Name: Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 7 min 13 s
Description: Learn quantitative analysis of neuronal dendritic arborization complexity in Drosophila using confocal microscopy and image tracing. This method reveals how SOX5 gene silencing reduces dendrite branching.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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
<ol>
	<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 for making the dissection dish, microscope slides and coverslips, a confocal microscope, and a computer with Fiji ImageJ software installed.</li>
</ol>
2. Larvae Collection
<ol>
	<li>Mate UAS-Sox102F-RNAi flies with UAS-GFP, ppk-GAL4, or W1118 wild type flies, respectively. Culture flies in standard conditions at 25 &#176;C.</li>
	<li>In ~5 - 6 days, collect the third instar larvae of UAS-Sox102F-RNAi/ ppk-GAL4, UAS-GFP or UAS-GFP, and ppk-GAL4 /+ control carefully with forceps for dissection.</li>
</ol>
3. Dissection of Larvae
NOTE: All procedures in this section are operated under a microdissection microscope. The magnification is up to the investigators. Try to adjust to the optimal sight view. ~4 - 6X magnification is recommended.
<ol>
	<li>Place a larva on a dissection dish made of silicone elastomer base and a tissue culture Petri dish.</li>
	<li>Pin the larva mouth hook to allow the dorsal side to face up, and then pin the tail of larva. Place the dorsal side in the middle as much as possible to expose the midline, which will be the open-up marker.</li>
	<li>Add 200 &#181;L of PBS to the larva to maintain body moisture.</li>
	<li>Make a cut with a pair of scissors along the dorsal midline between the two tracheas, from caudal to rostral. Make a small cut at each of the four corners of the larva body.</li>
	<li>Place a pin at each of the four corners of the body so the body lies flat.</li>
	<li>Fix the larva body wall in 4% PFA for 25 min at room temperature.</li>
	<li>Wash in PBST for 5 min. Repeat twice more.</li>
	<li>Remove the pins from the tissue. Then transfer the tissue onto a glass slide, cover with antifade mounting medium, and mount with a cover slip. Air dry for 1 h and seal the edges with fingernail polish.</li>
</ol>
4. Imaging Processing
NOTE: Images were taken using an inverted confocal microscope system. The user can photograph the sample using a 20X objective (recommended).
<ol>
	<li>Capture Z-series images. Open the &#34;Capture Z-series&#34; dialog box in the confocal microscope software. Determine the range for capturing the Z series images.
	<ol>
		<li>Click the &#34;Top and Bottom&#34; button. Determine the top and bottom position and input the values to &#34;Bottom:&#34; and &#34;Top:&#34; boxes. Set the &#34;Step&#34; in &#34;Capture Z-series&#34; dialog box as 0.5 &#181;m. Then click the &#34;Run now&#34; button to acquire the Z-series images.</li>
	</ol>
	</li>
	<li>Save the obtained images as *.tiff or *.nd2 files.</li>
	<li>Store the slides in a dark holder at 4 &#176;C after imaging.</li>
</ol>
5. Dendrite Evaluation
Note: GFP protein was co-overexpressed in UAS-GFP and ppk-GAL4 flies in the da neurons for GFP fluorescence imaging analysis. The length, branching, and structure of da neurons in the third instar larvae were quantified. Analysis parameters include length of dendrites (&#181;m), surface area (&#181;m2), total number of branches, and branching structure (%). Figure 1 shows the analysis parameters in detail.
<ol>
	<li>Length of Dendrites 
	NOTE: The length of dendrites is the sum of all dendrites labeled in tracer plugin.
	<ol>
		<li>Setup and run Fiji ImageJ (https://fiji.sc/)20&#160;software. Then split images into separate channels, if there are several channels of images. 
		&#8203;NOTE: The image in this protocol only has one channel of GFP.</li>
		<li>Run &#34;Image | Stacks | Z project&#34; to get a Z projection; a new window will appear with the name &#39;Z-projection.&#39;&#160;Click &#34;max intensity&#34; for projection type, and organize numbers between start slice and stop slice depending upon individual preferences. Click &#34;OK.&#34;&#160;A Z-projection image will appear presenting the dendrite projection clearly.</li>
		<li>Trace the neurites.
		<ol>
			<li>Select &#34;Plugins | Segmentation | Simple Neurites Tracer&#34; in the window to trace the dendrites. Find the neuron soma, and then click one point at a dendrite starting from the cell body, and another point at the tip of this dendrite. 
			NOTE: The program will connect the two points with a blue line.</li>
			<li>Press &#34;Y&#34; in the plug-in window if the path is clear; the dendrite path is traced until the endpoints of the selected path in the visible images. Click &#34;complete path&#34; on the same plugin window; the segment is completed (Figure 2). 
			NOTE: Some dendrites ended farther from the starting points, in which the pathway was divided into smaller segments. The segments were combined to provide a complete path for the tracer.</li>
		</ol>
		</li>
		<li>After a path is completed, go back to a new point where the branches are. The new path can be built on; the &#34;All paths&#34; window box will show the length values of all the paths (Figure 3). Save the tracing file, and continue with unfinished traces as shown in Figure 2.</li>
		<li>Export the dendrite length data by clicking, &#34;File | Export as *.cvs&#34; in the &#39;Plugin&#39; window. Then sum up all the path lengths, and export the data to a data analysis/spreadsheet software. (Figure 3).</li>
	</ol>
	</li>
	<li>Surface Area of the da Neurons
	<ol>
		<li>Select the Freehand drawing tool in the &#39;Fiji ImageJ&#39; window. Trace the path, and then connect the endpoints. From the &#39;Analyze&#39; menu, select &#34;Set Measurements | Area.&#34;&#160;Click &#34;measure&#34; from the &#39;Analyze&#39; menu. The result appears in a new box with the value for the area selected (Figure 4). Copy it to the data analysis software.</li>
	</ol>
	</li>
	<li>Total Number of Branches and Branching Structure of the da Neurons
	<ol>
		<li>Calculate the total number of branches by opening &#34;Analysis&#34; and then &#34;Render | Analyze Skeletonized Paths&#34; in the plugin window of tracing (Figure 5). 
		NOTE: The structure of the arbor is the sum of the levels of branches. The path was defined between the cell body and the dendrite tips, and one path can include multiple branches, such as the primary arbors are the dendrites from the neuron cell body. The secondary arbors are branches from the primary and so on with the tertiary, quaternary, and quinary arbors. The structure of dendrite branching is separated depending on the levels of branches. For instance, the primary % is the number of branches divided by total number of branching, and so on.</li>
	</ol>
	</li>
</ol>

<ol><li>Wassle, H., Boycott, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+architecture+of+the+mammalian+retina%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Functional architecture of the mammalian retina.</a> Physiol Rev. 71 (2), 447-480 (1991).</li>
<li>MacNeil, M. A., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extreme+diversity+among+amacrine+cells%3A+implications+for+function%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Extreme diversity among amacrine cells: implications for function.</a> Neuron. 20 (5), 971-982 (1998).</li>
<li>Losonczy, A., Makara, J. K., Magee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compartmentalized+dendritic+plasticity+and+input+feature+storage+in+neurons%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Compartmentalized dendritic plasticity and input feature storage in neurons.</a> Nature. 452 (7186), 436-441 (2008).</li>
<li>Spruston, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyramidal+neurons%3A+dendritic+structure+and+synaptic+integration%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Pyramidal neurons: dendritic structure and synaptic integration.</a> Nat Rev Neurosci. 9 (3), 206-221 (2008).</li>
<li>Jaworski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+microtubules+regulate+dendritic+spine+morphology+and+synaptic+plasticity%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity.</a> Neuron. 61 (1), 85-100 (2009).</li>
<li>Kweon, J. H., Kim, S., Lee, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cellular+basis+of+dendrite+pathology+in+neurodegenerative+diseases%5BTitle%5D)+AND+%22BMB+Rep%22%5BJournal%5D)">The cellular basis of dendrite pathology in neurodegenerative diseases.</a> BMB Rep. 50 (1), 5-11 (2016).</li>
<li>Baloyannis, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+pathology+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">Dendritic pathology in Alzheimer's disease.</a> J Neurol Sci. 283 (1-2), 153-157 (2009).</li>
<li>Masliah, E., Terry, R. D., Alford, M., DeTeresa, R., Hansen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+and+subcortical+patterns+of+synaptophysinlike+immunoreactivity+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Cortical and subcortical patterns of synaptophysinlike immunoreactivity in Alzheimer's disease.</a> Am J Pathol. 138 (1), 235-246 (1991).</li>
<li>Wayman, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity-dependent+dendritic+arborization+mediated+by+CaM-kinase+I+activation+and+enhanced+CREB-dependent+transcription+of+Wnt-2%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2.</a> Neuron. 50 (6), 897-909 (2006).</li>
<li>Rosso, S. B., Sussman, D., Wynshaw-Boris, A., Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt+signaling+through+Dishevelled%2C+Rac+and+JNK+regulates+dendritic+development%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+levels+of+the+homeodomain+protein+cut+regulate+distinct+dendrite+branching+patterns+of+Drosophila+multidendritic+neurons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Different levels of the homeodomain protein cut regulate distinct dendrite branching patterns of Drosophila multidendritic neurons.</a> Cell. 112 (6), 805-818 (2003).</li>
<li>Sugimura, K., Satoh, D., Estes, P., Crews, S., Uemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+morphological+diversity+of+dendrites+in+Drosophila+by+the+BTB-zinc+finger+protein+abrupt%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Development of morphological diversity of dendrites in Drosophila by the BTB-zinc finger protein abrupt.</a> Neuron. 43 (6), 809-822 (2004).</li>
<li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Branching+out%3A+mechanisms+of+dendritic+arborization%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">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/pubmed?term=((FoxO+regulates+microtubule+dynamics+and+polarity+to+promote+dendrite+branching+in+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Silencing+of+the+Drosophila+ortholog+of+SOX5+leads+to+abnormal+neuronal+development+and+behavioral+impairment%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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/pubmed?term=((A+Genome-Wide+Screen+for+Dendritically+Localized+RNAs+Identifies+Genes+Required+for+Dendrite+Morphogenesis%5BTitle%5D)+AND+%22G3+%28Bethesda%29%22%5BJournal%5D)">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/pubmed?term=((Control+of+dendritic+branching+and+tiling+by+the+Tricornered-kinase%2FFurry+signaling+pathway+in+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Extensive+use+of+RNA-binding+proteins+in+Drosophila+sensory+neuron+dendrite+morphogenesis%5BTitle%5D)+AND+%22G3+%28Bethesda%29%22%5BJournal%5D)">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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+microRNA+bantam+functions+in+epithelial+cells+to+regulate+scaling+growth+of+dendrite+arbors+in+drosophila+sensory+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in drosophila sensory neurons.</a> Neuron. 63 (6), 788-802 (2009).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
<li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecules+and+mechanisms+of+dendrite+development+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Knot%2FCollier+and+cut+control+different+aspects+of+dendrite+cytoskeleton+and+synergize+to+define+final+arbor+shape%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Control+of+multidendritic+neuron+differentiation+in+Drosophila%3A+the+role+of+Collier%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Importance+of+gene+dosage+in+controlling+dendritic+arbor+formation+during+development%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Importance of gene dosage in controlling dendritic arbor formation during development.</a> Eur J Neurosci. 42 (6), 2234-2249 (2015).</li>
<li>Rosso, S. B., Inestrosa, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((WNT+signaling+in+neuronal+maturation+and+synaptogenesis%5BTitle%5D)+AND+%22Front+Cell+Neurosci%22%5BJournal%5D)">WNT signaling in neuronal maturation and synaptogenesis.</a> Front Cell Neurosci. 7, 103(2013).</li>
<li>Engel, T., Hernandez, F., Avila, J., Lucas, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Full+reversal+of+Alzheimer%27s+disease-like+phenotype+in+a+mouse+model+with+conditional+overexpression+of+glycogen+synthase+kinase-3%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Full reversal of Alzheimer's disease-like phenotype in a mouse model with conditional overexpression of glycogen synthase kinase-3.</a> J Neurosci. 26 (19), 5083-5090 (2006).</li>
<li>Longair, M. H., Baker, D. A., Armstrong, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+Neurite+Tracer%3A+open+source+software+for+reconstruction%2C+visualization+and+analysis+of+neuronal+processes%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes.</a> Bioinformatics. 27 (17), 2453-2454 (2011).</li>
<li>Pool, M., Thiemann, J., Bar-Or, A., Fournier, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NeuriteTracer%3A+a+novel+ImageJ+plugin+for+automated+quantification+of+neurite+outgrowth%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">NeuriteTracer: a novel ImageJ plugin for automated quantification of neurite outgrowth.</a> J Neurosci Methods. 168 (1), 134-139 (2008).</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Phosphate buffered saline(PBS)</td><td>Gibco Life Sciences</td><td>10010-023</td><td></td></tr><tr><td>TritonX-100</td><td>Fisher Scientific</td><td>9002-93-1</td><td></td></tr><tr><td>Paraformaldehyde(PFA)</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer base and curing agent</td><td>Dow Corning Corportation</td><td>3097366-0516;3097358-1004</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>P36931</td><td></td></tr><tr><td>Fingernail polish&amp;nbsp;</td><td>CVS</td><td>72180</td><td></td></tr><tr><td>Stereo microscope</td><td>Nikon</td><td>SMZ800</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Eclipse Ti-E</td><td></td></tr><tr><td>Petri dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Forceps</td><td>Dumont</td><td>11255-20</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Roboz Surgical Instrument Co</td><td>RS-5611</td><td></td></tr><tr><td>Insect Pins&amp;nbsp;</td><td>Roboz Surgical Instrument Co</td><td>RS-6082-25</td><td></td></tr><tr><td>Microscope slides and cover slips</td><td>Fisher Scientific</td><td>15-188-52</td><td></td></tr></tbody></table>

quantitative analysis of neuronal dendritic arborization complexity in drosophila

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...

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

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

quantitative-analysis-neuronal-dendritic-arborization-complexity

Research

JoVE Journal

Neuroscience

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Source: Ting, C., et al., Analyzing Dendritic Morphology in Columns and Layers. J. Vis. Exp. (2017)This video demonstrates the procedure of preparing a Drosophila brain, capturing horizontal and frontal image stacks of GFP-labeled medulla dendrites under a confocal microscope, and creating a 3D reconstruction to analyze their morphology across layers and columns.

Analyzing Dendritic Morphology Across Drosophila Medulla Layers and Columns

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analyzing Dendritic Morphology in Columns and Layers

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Learn quantitative analysis of neuronal dendritic arborization complexity in Drosophila using confocal microscopy and image tracing. This method reveals how SOX5 gene silencing reduces dendrite branching.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Explore More Videos