Name: isolation and culture of hippocampal neurons from prenatal mice
Uploaded: 2012-07-26T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Hippocampal Neurons from Prenatal Mice at JoVE.com

We thank Dr. Michael Wooten for his help in preparing the manuscript. This work was supported by NIH 2RO1NS033661 (MWW).

1. Set-up Prior to Harvest
<ol>
 <li>To generate prenatal pups for neuron harvest, schedule breeding between adult mice 19 days prior to the day of neuron isolation. (C57BL/6 mice ages 2-8 months were used in matings for the purposes of developing this protocol). Successful mating can be confirmed by detection of a vaginal plug in the female, palpitation or visual confirmation of pregnancy.</li>
 <li>The day prior to neuron isolation:
 <ol class="ualpha">
 <li>For immunofluorescence applications, coat glass c...

<ol>
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Hippocampal cultures have been used in molecular biology for more than 20 years. While in principle, neuronal cultures can be made from any part of the brain, hippocampal cultures have proven to be the most popular due to the relatively simple architecture of the nerve cell population in the hippocampus7. Hippocampal cultures are typically made from late-stage embryonic tissue. This tissue is easier to dissociate and contains fewer glial cells than does mature brain tissue1. Isolation of hippocampal...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Vendor</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>Rat Tail Collagen 1</td>
 <td>BD Biosciences</td>
 <td>354236</td>
 </tr>
 <tr>
 <td>Poly-D-lysine Solution</td>
 <td>Chemicon</td>
 <td>A-003-E</td>
 </tr>
 <tr>
 <td>Hanks Balanced Salt Solution</td>
 <td>Invitrogen</td>
 <td>14175-095</td>
 </tr>
 <tr>
 <td>Trypsin Solution (1X) 0.25%, liquid</td>
 <td>Invitrogen</td>
 <td>15050-065</td>
 </tr>
 <tr>
 <td>NeuroBasal Medium (1X) liquid</td>
 <td>Invitrogen</td>
 <td>21103-049</td>
 </tr>
 <tr>
 <td>B27 Supplement (50X) liquid</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 </tr>
 <tr>
 <td>L-Glutamine 200 mM (100X) liquid</td>
 <td>Invitrogen</td>
 <td>25030-149</td>
 </tr>
 <tr>
 <td>Penicillin (10,000 units/ml) / Streptomycin (10,000 &mu;g/ml)</td>
 <td>Invitrogen</td>
 <td>15140-148</td>
 </tr>
 <tr>
 <td>HI-Donor Horse Serum</td>
 <td>Atlanta Biologicals</td>
 <td>S12150H</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Isolation and Culture of Hippocampal Neurons from Prenatal Mice at JoVE.com

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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.

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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