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

Summary

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.

Abstract

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Introduction

Dendrites are specialized neuronal compartments that receive and process sensory and synaptic input. The complex and stereotyped structure of dendritic arbors has been under intense investigation since their discovery. A number of model systems, including Xenopus optic tectal neurons, chick retinal ganglion cells, and dendritic arborization (da) neurons in the Drosophila system, have been established to study the development, remodeling and plasticity of neuronal dendrites^1,2,3,4. Drosophila ventral lateral neurons....

Protocol

CAUTION: This protocol involves the use of class IV lasers and will require proper training and safety guidelines to be followed. Avoid eye or skin exposure to direct or scattered laser light.
NOTE: The protocol includes six steps. The workflow is shown in Figure 1A.

1. Labeling Individual Neurons Using the Flip-out Technique

NOTE: The single labeling of LNvs is achieved by expressing mCD8::GFP in single LNvs using flippase -mediated stochastic labeling. The genotype of the fly line is: hs-flp; Pdf-Gal4; UAS-....

Results

Using the live imaging protocol described above, we capture high resolution image stacks for the subsequent analyses and quantification. Supplementary Video 1 shows the maximum intensity projected (MIP) image series collected from a representative individually labeled LNv. Figure 2B shows the corresponding montage of eight frames of the image series. In both panels, arrowheads mark retraction events and arrows mark extension .......

Discussion

Here, we describe a protocol we developed to record and quantify the dynamic behavior of dendritic branches in individually labeled neurons in Drosophila larval brains. Notably, our live imaging protocol contains specific parameters that enable us to capture the whole dendritic arbor of a larval LNv and provide a global view of the dynamic state of its dendrite branches. However, because our quantification methods heavily rely on the annotation of branch terminals, neurons with complex dendritic structures and c.......

Materials

Name	Company	Catalog Number	Comments
Chameleon Vision II multiphoton laser	Coherent
high vacuum grease	Dow Corning	79751-30
LSM 780 two-photon laser scanning confocal microscope	Carl Zeiss		upright configuration
Microscope Cover Glass	Fisher Scientific	12-544-E
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Software
Excel	Microsoft		for processing .csv files
Huygens Professional	Scientific Volume Imaging		for drift correction and deconvolution
Imaris	Oxford Instruments		for 3D visualization and image annotation
Reagents
Glucose
HEPES
KCl
MgCl2
NaCl
NaHCO3
PBS
Sucrose
TES