In-vivo and Ex-vivo Calcium Imaging of an Olfactory Circuit Neuron in the Third-Instar Drosophila melanogaster Larva

Summary

We present calcium imaging protocols for a Drosophila larval olfactory neuron, using a topical tissue adhesive for immobilization. This method enhances stability, facilitating reliable in-vivo and ex-vivo experiments. Custom R scripts analyze calcium signals, providing an efficient platform for detailed neurophysiological research.

Abstract

Calcium imaging is crucial for studying neural dynamics and brain function. However, current methods for imaging calcium in Drosophila larvae are challenging due to motion artifacts and specialized equipment needs. Here, we present a practical, low-cost method for performing both in vivo and ex vivo calcium imaging in third-instar Drosophila larvae, using a topical tissue adhesive for immobilizing samples. We also provide custom R scripts and a framework for data analysis, facilitating measurements of calcium changes under different experimental conditions. Using this approach, we successfully recorded calcium transients in a pair of larval CNS neurons with minimal motion artifacts, demonstrating reproducibility across preparations. The method is compatible with standard spinning disk confocal systems and can be adapted for DIY or commercial two-photon setups, making it accessible to laboratories with different resources. This approach may increase accessibility to larval calcium imaging studies and can be adapted to examine the effects of both external stimuli, such as odors, and internal stimuli, such as neuromodulators.

Introduction

Understanding calcium dynamics in neurons is crucial for unraveling the complex mechanisms underlying neural signaling and brain function, as calcium ions play a pivotal role in modulating neuronal activity and driving cellular responses to stimuli. Neuronal calcium dynamics can be monitored via calcium imaging, which provides real-time insights into calcium-mediated signaling across a wide range of organisms, including the Drosophila larva. The Drosophila larva is emerging as a powerful model system to study circuit function underlying behavior. It is one of only three organisms for which a complete synaptic-resolution brain connectome has been gene....

Protocol

1. Preparing third-instar Drosophila larvae for calcium imaging

1. Maintain flies on standard fly food (see Table of Materials) at 25 °C, with 50-60% relative humidity (RH) and a 12 h light/dark cycle.
2. To express Red Fluorescent Protein (RFP) for neuron identification and GCaMP6f (calcium indicator) in the neuron(s) of interest ('X'), cross virgin females of the X-Gal4 line with males carrying UAS-GCaMP6f and UAS-tdTomato²².
   NOTE: For this study, the Keystone-Gal4 line was used to drive expression of tdTomato and GCaMP6f in a single pair of inhibitory local neurons (Keystone-LNs....

Results

To demonstrate our larval calcium imaging approach, we successfully applied it to both in-vivo and ex-vivo preparations of the Drosophila larvae (Figure 2 and Figure 3, Video 1, Video 2, Video 3, and Video 4). We measured calcium dynamics in third-instar Drosophila larvae expressing GCaMP6f and Td-Tomato in a single pair of Keystone-LNs in the larval brain^18<.......

Discussion

Here, we describe an approach for both in-vivo and ex-vivo calcium imaging in third-instar Drosophila melanogaster larvae. We imaged calcium dynamics in Keystone-LN, an inhibitory local neuron in the Drosophila larva. We measured Keystone-LN calcium dynamics in whole larval preparations (in vivo) and in dissected preparations of the larval brain (ex vivo) (Figure 1).

We previously performed qualitative comparis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fly Stocks & Maintenance
Nutrifly fly food	Genesee Scientific	66-112
UAS-GCaMP6f, tdTomato	Gift from Dr. JH Kim
w;;Keystone-Gal4 (GMR27F08-GAL4)	BDSC	49232
Reagents for Starvation protocol
6 cm Petri dish	Genesee Scientific	32-106G
Sucrose	Millipore Sigma	S0389-500G
Reagents for Imaging and Dissecting buffer
Calcium chloride	J.T. Baker, Avantor Sciences	JT1311-1	CAS: 1311-01, 500g
D-(+)-Glucose	Thermo Fisher Scientific	A16828.36	CAS: 50-99-7, 500g
HEPES-2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid	J.T. Baker, Avantor Sciences	JT4018-1	CAS: 4018-01, 100g
Magnesium chloride	VWR	97064-150	CAS: 7786-30-3, 100g
Potassium chloride	Thermo Fisher Scientific	J64189.36	CAS: 7447-40-7, 500g
Sodium chloride	EMD Milipore Sigma	EMD-SX0420-1	CAS: 7647-14-5, 500g
Tools and Reagents for Dissection
Dissection Buffer			Composition provided in text
Olympus Petri Dishes, 60 x 15mm Vented	Genesee Scientific	32-105
Sylgard 184 Elastomer Base	Electron Microscopy Sciences	24236-10A
Sylgard 184 Elastomer Base Curing Agent	Electron Microscopy Sciences	24236-10B
Tweezers Style 5, EMS	Electron Microscopy Sciences	78320-5
Vannas Spring Scissors - 3mm Cutting Edge	Fine Science Tools	15000-00
Imaging and Mounting Equipment/Reagents
Confocal Scanner module	Yokogawa	CSU-W1	connected to microscope
Imaging Buffer			Composition provided in text
Inverted Microscope	Leica Microsystems	DMi8	Connected to a spinning disc confocal scanner module and a CCD camera
Microscope Cover Glass, 24mm x 50mm, # 1.5 Thickness	Globe Scientific Inc.	1415-15
MWI Veterinary GLUTURE Topical Adhesive	Fisher Scientific	NC0632797
Pure Petroleum Jelly	Walgreens	912556
Links to Publicly Available Resources
BioRender			https://www.biorender.com/
ImageJ	NIH	912556	https://imagej.net/ij/
Janelia Flylight protocol video	https://www.janelia.org/		https://www.youtube.com/watch?v=UVCPmuu2b6o
Mathew Lab Github			https://github.com/MathewLab/JoVE-Calcium-imaging