Name: primary dissociated midbrain dopamine cell cultures from rodent neonates
Uploaded: 2008-11-05T00:00:00
Description: Watch this Scientific Journal Video about Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates at JoVE.com

The work was supported by DK065872 (ENP), a Smith Family Foundation Award of Excellence in Biomedical Research (ENP), F31 DA023760 (BMG, ENP) and P30 NS047243 (Tufts Center for Neuroscience Research).

Preparation Preceding the Culture
Note: ** Glial cells must be plated well in advance so that they have time to proliferate and cover the bottom of the&nbsp; dishes. For mice with atypical or experimental genetic background, make sure to match glial and neuronal cultures in terms of genotype.
Note: ** 1-7 days before dissection, prepare fresh neuronal medium and replace the glial medium in the dishes with 2ml.
Note<...

<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>

The methods described here allow for fine resolution of morphological and neurochemical features of central dopamine neurons that are otherwise not available in a systemic or in vivo approach.

Materials are included in the protocol text.

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.

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

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.

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

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