Name: quantitative analysis of neuronal dendritic arborization complexity in drosophila
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila at JoVE.com

We would like to thank William A. Eimer for imaging technical assistance. This work was supported by the Cure Alzheimer's Fund [to R.E.T], the National Institute of Health [R01AG014713 and R01MH60009 to R.E.T; R03AR063271 and R15EB019704 to A.L.], and the National Science Foundation [NSF1455613 to A.L.].

1. Experimental Preparation
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	<li>Prepare the following reagents: Dulbecco&#39;s phosphate buffered saline (PBS); Triton X-100; 0.2% PBST (PBS + 0.2% Triton X-100); 32% paraformaldehyde (PFA), diluted into 4% before use; silicone elastomer base and curing agent; antifade mounting medium (e.g., ProLong Gold); and fingernail polish.</li>
	<li>Prepare the following equipment: dissection microscope, two sharp forceps and a pair of scissors for microdissection, a number of pins for microdissection, a Petri dish...

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+through+Dishevelled,+Rac+and+JNK+regulates+dendritic+development.">Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development.</a> Nat Neurosci. 8 (1), 34-42 (2005).</li><li>Grueber, W. B., Jan, L. Y., Jan, Y. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+out:+mechanisms+of+dendritic+arborization.">Branching out: mechanisms of dendritic arborization.</a> Nat Rev Neurosci. 11 (5), 316-328 (2010).</li><li>Sears, J. C., Broihier, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxO+regulates+microtubule+dynamics+and+polarity+to+promote+dendrite+branching+in+Drosophila+sensory+neurons.">FoxO regulates microtubule dynamics and polarity to promote dendrite branching in Drosophila sensory neurons.</a> Dev Biol. 418 (1), 40-54 (2016).</li><li>Li, A., 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=Silencing+of+the+Drosophila+ortholog+of+SOX5+leads+to+abnormal+neuronal+development+and+behavioral+impairment.">Silencing of the Drosophila ortholog of SOX5 leads to abnormal neuronal development and behavioral impairment.</a> Hum Mol Genet. 26 (8), 1472-1482 (2017).</li><li>Misra, M., 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=A+Genome-Wide+Screen+for+Dendritically+Localized+RNAs+Identifies+Genes+Required+for+Dendrite+Morphogenesis.">A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis.</a> G3 (Bethesda). 6 (8), 2397-2405 (2016).</li><li>Emoto, 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=Control+of+dendritic+branching+and+tiling+by+the+Tricornered-kinase/Furry+signaling+pathway+in+Drosophila+sensory+neurons.">Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons.</a> Cell. 119 (2), 245-256 (2004).</li><li>Olesnicky, E. 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=Extensive+use+of+RNA-binding+proteins+in+Drosophila+sensory+neuron+dendrite+morphogenesis.">Extensive use of RNA-binding proteins in Drosophila sensory neuron dendrite morphogenesis.</a> G3 (Bethesda). 4 (2), 297-306 (2014).</li><li>Parrish, J. Z., Xu, P., Kim, C. C., Jan, L. Y., Jan, Y. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136 (7), 1049-1061 (2009).</li><li>Jinushi-Nakao, S., 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=Knot/Collier+and+cut+control+different+aspects+of+dendrite+cytoskeleton+and+synergize+to+define+final+arbor+shape.">Knot/Collier and cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape.</a> Neuron. 56 (6), 963-978 (2007).</li><li>Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+multidendritic+neuron+differentiation+in+Drosophila:+the+role+of+Collier.">Control of multidendritic neuron differentiation in Drosophila: the role of Collier.</a> Dev Biol. 315 (1), 232-242 (2008).</li><li>Copf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+gene+dosage+in+controlling+dendritic+arbor+formation+during+development.">Importance of gene dosage in controlling dendritic arbor formation during development.</a> Eur J Neurosci. 42 (6), 2234-2249 (2015).</li><li>Rosso, S. 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Dendrites that innervate the epidermis are the input regions of neurons, and their morphologies determine how information is received and processed by individual neurons. Development dendrite morphology reflects gene modulation of dendrite organization. The Drosophila larval da neuron of the peripheral nervous system is an important model for studying dendrite development because of: 1) the functional similarity with mammals11,12; 2) four class distincti...

Dendrites, which are the branched projections of a neuron, cover the field that encompasses the neuron&#39;s sensory and synaptic inputs from other neurons1,2. Dendrites are an important component of synapse formation and play a critical role in integrating synaptic inputs, as well as propagating the electrochemical stimulation in a neuron. Dendritic arborization (da) is a process by which neurons form new dendritic trees and branches to create new synapses. The development and morphology of da, such as branch density and grouping patterns, result from multi-step biological processes and are highly correlated to neuronal function. The goal of this protocol is to provide a method for quantitative analysis of neuronal dendritric arborization complexity in Drosophila. 
The complexity of dendrites determines the synaptic types, connectivity, and inputs from partner neurons. Branching patterns and the density of dendrites are involved in processing the signals that converge onto the dendritic field3,4. Dendrites have the flexibility for adjustment in development. For instance, synaptic signaling has an effect on dendrite organization in the somatosensory neuron during the developmental phase and in the mature nervous system5. The establishment of neuronal connectivity relies on the morphogenesis and maturation of dendrites. Malformation of dendrites is associated with impaired neuronal function. Studies have shown that the abnormality of da neuron morphogenesis might contribute to the etiologies of multiple neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and Lou Gehrig&#39;s disease/Amyotrophic lateral sclerosis (ALS)6,7,8. Synaptic alterations appear in the early stage of AD, in concert with the decline and impairment of neuron function7,8. However, the specifics of how dendrite pathology contributes to pathogenesis in these neurodegenerative diseases remains elusive.
The development of dendrites is regulated by genes that encode a complex network of regulators, such as the Wnt family of proteins9,10, transcription factors, and ligands on cell surface receptors11,12. Drosophila da neurons consist of four classes (Class I, II III, IV), of which class IV da neurons have the most complex branching patterns and have been employed as a powerful experimental system for better understanding morphogenesis13,14. During early morphogenesis, overexpression and/or RNAi silencing of genes in class IV da neurons result in changes in branching patterns and dendrite pruning13. It is important to develop a practical method for quantitative analysis of the neuronal dendritic arborization.
We have previously shown that silencing of the Drosophila ortholog of SOX5, Sox102F, led to shorter dendrites of da neurons and reduced complexity in class IV da neurons15. Here, we present the procedure of quantitative analysis for the neuronal dendritic arborization complexity (NDAC) in Drosophila. This protocol, adapted from the previous described methodology, provides a brief method to assay the development of da sensory neurons. It illustrates the robust image labeling and the da neuron in the third instar larval body wall16,17,18,19. It is a valuable protocol for researchers who wish to investigate the NDAC and developmental differences in vivo.

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			<td height="21" style="height:21px;width:363px;">Phosphate buffered saline(PBS)</td>
			<td style="width:223px;">Gibco Life Sciences</td>
			<td style="width:197px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">TritonX-100</td>
			<td style="width:223px;">Fisher Scientific</td>
			<td style="width:197px;">9002-93-1</td>
			<td></td>
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			<td height="21" style="height:22px;width:363px;">Paraformaldehyde(PFA)</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:197px;">15714-S</td>
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			<td height="28" style="height:29px;width:363px;">Sylgard 184 silicone elastomer base and curing agent</td>
			<td style="width:223px;">Dow Corning Corportation</td>
			<td style="width:197px;">3097366-0516;3097358-1004</td>
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			<td height="24" style="height:25px;width:363px;">ProLong Gold Antifade Mountant</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:197px;">P36931</td>
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			<td height="21" style="height:21px;width:363px;">Fingernail polish&#160;</td>
			<td style="width:223px;">CVS</td>
			<td style="width:197px;">72180</td>
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			<td height="21" style="height:21px;width:363px;">Stereo microscope</td>
			<td style="width:223px;">Nikon</td>
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			<td height="21" style="height:21px;width:363px;">Confocal microscope</td>
			<td style="width:223px;">Nikon</td>
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			<td height="21" style="height:21px;width:363px;">Petri dish</td>
			<td style="width:223px;">Falcon</td>
			<td style="width:197px;">353001</td>
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			<td height="21" style="height:21px;width:363px;">Forceps</td>
			<td style="width:223px;">Dumont</td>
			<td style="width:197px;">11255-20</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Scissors&#160;</td>
			<td style="width:223px;">Roboz Surgical Instrument Co</td>
			<td style="width:197px;">RS-5611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Insect Pins&#160;</td>
			<td style="width:223px;">Roboz Surgical Instrument Co</td>
			<td style="width:197px;">RS-6082-25</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:363px;">Microscope slides and cover slips</td>
			<td style="width:223px;">Fisher Scientific</td>
			<td style="width:197px;">15-188-52</td>
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	</tbody>
</table>

quantitative analysis of neuronal dendritic arborization complexity in drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

The dendrites of da neurons were labeled by co-overexpressing GFP (UAS-GFP; ppk-GAL4) in the da neural soma and dendritic arbors for GFP fluorescence imaging analysis. The morphology of da neuron dendrites was imaged by an inverted confocal microscope (Figure 2).
The dendrites of da neurons were traced using Fiji ImageJ software. The file was used to estimate the dendrite length (<strong class="xfig...

Watch this Scientific Journal Video about Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila at JoVE.com

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

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