Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Summary

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Abstract

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors^1,2, ion channels^3-6, and ionotropic receptors⁷ that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Introduction

Animals rely on olfactory and gustatory information to mediate decisions essential for survival and reproduction. Understanding how chemosensory cues are detected and processed by the nervous system requires identification of the sensory receptor(s) and the corresponding chemical ligands. Drosophila detect a staggering variety of volatile and non-volatile compounds and are an excellent model in which to study the physiological mechanisms underlying chemosensation. While the olfactory organs perceive volatile molecules, the gustatory organs are specialized to detect low volatility compounds. Here, we present a method to directly measure neuronal responses from....

Protocol

1. Sample Preparation

1. Cross the Gal4^3,22 driver line to UAS-GCaMP5G¹⁹ flies (w¹¹¹⁸;P{20XUAS-IVS-GCaMP5G}attP40). Allow the cross to grow at 25 °C.
   Note: Generating flies with multiple copies of the Gal4 or UAS-GCaMP transgenes can help to enhance the fluorescent signal intensity. An earlier version of the UAS-GCaMP transgene (UAS-GCaMP3) showed very weak fluorescence intensity when expressed under the control of the Gr68a-Gal4 driver.
   1. Collect and separate by sex newly eclosed adult flies and raise at 25 °C.
      Note: The baseline leve....

Results

The GCaMP calcium indicator was genetically expressed using Gr68a-Gal4 or ppk23-Gal4 drivers. Distinct populations of foreleg neurons and non-neural support cells are labeled by each driver (Figure 3A-C). Ligand-specific responses to the lipophilic pheromone CH503 were observed in Gr68a-Gal4 and ppk23-Gal4 cells expressing GCaMP (Figure 3D). The relative change in fluorescence intensity of the GCaMP5G signal (ΔF/F) and increased with higher co.......

Discussion

We describe here a method to perform live calcium imaging of Drosophila peripheral neurons in 2 different sensory organs. The Ca²⁺-evoked GCaMP fluorescent responses in Gr68a-neurons induced by the pheromone ligand CH503 were dose-dependent and quantitative. It was also possible to discern different neural response patterns such as phasic and tonic responses.

Neurons showing phasic responses are believed to allow rapid adaptation to continuous stimuli. This type of response.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gr68a-Gal4			Gift from H. Amrein (Texas A&M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)
ppk23-Gal4			Gift from K. Scott (Univ. of California, Berkeley, CA, USA)
UAS-GCaMP5		42037	Bloomington Drosophila  Stock Center
0.17 mm coverslip (Gold-Seal coverslip)	Electron Microscopy Services	63790-10
Nail polish, "Hard as Nails Clear"	Sally Hansen
PAP pen	Sigma-Aldrich	Z377821
Paint brush			fine-tipped brush
Tape	Scotch brand
Triton X-100	Sigma-Aldrich	13021
Ethanol, lab grade	Merck	10094
Hexane, HPLC grade	Sigma-Aldrich	H303SK-4
DMSO	Sigma-Aldrich	472301
PBST			Recipe described in the protocol section
CH503			Synthesis described in Mori et al., 2010
sCMOS Camera (ORCA Flash4.0)	Hamamatsu	C11578-22U
Microscope (Ti-Eclipse)	Nikon	Ni-E
Spinning Disk Scan head	Yokogawa	CSU-X1-A1
Aquistion Software (MetaMorph Premier)	Molecular Devices	40002
Fiji software	open source		http://fiji.sc/Fiji