissuti per più di 6 settimane in queste condizioni. </li></ol>

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No conflict of interest declared.

Neuroni dopaminergici nel mesencefalo sono la principale fonte di dopamina nel sistema nervoso centrale. Sono divisi in tre gruppi, pars compacta della sostanza nera (SNPC), area ventrale tegmentale (VTA) e campo retrorubral (RRF) 10, 11. I neuroni in SNPC e VTA danno luogo a grandi vie dopaminergiche, mesocorticali, mesolimbico e nigrostriatale, coinvolte in funzioni come il controllo delle emozioni, la motivazione e il comportamento del motore. Scomparsa dei neuroni in SNPC e disgregazione funzionale del si...

La perdita di neuroni dopaminergici della substantia nigra pars compacta conduce ai sintomi motori cardinali della malattia di Parkinson (PD), la seconda malattia neurodegenerativa più diffusa. La causa della morte di questa popolazione neuronale mesencefalica non è nota. Per studiare i meccanismi biochimici responsabili dello sviluppo e modulando le proprietà neurofisiologiche e la sopravvivenza dei neuroni mesDA, diverse colture cellulari e sistemi modello animali sono stati utilizzati. Linee cellulari immortalizzate, compreso il ratto dopaminergica linea cellulare 1RB 3 AN 27 (N27), il dopaminergico umano linea cellulare di neuroblastoma SH-SY5Y, la linea cellulare di ibridoma del mouse dopaminergica MN9D e mesencefalico umana LUHMES cellule sono state usate per gli studi meccanicistici biochimici e limitate 1 -5. Per lo studio della perdita di neuroni specifica mesDA, vari neurotossina-based e modelli genetici sono stati sviluppati 6-8. Culture mesencefalo ventrale primarie, Fornire uno strumento indispensabile per lo studio delle proprietà neuronali e sinaptiche dei neuroni dopaminergici e i meccanismi coinvolti nella patogenesi di questa malattia comune. Qui vi presentiamo un protocollo dettagliato per l&#39;isolamento dei neuroni dopaminergici mesencefalici, che contiene le modifiche con conseguente maggiore sopravvivenza e una maggiore resa di vetrini per dell&#39;embrione. L&#39;uso di pre-maturo mesencefalo del mouse E12.5 (E14.5 nel ratto) migliora la sopravvivenza. A questa età i neuroni non hanno ancora sviluppato assoni, che lascia intatte le cellule durante la dissezione e riduce al minimo lo stress incrementando in modo significativo la redditività. Inoltre, attenta dissezione del mesencefalo ventrale, come descritto nella sezione 2 del presente protocollo, migliora ulteriormente la capacità di sopravvivenza. Per aumentare il numero di vetrini per embrione, un metodo alternativo di placcatura è presentato nella sezione 4 del presente protocollo. Questo porta ad un rendimento di fino a 10 vetrini per dell&#39;embrione rispetto a 4 coprioggetti sottocondizioni standard di placcatura riducendo così la quantità di animali per esperimento. I neuroni in coltura mostra conseguenza di assoni e dentrites, formano connessioni sinaptiche e rivelano la presenza di marcatori neuronali e sinaptiche rendono queste culture adatto per l&#39;imaging cellulare dal vivo, immunocitochimica e studi elettrofisiologici. Inoltre, l&#39;uso di colture neuronali facilita la manipolazione genetica e farmacologica. Escrescenza dei neuriti dal giorno 2 in vitro consente per gli studi di sviluppo. Inoltre, la sopravvivenza a lungo termine di culture (fino a sei settimane) li rende adatti per lo studio della lenta, progressiva degenerazione dei neuroni questi.

<table><tbody><tr><td>Dulbecco&#039;s modified Eagle medium nutrient mixture F-12</td><td>Invitrogen</td><td>11330</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) (1X), liquid</td><td>Invitrogen</td><td>24020-117</td><td></td></tr><tr><td>Fetal bovine serum, heat-inactivated (FBS)</td><td>Invitrogen</td><td>16140</td><td></td></tr><tr><td>N2 Supplement (100X), liquid</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>D-(+)-Glucose solution (45% (wt/vol) in water</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>Bovine serum albumin BSA</td><td>Sigma</td><td>A9430</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15070</td><td></td></tr><tr><td>Trypsin (0.05% (wt/vol)</td><td>Invitrogen</td><td>25300</td><td></td></tr><tr><td>Bovine serum albumin (BSA) cell culture tested</td><td>Sigma&amp;nbsp;</td><td>A9418</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma</td><td>P3813</td><td></td></tr><tr><td>Anti-Tyrosine hydroxylase (Th) antibody</td><td>Pel-Freez Biologicals</td><td>P60101-0&amp;nbsp;</td><td></td></tr><tr><td>Poly-L-ornithine, 0.01% solution</td><td>Sigma</td><td>P4957</td><td></td></tr><tr><td>Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td><td>Millipore</td><td>MAB3418</td><td></td></tr><tr><td>Anti-Synapsin-1 antibody</td><td>Millipore</td><td>AB1543P</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21206</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21207</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11015</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11016</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-mouse IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21203</td><td></td></tr><tr><td>Trypan blue solution (0.4% (wt/vol))</td><td>Biowhittaker</td><td>17-942E</td><td></td></tr><tr><td>Stereo Microscope</td><td>Carl Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>Inverted phase contrast microscope</td><td>Carl Zeiss</td><td>Axiovert 40 C</td><td></td></tr><tr><td>Dumont Forceps</td><td>Fine Scientific Tools</td><td>May-45</td><td></td></tr><tr><td>Cover Slip Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11251-33</td><td></td></tr><tr><td>Two Dumont #45 Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11245-30</td><td></td></tr><tr><td>Blade Holder/Breaker Flat Grip - 11cm</td><td>Fine Scientific Tools</td><td>10052-11</td><td></td></tr><tr><td>Student Iris Scissors - Straight 11.5 cm</td><td>Fine Scientific Tools</td><td>91460-11</td><td></td></tr><tr><td>Fiber optic halogen illuminator</td><td>Nikon</td><td>MKII</td><td></td></tr><tr><td>Disposable Borosilicate Glass Pasteur Pipettes</td><td>Fisherbrand</td><td>13-678-20C</td><td></td></tr><tr><td>Hemocytometer</td><td>Proscitech</td><td>SVZ2NIOU</td><td></td></tr><tr><td>0.2 &amp;micro;m sterile filter units</td><td>Nalgene</td><td>NL-CE-156-4020</td><td></td></tr><tr><td>100x20 mm Petri dishes</td><td>BD Biosciences</td><td>351005</td><td></td></tr><tr><td>Round Cover Slip #1 Thickness German Glass 12 mm</td><td>Bellco Glass</td><td>1943-10012&amp;nbsp;</td><td></td></tr></tbody></table>

isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Immunoistochimica contro tirosina idrossilasi (TH) mostra che tra 0,5-1% delle cellule in coltura sono dopaminergica. Proiezioni neuronali sembrano in 2 ore dopo la placcatura e il primo giorno, assoni e dendriti sono distinguibili (figura 2), utilizzando la tirosina idrossilasi (TH) e proteina 2 (Map2) anticorpi associata ai microtubuli (Figura 3). I neuroni sopravvivono per più di sei settimane e mostrano un&#39;ampia conseguenza. Il rapporto neurone-glia nelle colture è direttament...

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e lungo termine Manutenzione di primaria mesencefalico neuroni dopaminergici Da Brains embrionali dei roditori

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

isolation-culture-long-term-maintenance-primary-mesencephalic

Research

JoVE Journal

Neuroscience

13.9K Views.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Video: Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

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

Ricerca

Biologia

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e la coltura di neuroni ippocampali da topi prenatali

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Neuroscienze

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primario di topo neuroni dopaminergici

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Rapida genotipizzazione degli animali Seguito da Stabilire colture primarie di neuroni del cervello

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Generazione di neurosfere da neuroni ippocampali primari misti e corticali isolati da E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultura primaria dei neuroni isolata dal cerbellello del topo embrionale

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

 Protocollo di co-coltura primaria di microglia e neuroni per studi di interazione con il SNC

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Autore in primo piano: Modello di co-coltura in vitro per lo studio delle interazioni microglia-neuroni in condizioni di malattia

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Isolamento, cultura e lungo termine Manutenzione di primaria mesencefalico neuroni dopaminergici Da Brains embrionali dei roditori

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Capitoli in questo video

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

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Cultura primario di topo neuroni dopaminergici

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Generazione di neurosfere da neuroni ippocampali primari misti e corticali isolati da E14-E16 Sprague Dawley Rat Embryo

Cultura primaria dei neuroni isolata dal cerbellello del topo embrionale

Generazione e co-coltura di microglia primarie murine e neuroni corticali

Generazione e co-coltura di microglia primarie murine e neuroni corticali

Generazione e co-coltura di microglia primarie murine e neuroni corticali