Name: Dissection of Larval CNS in Drosophila Melanogaster (Video) | JoVE Journal
Uploaded: 2006-11-09T00:00:00.000+00:00
Duration: 6 min 29 s
Description: Learn CNS dissection in Drosophila melanogaster third instar larvae for neuroscience research. This method preserves internal structures for protein expression analysis and developmental neurobiology studies.

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<ol>
<li>Dissect 3rd instar larvae, leaving CNS attached to the cuticle. Place in a 1.5 ml tube.</li>
<li>Fix in PBS (1x) containing 2% formaldehyde for 40 min with rocking. ; Use 250ul for every 5-10 dissected CNS.</li>
<li>Remove formaldehyde and briefly wash 3 times with PBS + 0.1% Triton X-100 (PBST). Use a finely pulled Pasteur pipette to avoid loss of tissue.</li>
<li>Block non-specific protein binding sites and permeabilize cell membranes by incubating tissue 1-8 hrs in PBST + 5% BSA, rocking at 4&deg;C.</li>
<li>Incubate in primary antibody diluted in PBST + 5% BSA rocking at 4&deg;C overnight. Optimal antibody dilution will vary for different antibodies.</li>
<li>Rinse 3x in PBST followed by two 30min washes at 4&deg;C.</li>
<li>Incubate in secondary antibody diluted in PBST + 5% BSA for 1-3 hrs rocking at 4&deg;C. Rinse 3x in PBST followed by two 30min washes at 4&deg;C. If the secondary is conjugated to a light sensitive compound, wrap the tube in aluminum foil.</li>
<li>Dissect tissue further (if needed) and mount on slides.</li>
</ol>

<ol>
	<li>Lin, D. M., Goodman, C. S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+and+increased+expression+of+fasciclin+II+alters+motoneuron+growth+cone+guidance.">Ectopic and increased expression of fasciclin II alters motoneuron growth cone guidance.</a> Neuron. 13, 507-523 (1994).</li><li>Park, Y., Fujioka, M., Kobayashi, M., Jaynes, J., Datta, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=even+skipped+is+required+to+produce+a+trans-acting+signal+for+larval+neuroblast+proliferation+that+can+be+mimicked+by+ecdysone.+Development+128.">even skipped is required to produce a trans-acting signal for larval neuroblast proliferation that can be mimicked by ecdysone. Development 128.</a> , 1899-1909 (2001).</li></ol>

The authors have nothing to disclose.

This video demonstrates how to dissect the CNS from third instar Drosophila larvae. After dissection, the CNS can be stained using a standard immunohistochemistry protocol. Currently, much is still unknown about how the adult nervous system is generated as well as how it stores and transmits information. Studies of the developing Drosophila larval CNS should enhance our knowledge of nervous system wiring and function.

Drosophila larvae exhibit a number of stereotpyed behaviors, such as a...

<table><tbody><tr><td>Forceps</td><td>fine tipped</td><td></td><td></td><td></td></tr><tr><td>9-well Watch glass</td><td></td><td></td><td></td><td></td></tr><tr><td>Plastic Petri dish</td><td></td><td></td><td></td><td></td></tr></tbody></table>

dissection of larval cns in drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

My name's Nate Fer. I'm a grad student here at Princeton.Okay. The thing you wanna do is take find the worm and then transfer em to a dish with some PBS.So that's what they'll look like and you tear em in half and all the guts fly out. This is, so I have the cuticle here with a bunch, all the internal structures and trying to make sure it's turned inside out and make sure that the CNS is still attached, So.Got it.Yep. So it's right there.Right there. Which one? You can see on my right forceps.There You've Got two different lobes and then there's the, the CNS itself that kind of extends down like a tap root and it's right here off to the right side of the, the dark mouth hooks and the rest of the cuticle there. Would you like to separate the CNS like this? Yeah, sure.I can pull it off. And that's looking from the top down. So you see the two lobes and then you see the, the CNS itself.So why do you do this experiment? Well, we're interested in looking at the expression of protein in the nervous system, in the larvae. And so the best way to do it is just to take the larval tissue apart.And since it's difficult for antibodies to penetrate through the cuticle in a whole tissue, we dissect out the larva CNS first and then use the dissect dissected tissue to, to do the analysis we want to do as far as the protein stain. And why do you do it? In re why specifically?In re Well, because it really just depends on what your interests are as far as the analysis goes. The protein that I'm studying is expressed throughout development, and we want to see how the protein localization changes and see what types of cells are, are expressing our protein. And so we want to get a time course throughout development.What are the specific problem with this experiment? Well, one of the problems, like I mentioned first, is that you have to dissect the tissue. And so there's a lot of, sort of, at least initially, the technical expertise of getting, being able to hold the larvae steady, pull it apart carefully and, and do the inverted technique.To get the, the CNS exposed, you'll have to have a fine tips forceps. cause if you're, your forceps aren't fine tipped enough, you're really almost trying to work with mittens on rather than your fingers. And so it's a little tricky to get to learn the technique at first, but after that, it's not so bad.And, and the staining really is not all that difficult. It's just a standard antibody stain. But you have to be careful while you're pipetting your solutions up and down in your tube, but you don't lose the tissue because it can be pretty small.

dissection-of-larval-cns-in-drosophila-melanogaster

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JoVE Journal

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Learn CNS dissection in Drosophila melanogaster third instar larvae for neuroscience research. This method preserves internal structures for protein expression analysis and developmental neurobiology studies.

Dissection of Larval CNS in Drosophila Melanogaster

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