Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Summary

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

Abstract

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.

Introduction

Dendrites, which are the branched projections of a neuron, cover the field that encompasses the neuron's sensory and synaptic inputs from other neurons^1,2. 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 field^3,4. 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's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and Lou Gehrig'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 function^7,8. 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 proteins^9,10, transcription factors, and ligands on cell surface receptors^11,12. 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 morphogenesis^13,14. 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 wall^16,17,18,19. It is a valuable protocol for researchers who wish to investigate the NDAC and developmental differences in vivo.

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Protocol

1. Experimental Preparation

1. Prepare the following reagents: Dulbecco'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.
2. 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.

2. Larvae Collection

1. Mate UAS-Sox102F-RNAi flies with UAS-GFP, ppk-GAL4, or W¹¹¹⁸ wild type flies, respectively. Culture flies in standard conditions at 25 °C.
2. 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.

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.

1. Place a larva on a dissection dish made of silicone elastomer base and a tissue culture Petri dish.
2. 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.
3. Add 200 µL of PBS to the larva to maintain body moisture.
4. 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.
5. Place a pin at each of the four corners of the body so the body lies flat.
6. Fix the larva body wall in 4% PFA for 25 min at room temperature.
7. Wash in PBST for 5 min. Repeat twice more.
8. 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.

4. Imaging Processing

NOTE: Images were taken using an inverted confocal microscope system. The user can photograph the sample using a 20X objective (recommended).

1. Capture Z-series images. Open the "Capture Z-series" dialog box in the confocal microscope software. Determine the range for capturing the Z series images.
   1. Click the "Top and Bottom" button. Determine the top and bottom position and input the values to "Bottom:" and "Top:" boxes. Set the "Step" in "Capture Z-series" dialog box as 0.5 µm. Then click the "Run now" button to acquire the Z-series images.
2. Save the obtained images as *.tiff or *.nd2 files.
3. Store the slides in a dark holder at 4 °C after imaging.

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 (µm), surface area (µm²), total number of branches, and branching structure (%). Figure 1 shows the analysis parameters in detail.

1. Length of Dendrites
   NOTE: The length of dendrites is the sum of all dendrites labeled in tracer plugin.
   1. Setup and run Fiji ImageJ (https://fiji.sc/)²⁰ software. Then split images into separate channels, if there are several channels of images.
      ​NOTE: The image in this protocol only has one channel of GFP.
   2. Run "Image | Stacks | Z project" to get a Z projection; a new window will appear with the name 'Z-projection.' Click "max intensity" for projection type, and organize numbers between start slice and stop slice depending upon individual preferences. Click "OK." A Z-projection image will appear presenting the dendrite projection clearly.
   3. Trace the neurites.
      1. Select "Plugins | Segmentation | Simple Neurites Tracer" 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.
      2. Press "Y" 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 "complete path" 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.
   4. After a path is completed, go back to a new point where the branches are. The new path can be built on; the "All paths" 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.
   5. Export the dendrite length data by clicking, "File | Export as *.cvs" in the 'Plugin' window. Then sum up all the path lengths, and export the data to a data analysis/spreadsheet software. (Figure 3).
2. Surface Area of the da Neurons
   1. Select the Freehand drawing tool in the 'Fiji ImageJ' window. Trace the path, and then connect the endpoints. From the 'Analyze' menu, select "Set Measurements | Area." Click "measure" from the 'Analyze' menu. The result appears in a new box with the value for the area selected (Figure 4). Copy it to the data analysis software.
3. Total Number of Branches and Branching Structure of the da Neurons
   1. Calculate the total number of branches by opening "Analysis" and then "Render | Analyze Skeletonized Paths" 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.

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Results

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 (

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Discussion

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 mammals^11,12; 2) four class distincti...

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Materials

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