Name: preparation of neuronal cultures from midgastrula stage drosophila embryos
Uploaded: 2007-07-04T00:00:00
Description: Watch this Scientific Journal Video about Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos at JoVE.com

This work was supported by NIH grant NS27501 to DKOD. Additional support for this work from an HHMI Professor Grant to DKOD.

I. Drosophila Embryo Collection
<ol>
<li>Prepare egg collection plates <ol>
<li>Boil 200 ml dH2O with 8 g agar</li>
<li>Cool to 50&deg;C</li>
<li>Add 2 ml EtOH and 2 ml Acetic acid</li>
<li>Pour into 35 mm plastic petri dishes, tops </li>
<li>Cool and store in air tight container at 4&deg;C</li>
<li>Makes approximately 80 plates </li>
</ol></li>
<li>Prepare Yeast paste <ol>
<li>Dissolve Fleishmans active baking yeast in water to form paste</li>
<li>Microwave for 20-30 seconds to kill yeast (watch out for boil...

<ol>
	<li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic-AMP-dependent+plasticity+at+excitatory+cholinergic+synapses+in+Drosophila+neurons:+Alterations+in+the+memory+mutant+dunce.">Cyclic-AMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: Alterations in the memory mutant dunce.</a> J. Neurosci. 20, 2104-2111 (2000).</li><li>Hodges, D. D., Lee, D., Boswell, K., Preston, C. F., Hall, L. M., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tipE+regulates+sodium-dependent+repetitive+firing+in+Drosophila+neurons.">tipE regulates sodium-dependent repetitive firing in Drosophila neurons.</a> Mol. Cell Neurosci. 19, 402-416 (2002).</li><li>Lee, D., Su, H., Dowd, O., K, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+receptors+containing+Rdl+subunits+mediate+fast+inhibitory+synaptic+transmission+in+Drosophila+neurons.">GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons.</a> J. Neurosci. 23, 4625-4634 (2003).</li></ol>

In Drosophila cultures prepared from midgastrula stage embryos the neurons arise from neuroblast precursors, many of which divide prior to differentiation in vitro. This system thus provides a unique opportunity for exploration of genetic and environmental factors important in very early phases of neuronal development (Rohrbaugh et al., 2003). We have shown that neurons grown in a simple defined medium differentiate into electrically excitable neurons that also form functional synaptic connections (O Dowd, 1995; Lee and ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Drosophila melanogaster</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Fruit flies</td>
</tr>
<tr>
<td>Transferrin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T-1147</td>
<td>100x Stock: 10 mg/ml in water. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C.
</td>
</tr>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>200x Stock: 10 mg/ml in 0.05N HCl. Filter through 0.2 um syringe filter (cellulose acetate). Store in 120 ul aliquots (for 20 ml DMEM) at -20C.
</td>
</tr>
<tr>
<td>Putrescine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5780</td>
<td>100x Stock: 10 mM in ddH2O. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20 C
</td>
</tr>
<tr>
<td>Selenium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-5261</td>
<td>100x Stock: 3 uM in ddH2O. Put 0.0051 g Selenium in a 15 ml tube labeled A (3 mM stock). Add 10 ml of sterile water. Take 10 ul from Tube A to Tube B with 10 ml ddH2O (3 uM stock). Filter Tube B through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C
</td>
</tr>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-6149</td>
<td>100x Stock: 2 ug/ml
1.	Add 1ml of 100% EtOH to 0.001 g Progesterone bottle
2.	Add 49 ml ddH2O
3.	Transfer 1 ml of this to second tube with 9 ml ddH2O
4.	Filter through 0.2 um syringe filter (cellulose acetate)
5.	Store in 220 ul aliquots (for 20 ml DMEM) at -20C
</td>
</tr>
<tr>
<td>DDM1</td>
<td>Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To 10 mls of DMEM add from stocks:
	100 ul Transferrin, 
	100 ul Putrescine,
	100 ul Selenium,
	100 ul Progesterone,
	 50 ul Insulin
</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cover-slips</td>
<td>&nbsp;</td>
<td>Bellco Biological Glassware</td>
<td>1943-00012</td>
<td>Low lead glass, autoclaved.</td>
</tr>
<tr>
<td>Paper filter</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>#1</td>
<td>&nbsp;</td>
<tr>
<td>10cc syringe</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			The cultures made in this media must be maintained in a 5% CO2 incubator at about 22-24&deg;C. We use a standard mammalian tissue culture incubator placed in a cold room and heated to 23&deg;C. Always used autoclaved filtered water and make in containers that are reserved for media and supplements only. Never put a pH meter or stir bar used for other purposes in media.		</div>

preparation of neuronal cultures from midgastrula stage drosophila embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

Watch this Scientific Journal Video about Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos at JoVE.com

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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