An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

Summary

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

Abstract

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

Introduction

Neurons are precisely interconnected to form circuits essential for the proper function of the brain. For over 100 years, neuroscientists have been trying to understand how neurites extend toward their intermediate and final targets with extreme precision. As a result, they have identified important genes that encode guidance cues for developing neuronal processes¹. The Drosophila olfactory system provides an excellent model to investigate this process since olfactory receptor neurons (ORNs, the primary sensory neurons) project to 50 identifiable glomeruli with stereotypical size, shape, and relative position, where they form synaptic ....

Protocol

1. Preparation of reagents

NOTE: All the steps in this protocol are carried out at room temperature (20-25 °C) unless explained otherwise.

1. To prepare the culture dish for immobilizing the explant during time lapse imaging, lay 0.5 cm thick Sylgard (thoroughly mix two liquid components at 10:1 ratio before use) on the bottom surface of a 60 mm x 15 mm Petri dish and let it cure for 48 h at room temperature (Figure 2A, referred as Sylgard plate in the following text).
2. To prepare micro pins for immobilizing explant on this plate, use a pair of forceps to stick multiple m....

Results

ORN axons arrive at the antennal lobe between 18 h and 36 h APF. They then navigate the antennal lobe, cross the midline, and innervate the glomeruli. Video 1 is a representative video showing the entire process for several individually identifiable axons, taken at the frequency of every 20 min for 24 h. Before registration using TurboReg, the axons exhibit some lateral drifting as the brain develops (first half of the video). After registration, the drifting is corrected (second half of the video).

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Discussion

The Drosophila antennae-brain explant retains normal targeting of the olfactory circuit. We did notice that the development is 2 times slower ex vivo compared to in vivo. It is noted that the explant system does not retain maxillary palp, which hosts six types of ORNs. To ensure normal development is recapitulated ex vivo, stretching of the antennal nerves needs to be avoided during explant dissection. During ex vivo culture bacteria growth usually causes arrested development .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20-hydroxyecdysone	Sigma	H5142
Chameleon Ti:Sapphire laser	Coherent	Coherent MRU X1
Fetal Bovine Serum	Thermo Fisher Scientific	10082147
Human insulin	Thermo Fisher Scientific	12585014
Imaging software	Prairie
Micro Scissors	World Precision Instruments	501778
Minutien Pins	Fine Science Tools	26002-10
Oxygen cylinder	Praxair	OX M-E
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122
Schneider’s Drosophila Medium	Thermo Fisher Scientific	21720024
SYLGARD 184 Silicone Elastomer	Thermo Fisher Scientific	NC0162601
Two-photon microscopy	Bruker
water immerse objective (20X)	Zeiss	421452-9800-000