Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Summary

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Abstract

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Introduction

Olfactory information in a Drosophila larva's environment is sensed by only 21 functionally distinct ORNs, the activities of which ultimately determine larval behavior^1,2,3,4. Yet, relatively little is known about the logic by which sensory information is encoded in the activities of these 21 ORNs. There is thus a need to experimentally measure the functional contributions of each larval ORN to behavior.

Although the sensory response profile of the entire repertoire of Drosophila larval ORNs ....

Protocol

1. Building a Behavior Arena and Preparing Hardware to Enable Optogenetic Stimulation in the Behavior Arena

1. To build a light-deprived behavior arena, construct a box with a dimension of 89 x 61 x 66 cm³ (35" L x 24" W x 26" H) made of black colored plexiglass acrylic sheets (3 mm thick) (see Table of Materials). Materials to build such a box should be available at local hardware stores. Place this box on a table-top in the behavior room (Figure 1A).
2. Mount a monochrome USB 3.0 CCD camera fitted with an IR long-pass 830 nm filter and an 8 mm F1.4 C-mount lens (se....

Results

To demonstrate the specificity of ORN activation, our method was successfully applied to determine the impact of two different ORN (ORN::7a & ORN::42a) (ORNs expressing either Or7a or Or42a) activation on larval behavior (Figure 3). Consistent with recent studies that individual larval ORNs are functionally distinct^1,10,13, our representative data demonstrates th.......

Discussion

Here, we described a method that allows for the measurement of Drosophila larval behavior in response to simultaneous optogenetic activation of olfactory neurons. Previously described larval tracking methods^1,8 use different odor delivery technology to activate ORNs. However, these methods cannot control for either the specificity or temporal patterns of ORN activation. Our method overcomes these deficits by using light stimuli instead of odor stimuli fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Video camera to capture larval movement
CCD Camera	Edmund Optics	106215
M52 to M55 Filter Thread Adapter	Edmund Optics	59-446
2" Square Threaded Filter Holder for Imaging Lenses	Edmund Optics	59-445
RG-715, 2" Sq. Longpass Filter	Edmund Optics	46-066
Electronics for optogenetic setup
Raspberry Pi 2B	RASPBERRY-PI.org	RPI2-MODB-V1.2
3 Channel programmable power supply	newegg.com	9SIA3C62037092
8 Channel optocoupler relay	amazon.com	6454319
630nm Quad-row LED strip lights	environmentallights.com	red3528-450-reel
850nm LED strips	environmentallights.com	wp-4000K-CC5050-60x2-kit
Software
Matlab	Mathworks Inc.
Ubuntu MATE v16.04	Nubuntu	https://github.com/yslo/nubuntu
Other items
Plexiglass black acrylic	Home Depot	MC1184848bl
Fly food and other reagents
Nutrifly fly food	Genesee Scientific	66-112
Agarose powder	Genesee Scientific	20-102
22cm X 22cm square petri-dish	VWR Inc.	25382-327
DMSO	Sigma-Aldrich	D2650
Sucrose	Sigma-Aldrich	84097
All trans-retinal	Sigma-Aldrich	R2500
Flies
UAS-IVS-CsChrimson	Bloomington Drosophila Stock Center	55134
Orco-Gal4	Bloomington Drosophila Stock Center	26818
Or42a-Gal4	Bloomington Drosophila Stock Center	9970
Or7a-Gal4	Bloomington Drosophila Stock Center	23907