<p class="jove_content">This work was supported by NIH grant NS27501 to DKOD. Additional support for this work was provided by a grant to UC Irvine in support of DKOD through the HHMI Professor Program.</p>

<p class="jove_title">Preparations before day of culturing:</p>
<ol>
<li>Make sterile dissecting solution.</li>
<li>Make sterile DMEM and keep in 10 ml aliquots at 4&deg;C for 2 weeks.</li>
<li>Make sterile DDM2 supplements and freeze in 50 or 100 &micro;l aliquots for 1 month.</li>
<li>Make ConA/laminin.</li>
<li>Coat coverslips.<br />&nbsp;&nbsp;&nbsp;&nbsp; Optional: Make sterile CNBM and store frozen for up to 4 months. </li>
</ol>
<p class="jove_title">On day of culturing</p>
<p class="jove_step">I.&nbsp; Prepare Enzyme Solution (ES) in la...

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Culturing Nerve Cells</em>. , (1991).</li><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> <em style='font-style: italic'>J. Neurobiol</em>. <b>54</b>, 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> <em style='font-style: italic'>J. Neurobiol</em>. <b>27</b>, 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> <em style='font-style: italic'>J. Neurosci</em>. <b>19</b>, 5311-5321 (1999).</li><li>Su, H., 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+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> <em style='font-style: italic'>J. Neurosci</em>. <b>23</b>, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., 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=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> <em style='font-style: italic'>J. Neurophysiol</em>. <b>94</b>, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., 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=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> <em style='font-style: italic'>Develop Neurobiol</em>. <b>67</b>, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., 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=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> <em style='font-style: italic'>Develop Neurobiol</em>. , (2007).</li></ol>

<p class="jove_content">Neurons harvested from the brains of embryonic/postnatal rodents can be grown in primary cell culture where they extend neurites and form functional synaptic connections. Methods for preparation of these cultures are well established and studies in rodent neuronal cultures have played a critical role in identifying genes and environmental factors involved in regulation of synapse formation and function (Banker and Goslin, 1991). While insect neurons from a variety of species can also be grown in culture, the only insect ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Concanavalin A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-2010</td>
<td>To 2.5 ml DS, add 25 mg Concanavalin A bottle. This is 10 mg/ml concentration.
Make aliquots of 90 ul and store at -20 C, no longer than 3 months.</td>
</tr>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-2020</td>
<td>Add 1ml DS to 1mg Laminin bottle. This is 0.5 mg/ml concentration.
Make aliquots of 10  ul and store at -20 C, no longer than 3 months.</td>
</tr>
<tr>
<td>Coverslips</td>
<td>&nbsp;</td>
<td>Bellco Biological Glassware</td>
<td>1943-00012</td>
<td>12 mm glass coverslips</td>
</tr>
<tr>
<td>ConA + Laminin solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Stock solution, from Con A stock and laminin stock, for coverslip coating: Add in 5 ml DS, 83.5 ul of ConA  (167 ug/ml) and 8.35 ul of Laminin (0.835 ug/ml). Mix.
Make aliquots of 100  ul and store at -20 C, not longer than a month.</td>
</tr>
<tr>
<td>Dissecting Solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A +                            14 ml Stock Solution B +  3.0 g (33.3 mM) D (+)-Glucose  (Sigma G-8270) + 7.5 g (43.8  mM) Sucrose  (Sigma S-0389).                  

Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label "Dissecting Solution" and store at 4?C. </td>
</tr>
<tr>
<td>Dissecting Solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A +                            14 ml Stock Solution B +  3.0 g (33.3 mM) D (+)-Glucose  (Sigma G-8270) + 7.5 g (43.8  mM) Sucrose  (Sigma S-0389).                  

Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label "Dissecting Solution" and store at 4?C. </td>
</tr>
<tr>
<td>Solution B: HEPES</td>
<td>Buffer</td>
<td>Sigma</td>
<td>H-3375</td>
<td>20.97g (9.9mM). 

Add ultra filtered water up to 200 ml. Mix until dissolve and bring final volume to 250 ml. Place in a clean bottle and autoclave. Label "Solution B" and store at 4 C. </td>
</tr>
<tr>
<td>Solution A</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 500 ml: 80.0g (137 mM) NaCl (Sigma S-9625) +  4.0g (5.4mM) KCl  (Sigma P-4504) +  0.24g (0.17mM) Na2HPO4  (Sigma S-0876) + 0.3g (0.22 mM) KH2PO4  (Sigma P-5379).

Weigh out all ingredients and mix until dissolved with 400 ml ultra filtered water. Bring final volume to 500 ml. Place in a clean bottle and autoclave. Label "Solution A" and store at 4&#176;C. 

</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			Coating Coverslips:

Put autoclaved coverslips in a 60 mm petri dish.
Pipet 5 ul of Con A/Laminin mix onto center of each coverslip.
Place in 37 C incubator for 2 hours.
Rinse 3x coverslips with 100 ul of autoclaved water each. Use vacuum attached to a                                          
sterile Pasteur pipet.
During the last rinse, pick up the coverslip with forceps and dry both sides.
Transfer the coverslip to a 35mm Petri dish.
Store at room temperature for up to a month.		</div>
		

primary neuronal cultures from the brains of late stage drosophila pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

<p class="jove_content">This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.</p>

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

primary-neuronal-cultures-from-brains-late-stage-drosophila

Research

JoVE Journal

Biology

14.9K visualizações.  University of California, Irvine (UCI). Este vídeo demonstra a preparação de culturas primárias de neurônios do cérebro de final de estágio Drosophila pupas. Pontos de vista de culturas vivas mostram crescimento de neuritos e imagem dos níveis de cálcio usando Fura-2.