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 ....

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.

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 (

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.......

Materials

List of materials used in this article

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