Name: a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains
Uploaded: 2016-12-01T13:30:00
Description: Watch this Scientific Journal Video about A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains Video at JoVE.com

We acknowledge Mr. Enes Mehmet, Ms. Kiara Andrade, Ms. Pilar Rodriguez, Chris Kwok, and Ms. Danna Ghafir for their immense support to the project.

1. Solutions Used for Brain Dissection and Immunofluorescent Staining
<ol>
	<li>Dissect the adult fly brains in artificial Cerebral Spinal Fluid (aCSF): 119 mM NaCl, 26.2 mM NaHCO3, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgCl2, and 10 mM glucose. Before use, gas the aCSF with 5% CO2/95% O2 for 10 - 15 min and spike with 2.5 mM CaCl2. Sterilize the aCSF solution by filtering through a 0.22 &#181;m membrane filter. 
	Note: Store ...

<ol>
	<li>Wangler, M. F., Yamamoto, S., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+flies+in+biomedical+research.">Fruit flies in biomedical research.</a> Genetics. 199, 639-653 (2015).</li><li>Bellen, H. J., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morgan's+legacy:+fruit+flies+and+the+functional+annotation+of+conserved+genes.">Morgan's legacy: fruit flies and the functional annotation of conserved genes.</a> Cell. 163, 12-14 (2015).</li><li>Aso, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuronal+architecture+of+the+mushroom+body+provides+a+logic+for+associative+learning.">The neuronal architecture of the mushroom body provides a logic for associative learning.</a> Elife. 3, 04577 (2014).</li><li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster.">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Res. 11, 1114-1125 (2001).</li><li>Yamagata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dopamine+neurons+mediate+reward+signals+for+short-+and+long-term+memories.">Distinct dopamine neurons mediate reward signals for short- and long-term memories.</a> Proc Natl Acad Sci U S A. 112, 578-583 (2015).</li><li>Nern, A., Pfeiffer, B. D., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+tools+for+multicolor+stochastic+labeling+reveal+diverse+stereotyped+cell+arrangements+in+the+fly+visual+system.">Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system.</a> Proc Natl Acad Sci U S A. 112, 2967-2976 (2015).</li><li>Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Plasticity:+Dopamine+Tunes+the+Mushroom+Body+Output+Network.">Neural Plasticity: Dopamine Tunes the Mushroom Body Output Network.</a> Curr Biol. 26, 109-112 (2016).</li><li>Wolff, T., Iyer, N. A., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroarchitecture+and+neuroanatomy+of+the+Drosophila+central+complex:+A+GAL4-based+dissection+of+protocerebral+bridge+neurons+and+circuits.">Neuroarchitecture and neuroanatomy of the Drosophila central complex: A GAL4-based dissection of protocerebral bridge neurons and circuits.</a> J Comp Neurol. 523, 997-1037 (2015).</li><li>Sweeney, S. T., Hidalgo, A., de Belle, J. S., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+adult+Drosophila+brains.">Dissection of adult Drosophila brains.</a> Cold Spring Harb Protoc. 2011, 1472-1474 (2011).</li><li>Wu, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat Protoc. 1, 2110-2115 (2006).</li><li>Mao, Z., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+different+types+of+dopaminergic+neurons+innervate+the+Drosophila+mushroom+body+neuropil:+anatomical+and+physiological+heterogeneity.">Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity.</a> Front Neural Circuits. 3, 5 (2009).</li><li>White, K. E., Humphrey, D. M., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopaminergic+system+in+the+aging+brain+of+Drosophila.">The dopaminergic system in the aging brain of Drosophila.</a> Front Neurosci. 4, 205 (2010).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathology+and+muscle+and+dopaminergic+neuron+degeneration+caused+by+inactivation+of+Drosophila+Pink1+is+rescued+by+Parkin.">Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin.</a> Proc Natl Acad Sci U S A. 103, 10793-10798 (2006).</li><li>Greene, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathology+and+apoptotic+muscle+degeneration+in+Drosophila+parkin+mutants.">Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants.</a> Proc Natl Acad Sci U S A. 100, 4078-4083 (2003).</li><li>Whitworth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutathione+S-transferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proc Natl Acad Sci U S A. 102, 8024-8029 (2005).</li><li>Pesah, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+parkin+mutants+have+decreased+mass+and+cell+size+and+increased+sensitivity+to+oxygen+radical+stress.">Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress.</a> Development. 131, 2183-2194 (2004).</li><li>Trinh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decaffeinated+coffee+and+nicotine-free+tobacco+provide+neuroprotection+in+Drosophila+models+of+Parkinson's+disease+through+an+NRF2-dependent+mechanism.">Decaffeinated coffee and nicotine-free tobacco provide neuroprotection in Drosophila models of Parkinson's disease through an NRF2-dependent mechanism.</a> J Neurosci. 30, 5525-5532 (2010).</li><li>Kim, K., Kim, S. H., Kim, J., Kim, H., Yim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+s-transferase+omega+1+activity+is+sufficient+to+suppress+neurodegeneration+in+a+Drosophila+model+of+Parkinson+disease.">Glutathione s-transferase omega 1 activity is sufficient to suppress neurodegeneration in a Drosophila model of Parkinson disease.</a> J Biol Chem. 287, 6628-6641 (2012).</li></ol>

With an increasing interest in using adult Drosophila brain to study human brain diseases, neuronal circuitry, and higher brain functions, it is necessary to develop simple and quick methods to obtain intact fly brains for whole-mount analyses, which is especially important for large-scale brain-based screens. Our method provides a simple and easy-to-learn approach to dissect out a fly head (often in less than 10 s&#160;with experience) with well-preserved morphology that is largely cleared of associated tissues...

The model organism Drosophila, commonly known as the fruit fly, has long been valued for its elegant genetic tools, short reproductive times, and highly conserved molecular and cellular pathways. The fruit fly has been successfully employed to dissect basic signaling pathways, the patterning mechanisms of multicellular organisms, as well as the mechanisms underlying neuronal development, functions, and diseases1,2. With recent advances in cell labeling and imaging technologies, the fruit fly brain has become especially powerful in fine mapping of neuronal circuitry and in dissecting the molecular and cellular basis of higher brain functions, such as learning and memory, and circadian rhythm1,3,4,5,6,7,8.
One particular advantage of the Drosophila system is its relatively small size, allowing whole-mount preparation and examination of the brain using a regular compound or confocal microscope. This feature enables detailed anatomic and functional analyses of neuronal circuitry, or even a single neuron, at cellular and subcellular levels, in the context of a whole brain tissue, thus providing both a holistic view of the studied subject and its exact geometry within the whole brain. However, given the rather miniature size of the brain, it also presents a technical challenge in efficiently dissecting an intact brain tissue out of the protective exoskeleton head case in an adult fly. Various effective and relatively simple dissection methods have been described in detail, which usually involve careful and step-wise removal of the head case and the associated tissues including the eyes, trachea, and fat from the brain proper9, 10. These microsurgical dissection methods often place rather stringent demands on the quality of the dissection forceps, relying on forceps with fine well-aligned tips that can be easily damaged. Moreover, as the dissected brains are often separated from the rest of the body, the brains can be easily lost during the subsequent staining and washing processes because of their small sizes and their transparency in the processing buffer. Here, we describe a relatively simple and easy-to-learn, one-step dissection protocol for adult brains that keeps the dissected brains attached to the torso. The dissection process often easily clears away most of the brain-associated tissues such as the eye and trachea and reduces the demand for good quality dissection forceps.
Additionally, when imaging the brain under the fluorescent compound microscope or confocal microscope, the side of the brain that is away from the fluorescent light source often produces a weaker signal and less clear images due to the thickness of the whole-mount brain. Here, we also describe a simple mounting method that allows easy flipping of the brain samples, enabling convenient imaging of both sides of the brain with similar signal intensity and quality.
As a proof-of-concept for the application of this method to study the adult brain, we further examined the presence of DA neurons in the brains of w1118 flies; a genotype that is often used as the parental line for generating transgenic flies and the wildtype control in many Drosophila studies.

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			<td height="54" style="height:54px;width:573px;">w*;&#160;park&#916;21/TM3,&#160;P{GAL4-Kr.C}DC2,&#160;P{UAS-GFP.S65T}DC10,&#160;Sb1</td>
			<td style="width:311px;">Bloomington Drosophila Stock Center</td>
			<td style="width:151px;">51652</td>
			<td style="width:287px;">Balancer was switched to TM6B</td>
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		<tr height="39">
			<td height="39" style="height:39px;">PBac{WH}parkf01950</td>
			<td>Exelixis at Harvard Medical School</td>
			<td>f01950</td>
			<td>Balancer was switched to TM6C</td>
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			<td height="21" style="height:21px;width:573px;">NaCl</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>S640-500</td>
			<td></td>
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			<td height="25" style="height:25px;width:573px;">Sodium Bicarbonate (NaHCO3)&#160;</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td style="width:151px;">02-003-990</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Potassium Chloride (KCl)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:573px;">Sodium phosphate, monobasic monohydrate (NaHCO3)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-004-198</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-003-265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">D-Sorbitol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>S1876-500G</td>
			<td>Replaces glucose</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:573px;">Calcium chloride dihydrate (CaCl2)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>C5670-500G</td>
			<td></td>
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			<td height="21" style="height:21px;">EMD Millipore&#160;Durapore PVDF Membrane Filters: Hydrophilic: 0.22&#181; Pore Size</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>GVWP14250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Formalin Solution, 10% (Histological)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>SF98-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Potassium Phosphate, Dibasic, Powder, Ultrapure Bioreagent</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-003-823</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">Tween 20</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">Excelta Precision Tweezers with Very Fine Points</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>17-456-055</td>
			<td>Protocol does not require very fine points.&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Anti-Tyrosine Hydroxylase Antibody</td>
			<td>Pel-Freez Biologicals</td>
			<td style="width:151px;">P40101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Rat-Elav-7E8A10 anti-elav</td>
			<td>The Developmental Studies Hybridoma Bank</td>
			<td>Clone 7E8A10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">A-11037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">DAPI Solution (1 mg/mL)</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propyl gallate powder</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>P3130-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Glycerol ACS reagent, &#8805;99.5%</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>G7893-500ML</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Zeiss Axioimager Z1</td>
			<td style="width:311px;">Zeiss</td>
			<td style="width:151px;">Quote</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Zeiss Apotome.2</td>
			<td style="width:311px;">Zeiss</td>
			<td style="width:151px;">Quote</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Zen lite software</td>
			<td style="width:311px;"></td>
			<td style="width:151px;">Quote</td>
			<td></td>
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a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

Figure 1 illustrates the main procedures for adult brain dissection, as described above. Figures 2 and 3 are representative images of 3-day-old WT (genotype: w1118) adult fly brains, which were costained with an antibody against Tyrosine Hydroxylase (TH, colored in red in Figure 2 and white in Figure 3), a marker commonly used to label DA neurons11, in addition to the DNA dy...

Watch this Scientific Journal Video about A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains Video at JoVE.com

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains Video (Video) | JoVE

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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