Name: isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains
Uploaded: 2015-02-19T15:00:00
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.

OBS: Djuren underhålls och hanteras i enlighet med de institutionella riktlinjer och alla djurförsök godkändes av Imperial College Animal Welfare och etiska granskningsorganet (AWERB) och Home Office och Harvard University Institutional Animal Care och användning kommittén (IACUC), i enlighet med federala och statliga regleringar. 1. Reagens och utrustning Setup <ol><li> Tina, alikvot och förvara 1 mg / ml laminin lösning vid -80 ºC. Lös 2 ul i 1 ml DMEM / F12, vilket resulterar ...

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Dopaminerga neuron i mitthjärnan är den viktigaste källan till dopamin i det centrala nervsystemet. De är uppdelade i tre grupper, substantia nigra pars compacta (SNPC), ventrala tegmentala arean (VTA) och retrorubral fält (RRF) 10, 11. De nervceller i SNPC och VTA ge upphov till stora dopaminerga vägar, mesocortical, mesolimbiska och nigro-striatala, deltar i funktioner såsom kontroll av känslor, motivation och motorik. Nedläggningen av nervceller i SNPC och funktionella störningar i det nigrostria...

Förlust av dopaminerga neuroner från substantia nigra pars compacta leder till kardinal motoriska symtomen vid Parkinsons sjukdom (PD), den näst vanligaste neurodegenerativa sjukdomen. Den underliggande orsaken till nedläggningen av denna mesencefal neuronala befolkningen är inte känt. För att studera de biokemiska vägar som ansvarar för utvecklingen och modulera neurofysiologiska egenskaper och överlevnad mesDA neuroner, flera cellodling och djurmodellsystem har använts. Odödliggjorda cellinjer, inklusive råtta dopaminerga cellinjen 1RB 3 AN 27 (N27), den humana dopaminerga neuroblastom-cellinjen SH-SY5Y, mus dopaminerga hybridcellinje MN9D och humant mesencefal LUHMES celler har använts för biokemiska och begränsade mekanistiska studier 1 -5. För studien av den specifika förlusten av mesDA neuroner, flera neurotoxin baserade och genetiska modeller har utvecklats 6-8. Primära ventrala mitthjärnan kulturer, Ger ett oumbärligt verktyg för att studera de neuronala och synaptiska egenskaper hos dopaminerga nervceller och de vägar som är involverade i patogenesen av denna gemensamma sjukdom. Här presenterar vi ett detaljerat protokoll för isolering av mesencefala dopaminerga neuroner, som innehåller ändringar som följer i högre över och ökat utbyte av täck per embryo. Användning av pre-mogna E12.5 mus mesencephalon (E14.5 i råtta) förbättrar överlevnadsförmåga. Vid denna ålder nervceller har inte utvecklat axoner ännu, som lämnar cellerna intakta under dissekering och minimerar stressen vilket avsevärt ökar lönsamheten. Dessutom noggrann dissektion av den ventrala mitthjärnan, som beskrivs i avsnitt 2 i detta protokoll, förbättrar ytterligare överlevnadsförmåga. För att öka antalet täck per embryo, är ett alternativt plätering metod som presenteras i avsnitt 4 i detta protokoll. Detta leder till en direktavkastning på upp till 10 täck per embryo jämfört med fyra täck enligtstandard Pläteringsförhållandena sålunda minska mängden djur per försök. Nervceller i kultur uppvisar utväxt av axoner och dentrites, bildar synapsförbindelser och avslöja förekomsten av neuronala och synaptiska markörer gör dessa kulturer lämplig för levande cell imaging, immuncytokemiska och elektrofysiologiska studier. Vidare användning av neuronala kulturer underlättar genetisk och farmakologisk manipulation. Utväxt av neuriter från dag 2 in vitro möjliggör utvecklingsstudier. Vidare, den långsiktiga överlevnaden av kulturer (upp till sex veckor) gör dem lämpliga för studier av den långsamma, progressiv degeneration av dessa neuroner.

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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.

Immunohistokemi mot tyrosinhydroxylas (TH) visar att mellan 0,5-1% av cellerna i odling är dopaminerga. Neuronala prognoser verkar inom 2 timmar efter bordläggning och av den första dagen, axoner och dendriter kan särskiljas (Figur 2), med hjälp av tyrosinhydroxylas (TH) och mikrotubuli-associerat protein 2 (MAP2) antikroppar (Figur 3). De nervceller överlever mer än sex veckor och visar omfattande utväxt. Neuronen-Glia förhållandet i odlingarna är direkt relaterad till konce...

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.

Isolering, kultur och långsiktigt underhåll av primär mesencefala dopaminerga neuron från embryonala Gnagare Brains

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

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Neuroscience

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Dissektion och kultur commissural neuroner från embryonala ryggmärg

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Nucleofection och Primary kultur av embryonala Mouse hippocampus och kortikala Neuroner

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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

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Isolering och odling av neurallistceller från embryonala Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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Generering och långvarigt underhåll av nervfria<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...