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Method Article

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

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DOI:

10.3791/68956

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October 31st, 2025

 ,  ,  ,  , 
1Department of Biology, University of Nevada, Reno, 2Integrative Neuroscience Program, University of Nevada, Reno, 3Molecular Biosciences Program, University of Nevada, Reno, 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno

Corresponding Authors: Dennis Mathew <dennismathew@unr.edu>

* These authors contributed equally

In This Article

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 generated1. The larva is amenable to a range of sophisticated tools that enable imaging and manipulation of neurons at both the whole-brain and single-neuron levels2,3.

The ability to measure calcium dynamics in Drosophila larval neurons via calcium imaging has been crucial to advancing neuroscience research4. Recent advances in calcium imaging have allowed for measuring calcium dynamics at the level of single synapses in the larva5 to multi-neuronal recordings in free-moving larvae6. Calcium imaging can be performed in vivo, where calcium events are captured under natural physiological contexts of the whole larva4,7,8,9, or ex vivo, where calcium events are recorded in dissected larval tissues preserved under simulated physiological conditions10. Overall, researchers have used various iterations of the calcium imaging method in Drosophila larvae to uncover fundamental mechanisms of neural circuitry, sensations, learning and memory, and behavior8,11,12,13,14,15,16,17. However, larval calcium imaging studies are limited due to the need for specialized equipment and problems in data interpretation due to motion artifacts.

Here, we introduce a cost-effective and easily implementable methodology for both in vivo and ex vivo calcium imaging in third-instar Drosophila melanogaster larvae. We image calcium dynamics in Keystone-LN, an inhibitory olfactory circuit neuron18,19, within whole larval preparations to showcase in vivo calcium imaging, and in dissected larval brains to showcase ex vivo calcium imaging. Whole larvae and dissected brains remain viable and responsive for approximately 10-15 min under our imaging conditions, allowing acquisition of multiple stimulus trials per sample. To address the challenge of immobilizing whole larvae or tissue samples while maintaining tissue viability and reducing experimental variability, we optimized the use of GLUture, a biocompatible cyanoacrylate topical tissue adhesive.

Compared with traditional larval immobilization methods, such as agarose embedding or pinning, this adhesive-based approach reduces motion artifacts while preserving tissue viability20,21; relative to two-photon imaging systems, the spinning-disk setup lowers instrumentation costs and improves accessibility for resource-limited laboratories. Finally, we describe straightforward imaging approaches, coupled with custom-developed R scripts for data analyses. Our 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.

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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) in the larval brain18,19.
  3. After allowing male and female flies to mate and lay eggs for 48 h, transfer the adults to a fresh vial.
  4. Extract third-instar larvae (approximately 120 h after egg laying) by floating them on a 15% sucrose solution. Use a P1000 micropipette to transfer the floating larvae into a 1,000 mL glass beaker.
  5. Wash the larvae 3-4x by replacing the water in the beaker with 800 mL of fresh distilled water each time. Allow larvae to rest for 10 min before proceeding with the rest of the protocol.

2. Starvation protocol for third-instar Drosophila larvae (if testing starved vs. non-starved conditions)

  1. Prepare two feeding chambers by placing a small square of laboratory wipe in 6 cm Petri dishes. For non-starved conditions, add 350 µL of 0.2 M sucrose solution; for starved conditions, add 350 µL of distilled water.
  2. Transfer an equal number of washed larvae to each feeding chamber. Allow the larvae to feed on sucrose (non-starved) or distilled water (starved) for 2 h at room temperature.

3. In-vivo calcium imaging

NOTE: All experiments involving the tissue adhesive should be conducted using standard laboratory safety practices. Personal protective equipment (PPE), including nitrile gloves, a lab coat, and safety goggles or a face shield, is required; cotton gloves should be avoided due to exothermic reactions with cyanoacrylate. Avoid contact of the adhesive with skin, eyes, or mucous membranes. In case of skin bonding, soak the area in warm, soapy water and gently separate; in case of eye exposure, rinse with lukewarm water for ≥5 min and seek immediate medical attention. The adhesive should be kept away from open flames and high heat, as cured adhesive may release irritating fumes if burned. Store adhesive at ≤30 °C, away from moisture and direct sunlight, in a tightly sealed container when not in use.

  1. Prepare the calcium imaging buffer with the following composition: NaCl 110 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM, D-glucose 10 mM, and HEPES 10 mM. Store at 4 °C when not in use.
    NOTE: The modified saline containing 110 mM NaCl is a widely accepted formulation for larval CNS dissection and imaging and falls within the typical range used in electrophysiology and imaging studies (≈108-128 mM NaCl)23,24,25,26.
  2. On a 24 x 50 mm (1.5 mm-thick) microscope cover glass, create a well using petroleum jelly dispensed from a syringe.
  3. Place a small drop of topical tissue adhesive (60% 2-octyl and 40% n-butyl cyanoacrylate) in the center of the cover glass. Spread the adhesive evenly into a thin, uniform layer using a glass rod.
  4. Carefully transfer a single larva onto the adhesive using a brush or thin wire. Gently press the ventral side of the larva onto the adhesive. Allow the adhesive to dry, ensuring the larva is fully immobilized.
  5. Once the larva is immobilized, place the cover glass on the inverted microscope stage (Figure 1A).

4. Ex-vivo calcium imaging

  1. Prepare the larval dissection buffer with the following composition: NaCl 110 mM, KCl 5.4 mM, CaCl2 0.3 mM, MgCl2 0.8 mM, D-glucose 10 mM, and HEPES 10 mM. Store at 4 °C when not in use.
  2. Dissect the brain dissections as described by Ishimoto and Sano10. Briefly, using a brush or thin wire, transfer a single larva to a 3.5 cm Petri dish with Silicone Elastomer layered at the bottom.
  3. Under a dissection microscope, position the larva with its ventral side facing up and locate the brain. Using dissection scissors, make a cut about one-third of the way down the anterior end of the larva. Make another cut at the mouth hooks. Preserve the brain while removing the body and mouth hooks (adapted from Janelia Flylight Protocol27).
  4. Identify the cluster of motor neuron-containing nerves from the ventral cord. Using forceps, gently pull the brain away from the rest of the dissected larva. Trim any excess tissue from the brain.
    NOTE: Handle gently-excessive force can cause tissue damage as the brain is fragile.
  5. Prepare a microscope cover glass with the tissue adhesive for calcium imaging as described in steps 3.2 to 3.3.
  6. Using a P10 micropipette, carefully aspirate the dissected brain with 5 µL of dissection solution from the Petri dish. Slowly expel the brain onto the adhesive. Before the adhesive dries, orient the brain in a dorsal-side-up position (with the two brain lobes facing down and the dorsal ventral cord facing up).
  7. Using a P200 micropipette, transfer 100 µL of the calcium imaging buffer over the immobilized larval brain on the cover glass.
  8. Place the cover glass with the brain onto the inverted microscope, connected to a spinning disk confocal scanner module and a CCD camera for calcium imaging (Figure 1B).
    NOTE: For ex-vivo preparations, be sure to orient the brain correctly while the glue is still wet. For optimal image quality, position the brain such that the ROI is closest to the microscope objective and avoid applying excess glue near the ROI.

5. Imaging protocol

  1. Open the software for calcium imaging using the confocal microscope. Capture images using a 10x/1.4 NA objective with filter switching between GFP laser (excitation 488 nm, intensity 70%, emission 525 nm/50 m, OD8) and RFP laser (excitation 561 nm, intensity 50%, emission 610 nm/75 m, OD8). Set the exposure between 100 ms and 1,000 ms, the gain at 50% of the corresponding exposure value, and a binning factor of 2 during image capture.
  2. Adjust the microscope to 10x magnification and locate the brain under brightfield illumination.
  3. Identify the neuron of interest by searching for the tdTomato signal with the RFP laser (excitation set to 561 nm).
    NOTE: Select saturation and reduce gamma to set the correct exposure value.
  4. Once the region of interest (ROI) is identified, switch to the GFP laser (excitation set to 488 nm) to capture GCaMP6f signals. Ensure that both tdTomato and GCaMP signals colocalize before capturing stack images of both channels.
    NOTE: Adjust image settings for tdTomato and GCaMP6f stack recordings to maximize fluorescence.
  5. Capture two separate time-series recordings of tdTomato and GCaMP6f signals simultaneously at a rate of 0.5 frames/s (for a total of 2 min).
    NOTE: The dissected larval brain remains viable for approximately 10 min under these buffering conditions; after this time, the signals will degrade.
  6. Once the sample is ready to image, capture two separate time-series recordings of tdTomato and GCaMP6f signals simultaneously at a rate of 0.5 frames/s (for a total of 2 min).
    NOTE: The dissected larval brain remains viable for approximately 10 min under these buffering conditions; after this time, the signals will degrade.
    NOTE: While conducting quantitative analysis, we recommend increasing laser power and adjusting the detector exposure gain as needed while maintaining linear intensity scaling.

6. Data processing and analysis

  1. Open and import the tdTomato and GCaMP6f signal stacks into ImageJ (NIH)28.
  2. To identify the correct region of interest (ROI), merge the tdTomato and GCaMP6f signals to confirm colocalization. After verification, split the tdTomato and GCaMP6f channels.
  3. In the top menu, select Plugins | New, and click on Macros to initiate a new macro. Next, import the custom ROI based macros into ImageJ. Select the GCaMP6f stack and hit Run for calcium analysis.
  4. Process the fluorescence signals using a custom ImageJ macro (https://github.com/MathewLab/JoVE-Calcium-imaging). The macro first generates maximum intensity projections, performs motion correction using the StackReg plugin (Rigid Registration mode)29, and applies bleach correction.
  5. An "Action Required" pop-up will appear requesting manual ROI selection, which allows users to manually draw a broad ROI. Click OK to continue the analysis. To carry out motion correction, ensure that the rectangular box covers the entire ROI in all frames for accurate motion correction while retaining local spatial and ROI shape across frames.
    NOTE: Recordings were registered in ImageJ using StackReg. When the in-plane motion was large (cumulative XY displacement > 1 µM) or axial Z-drift led to loss of focus, registration became unreliable, or the sample was lost entirely from the imaging window. Thus, samples showing extreme drift or declining calcium signals (>30%) were automatically tagged and excluded from downstream analysis to ensure accurate measurements of neuronal activity. These thresholds are consistent with commonly applied quality control practices in calcium imaging studies30,31,32,33. Exclusion also ensured that only stable recordings with reliable stimulus-linked calcium transients were used for analyses.
  6. For the analysis, extract fluorescence traces from manually defined circular ROIs, guided by pixel autocorrelation; these ROIs will encompass the entire keystone neuron field.
    1. Extract the raw fluorescence intensity data for two fluorophores: GCaMP6f/GFP and tdTomato/RFP, using custom macros in ImageJ (v1.54p) (https://github.com/MathewLab/JoVE-Calcium-imaging, see Table of Materials).
    2. Apply a sliding window with a 20 x 20-pixel ROI and a step size of 10 pixels across the field of view. For each ROI, the macro will calculate: (1) the intensity across all frames, (2) the baseline fluctuation difference (i.e., the difference between the maximum and minimum intensity values during the first 10 frames), and (3) the maximum frame-to-frame intensity change (ΔF). ROIs showing intensity fluctuations less than 10 units will be eliminated, and only the sharpest ΔF values will be selected for further analysis.
    3. Normalize the fluorescence traces using the following methods:
      1. Fmax and Fmin normalization: Calculate normalized Δ using the formula:
        Delta F normalization formula; equation for force standardization; data analysis method.
        Where F is the raw fluorescence at each time point, Fmin is the minimum fluorescence (baseline), and Fmax is the maximum fluorescence during the recording.
      2. ΔF/F 0 normalization: Additionally, perform ΔF/Fo normalization, where Fo (baseline) is determined by the first 10 frames of each recording, using the formula:
        Fluorescence quenching formula Ft-F0/F0, related to protein-ligand interaction analysis.
        Where Ft is the fluorescence at time t, and F0 is the baseline value calculated from the first 10 frames.
  7. After extracting GCaMP6f signals, left click and Select All, Cut, and Paste to transfer all values to a new Excel spreadsheet. In the top menu bar, select Data | Text to Columns. A "Text-to-Column" wizard pop-up will appear. In step 1, select Delimited, then click Next. In step 2, under Delimiters, select Tab and Comma, then click Next. In step 3, select Finish.
    OPTIONAL: To plot the normalized calcium signals, highlight Fnorm_FminFmax, and select Scatterplot.
  8. Look for a .csv file that is generated containing: (1) raw fluorescence values F, (2) ΔF/Fo, and (3) Fnorm (normalized fluorescence intensity). Organize the final dataset into four columns: (1) time (frames), (2) sample condition (starved or fed), (3) replicate ID (individual sample), and (4) normalized intensity (Fnorm values scaled from 0 to 1).
  9. Finally, perform data analysis and visualization in R using custom R scripts (https://github.com/MathewLab/JoVE-Calcium-imaging. see Table of Materials).
    1. Import the custom R script into a new R project. Ensure that the required R packages, including ggplot2, dplyr, and readr, are installed and updated prior to running the analysis.
    2. Starting from line 1 of the custom R script, hit Run continuously to conduct the calcium analysis. Also ensure that the output includes heatmaps illustrating the normalized calcium intensity for the first 30 frames, 60 frames, and all frames of the time-series recording, and boxplots to show the median of normalized intensities.
    3. Assess the significance of normalized calcium intensity using the Mann-Whitney U test.

7. Waste disposal and decontamination

  1. After experiments, remove gross debris from tools and surfaces under running water, then disinfect with 70% ethanol or 10% bleach followed by sterile water. Work on a disinfected surface, and wash hands after glove removal; do not eat or drink in the lab.
  2. Place larval tissue and contaminated disposables in biohazard bags and autoclave prior to disposal.
  3. Discard fully cured adhesive waste in general trash; uncured adhesive and contaminated disposables must be collected in a sealed, labeled hazardous chemical waste container.
  4. Place all sharps (blades, needles, broken glass) in a puncture-resistant sharps container.
  5. Collect flammable solvents (ethanol, acetone) in labelled, flammable waste containers.

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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 brain18<...

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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...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

We are grateful to Dr. Jeffrey Harper, who made his lab's spinning disk confocal microscope available to us for these experiments. We are grateful for the gift of the fly line: w; UAS-GCaMP6f/CyO, weep; UAS-tdTomato from Dr. Jung Hwan Kim. This work was supported by funds from the National Science Foundation under award numbers 2341202 and 2129234. Figure 1 was created using Biorender.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Fly Stocks & Maintenance
Nutrifly fly foodGenesee Scientific66-112
UAS-GCaMP6f, tdTomatoGift from Dr. JH Kim 
w;;Keystone-Gal4 (GMR27F08-GAL4)BDSC49232
Reagents for Starvation protocol
6 cm Petri dishGenesee Scientific32-106G
SucroseMillipore SigmaS0389-500G
Reagents for Imaging and Dissecting buffer
Calcium chlorideJ.T. Baker, Avantor SciencesJT1311-1CAS: 1311-01, 500g
D-(+)-GlucoseThermo Fisher ScientificA16828.36CAS: 50-99-7, 500g
HEPES-2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acidJ.T. Baker, Avantor SciencesJT4018-1CAS: 4018-01, 100g
Magnesium chlorideVWR97064-150CAS: 7786-30-3, 100g
Potassium chlorideThermo Fisher ScientificJ64189.36CAS: 7447-40-7, 500g
Sodium chlorideEMD Milipore SigmaEMD-SX0420-1CAS: 7647-14-5, 500g
Tools and Reagents for Dissection
Dissection BufferComposition provided in text
Olympus Petri Dishes, 60 x 15mm VentedGenesee Scientific32-105
Sylgard 184 Elastomer BaseElectron Microscopy Sciences24236-10A
Sylgard 184 Elastomer Base Curing AgentElectron Microscopy Sciences24236-10B
Tweezers Style 5, EMSElectron Microscopy Sciences78320-5
Vannas Spring Scissors - 3mm Cutting EdgeFine Science Tools15000-00
Imaging and Mounting Equipment/Reagents
Confocal Scanner moduleYokogawaCSU-W1connected to microscope
Imaging BufferComposition provided in text
Inverted MicroscopeLeica MicrosystemsDMi8 Connected to a spinning disc confocal scanner module and a CCD camera
Microscope Cover Glass, 24mm x 50mm, # 1.5 ThicknessGlobe Scientific Inc.1415-15
MWI Veterinary GLUTURE Topical AdhesiveFisher ScientificNC0632797
Pure Petroleum JellyWalgreens912556
Links to Publicly Available Resources
BioRenderhttps://www.biorender.com/
ImageJNIH912556https://imagej.net/ij/
Janelia Flylight protocol videohttps://www.janelia.org/https://www.youtube.com/watch?v=UVCPmuu2b6o
Mathew Lab Githubhttps://github.com/MathewLab/JoVE-Calcium-imaging

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