Summary

भ्रूणीय ventral midbrain के Organotypic स्लाइस संस्कृति: एक डोपामिनर्जिक neuronal विकास अध्ययन प्रणाली इन विट्रो में</em

Published: January 31, 2012
doi:

Summary

E12.5 murine भ्रूण midbrain से organotypic स्लाइस उत्पन्न विधि वर्णित है. organotypic टुकड़ा संस्कृतियों डोपामिनर्जिक न्यूरॉन्स या अन्य वेंट्रल मध्यमस्तिष्क न्यूरॉन्स के व्यवहार का निरीक्षण करने के लिए इस्तेमाल किया जा सकता है.

Abstract

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Protocol

इस प्रोटोकॉल के पार्ट्स Daza एट अल से संशोधित कर रहे हैं. 7 2007. 1. तैयारी एक दिन अग्रिम में तैयार किया जा सकता है 1X क्रेब्स बफर (1.5 एल) तैयार: 126 मिमी NaCl, 2.5, 1.2 मिमी मिमी KCl नाह 2 4 प?…

Discussion

organotypic टुकड़ा संस्कृति विधि यहाँ प्रस्तुत और भ्रूण वेंट्रल midbrain में डोपामिनर्जिक न्यूरॉन्स उनके प्रवासी और प्रक्षेपण मार्गों के विकास के इन विट्रो विश्लेषण में अल्पावधि के लिए एक प्रणाली उपलब्?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

हम Martine Emond और इसाबेल Brachmann organotypic टुकड़ा संस्कृति प्रणाली और वोल्फगैंग Hübner और Liviu गेब्रियल Bodea पांडुलिपि के महत्वपूर्ण पढ़ने के लिए स्थापित करने में उनकी मदद के लिए धन्यवाद. हम R26 संवाददाता चूहों श्श्श CreER चूहों के लिए और क्लिफ Tabin के लिए फ्रैंक COSTANTINI धन्यवाद देना चाहूंगा. उत्तर राइन वेस्टफेलिया (Programm zur Förderung der Rückkehr डेस wissenschaftlichen Spitzennachwuchses aus dem Ausland) के विज्ञान और अनुसंधान मंत्रालय से इस अध्ययन में एक अनुसंधान पुरस्कार द्वारा वित्त पोषित किया गया था.

Materials

Table of specific reagents and equipment

Name of the reagent Company Catalogue number Comments (optional)
DMEM Sigma-Aldrich D6429  
Glucose 30% Sigma-Aldrich G7528-250  
Horse Serum Invitrogen 26050-088  
DMEM (4,5g/L Glc., with L-Gln, Na Pyr, NaHCO3) Sigma-Aldrich D6429-500  
Penicillin/Streptomycin 100x Sigma-Aldrich P4333-20  
L-ascorbic acid Sigma-Aldrich A4403 prepare 200mM stock and store at -20°C
UltraPure LMP agarose Invitrogen 15517-022  
Millicel inserts Millipore PICMORG50  
μ-dish 35 mm, low Ibidi 80136  
Vibratome Microm HM 650V  
Razor Blade Plano GmbH 121-6  
Histoacryl  glue BRAU9381104 Braun Aesculap  
Perforated Spoon Dia
diameter 15 mm
Fine Science Tools 10370 -18
Forceps 5 Dumoxel Fine Science Tools 11252 – 30  

Antibodies used for immunostainings:

Name of the antibody Company Catalogue number Comments (optional)
Rabbit anti-tyrosine hydroxylase Millipore AB152 Dilution 1:500
Mouse anti-tyrosine hydroxylase Millipore MAB318 Dilution 1:500
Mouse anti-BrdU BD Pharmingen 555627 Dilution 1:200
Rabbit anti-cleaved caspase 3 Cell Signaling Technology 9661 Dilution 1:200
Donkey anti-rabbit IgG-Alexa 488 Invitrogen A21206 Dilution 1:500
Donkey anti-mouse IgG-Cy3 Jackson ImmunoResearch 715-165-150 Dilution 1:200

References

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Cite This Article
Bodea, G. O., Blaess, S. Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro. J. Vis. Exp. (59), e3350, doi:10.3791/3350 (2012).

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