Name: In-vivo and Ex-vivo Calcium Imaging of an Olfactory Circuit Neuron in the Third-Instar Drosophila melanogaster Larva (Video) | JoVE Journal
Uploaded: 2025-10-31T14:55:00.000+00:00
Duration: 11 min 58 s
Description: Learn calcium imaging protocols for Drosophila larval olfactory neurons using GLUture tissue adhesive immobilization. This method enables stable in-vivo and ex-vivo experiments with custom R scripts for quantitative analysis.

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5 minutes

We are grateful to Dr. Jeffrey Harper, who made his lab&#39;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.

1. Preparing third-instar Drosophila larvae for calcium imaging
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>After allowing male and female flies to mate and lay eggs for 48 h, transfer the adults to a fresh vial.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Starvation protocol for third-instar Drosophila larvae (if testing starved vs. non-starved conditions)
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>On a 24 x 50 mm (1.5 mm-thick) microscope cover glass, create a well using petroleum jelly dispensed from a syringe.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Once the larva is immobilized, place the cover glass on the inverted microscope stage (Figure 1A).</li>
</ol>
4. Ex-vivo calcium imaging
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Prepare a microscope cover glass with the tissue adhesive for calcium imaging as described in steps 3.2 to 3.3.</li>
	<li>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).</li>
	<li>Using a P200 micropipette, transfer 100 µL of the calcium imaging buffer over the immobilized larval brain on the cover glass.</li>
	<li>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.</li>
</ol>
5. Imaging protocol
<ol>
	<li>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.</li>
	<li>Adjust the microscope to 10x magnification and locate the brain under brightfield illumination.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Data processing and analysis
<ol>
	<li>Open and import the tdTomato and GCaMP6f signal stacks into ImageJ (NIH)28.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &gt; 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 (&gt;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.</li>
	<li>For the analysis, extract fluorescence traces from manually defined circular ROIs, guided by pixel autocorrelation; these ROIs will encompass the entire keystone neuron field.
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>Normalize the fluorescence traces using the following methods:
		<ol>
			<li>Fmax and Fmin normalization: Calculate normalized Δ using the formula: 
			<img alt="Delta F normalization formula; equation for force standardization; data analysis method." src="/files/ftp_upload/68956/68956eq1.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 1"> 
			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.</li>
			<li>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: 
			<img alt="Fluorescence quenching formula Ft-F0/F0, related to protein-ligand interaction analysis." src="/files/ftp_upload/68956/68956eq2.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 2"> 
			Where Ft is the fluorescence at time t, and F0 is the baseline value calculated from the first 10 frames.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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).</li>
	<li>Finally, perform data analysis and visualization in R using custom R scripts (https://github.com/MathewLab/JoVE-Calcium-imaging. see Table of Materials).
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Assess the significance of normalized calcium intensity using the Mann-Whitney U test.</li>
	</ol>
	</li>
</ol>
7. Waste disposal and decontamination
<ol>
	<li>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.</li>
	<li>Place larval tissue and contaminated disposables in biohazard bags and autoclave prior to disposal.</li>
	<li>Discard fully cured adhesive waste in general trash; uncured adhesive and contaminated disposables must be collected in a sealed, labeled hazardous chemical waste container.</li>
	<li>Place all sharps (blades, needles, broken glass) in a puncture-resistant sharps container.</li>
	<li>Collect flammable solvents (ethanol, acetone) in labelled, flammable waste containers.</li>
</ol>

<ol><li>Winding, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+connectome+of+an+insect+brain%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The connectome of an insect brain.</a> Science. 379 (6636), eadd9330(2023).</li>
<li>Eschbach, C., Zlatic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Useful+road+maps%3A+studying+Drosophila+larva%27s+central+nervous+system+with+the+help+of+connectomics%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Useful road maps: studying Drosophila larva's central nervous system with the help of connectomics.</a> Curr Opin Neurobiol. 65, 129-137 (2020).</li>
<li>Aimon, S., Grunwald Kadow, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+complex+brain+dynamics+using+Drosophila%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">Studying complex brain dynamics using Drosophila.</a> J Neurogenet. 34 (1), 171-177 (2020).</li>
<li>Riemensperger, T., Pech, U., Dipt, S., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+calcium+imaging+in+the+nervous+system+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Optical calcium imaging in the nervous system of Drosophila melanogaster.</a> Biochim Biophys Acta. 1820 (8), 1169-1178 (2012).</li>
<li>Fukumasu, K., Nose, A., Kohsaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraction+of+bouton-like+structures+from+neuropil+calcium+imaging+data%5BTitle%5D)+AND+%22Neural+Netw%22%5BJournal%5D)">Extraction of bouton-like structures from neuropil calcium imaging data.</a> Neural Netw. 156, 218-238 (2022).</li>
<li>Yamaguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-neuronal+recording+in+unrestrained+animals+with+all+acousto-optic+random-access+line-scanning+two-photon+microscopy%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Multi-neuronal recording in unrestrained animals with all acousto-optic random-access line-scanning two-photon microscopy.</a> Front Neurosci. 17, 1135457(2023).</li>
<li>Marescotti, M., Lagogiannis, K., Webb, B., Davies, R. W., Armstrong, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+brain+activity+and+behaviour+in+freely+moving+Drosophila+larvae+using+bioluminescence%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Monitoring brain activity and behaviour in freely moving Drosophila larvae using bioluminescence.</a> Sci Rep. 8 (1), 9246(2018).</li>
<li>Si, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structured+odorant+response+patterns+across+a+complete+olfactory+receptor+neuron+population%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Structured odorant response patterns across a complete olfactory receptor neuron population.</a> Neuron. 101 (5), 950-962.e7 (2019).</li>
<li>Ji, H., Han, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Larvaspa%2C+a+method+for+mounting+Drosophila+larva+for+long-term+time-lapse+imaging%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Larvaspa, a method for mounting Drosophila larva for long-term time-lapse imaging.</a> J Vis Exp. (156), e60792(2020).</li>
<li>Ishimoto, H., Sano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+calcium+imaging+for+visualizing+brain+responses+to+endocrine+signaling+in+Drosophila%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Ex vivo calcium imaging for visualizing brain responses to endocrine signaling in Drosophila.</a> J Vis Exp. (136), e57701(2018).</li>
<li>Maier, G. L., Komarov, N., Meyenhofer, F., Kwon, J. Y., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Taste+sensing+and+sugar+detection+mechanisms+in+Drosophila+larval+primary+taste+center%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Taste sensing and sugar detection mechanisms in Drosophila larval primary taste center.</a> Elife. 10, (2021).</li>
<li>Carreira-Rosario, A., York, R. A., Choi, M., Doe, C. Q., Clandinin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanosensory+input+during+circuit+formation+shapes+Drosophila+motor+behavior+through+patterned+spontaneous+network+activity%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity.</a> Curr Biol. 31 (23), 5341-5349.e4 (2021).</li>
<li>Yoon, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((System+level+analysis+of+motor-related+neural+activities+in+larval+Drosophila%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">System level analysis of motor-related neural activities in larval Drosophila.</a> J Neurogenet. 33 (3), 179-189 (2019).</li>
<li>Vaadia, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+proprioceptive+system+dynamics+in+behaving+Drosophila+larvae+using+high-speed+volumetric+microscopy%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Characterization of proprioceptive system dynamics in behaving Drosophila larvae using high-speed volumetric microscopy.</a> Curr Biol. 29 (6), 935-944.e4 (2019).</li>
<li>Utashiro, N., Williams, C. R., Parrish, J. Z., Emoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prior+activity+of+olfactory+receptor+neurons+is+required+for+proper+sensory+processing+and+behavior+in+Drosophila+larvae%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Prior activity of olfactory receptor neurons is required for proper sensory processing and behavior in Drosophila larvae.</a> Sci Rep. 8 (1), 8580(2018).</li>
<li>Gillespie, J. M., Hodge, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CASK+regulates+CaMKII+autophosphorylation+in+neuronal+growth%2C+calcium+signaling%2C+and+learning%5BTitle%5D)+AND+%22Front+Mol+Neurosci%22%5BJournal%5D)">CASK regulates CaMKII autophosphorylation in neuronal growth, calcium signaling, and learning.</a> Front Mol Neurosci. 6, 27(2013).</li>
<li>Francis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitory+circuit+motifs+in+Drosophila+larvae+generate+motor+program+diversity+and+variability%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Inhibitory circuit motifs in Drosophila larvae generate motor program diversity and variability.</a> PLoS Biol. 23 (4), e3003094(2025).</li>
<li>Berck, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+wiring+diagram+of+a+glomerular+olfactory+system%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">The wiring diagram of a glomerular olfactory system.</a> Elife. 5, (2016).</li>
<li>Odell, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Internal+state+affects+local+neuron+function+in+an+early+sensory+processing+center+to+shape+olfactory+behavior+in+Drosophila+larvae%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Internal state affects local neuron function in an early sensory processing center to shape olfactory behavior in Drosophila larvae.</a> Sci Rep. 12 (1), 15767(2022).</li>
<li>Worrell, J. W., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+voltage-dependent+Ca2%2B+currents+in+identified+Drosophila+motoneurons+in+situ%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ.</a> J Neurophysiol. 100 (2), 868-878 (2008).</li>
<li>Marley, R., Baines, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+of+third-instar+Drosophila+larvae+for+electrophysiological+recording+from+neurons%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Dissection of third-instar Drosophila larvae for electrophysiological recording from neurons.</a> Cold Spring Harb Protoc. 2011 (9), (2011).</li>
<li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li>
<li>Held, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aminergic+and+peptidergic+modulation+of+insulin-producing+cells+in+Drosophila%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Aminergic and peptidergic modulation of insulin-producing cells in Drosophila.</a> Elife. 13, (2025).</li>
<li>Kim, S. M., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+imaging+of+pheromone+responses+in+the+insect+antennal+lobe%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Calcium imaging of pheromone responses in the insect antennal lobe.</a> Methods Mol Biol. 1068, 179-187 (2013).</li>
<li>Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions%5BTitle%5D)+AND+%22J+Comp+Physiol+%5BA%5D%22%5BJournal%5D)">Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.</a> J Comp Physiol [A]. 175 (2), 179-191 (1994).</li>
<li>Macleod, G. T., Marin, L., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+vesicles%3A+test+for+a+role+in+presynaptic+calcium+regulation%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Synaptic vesicles: test for a role in presynaptic calcium regulation.</a> J Neurosci. 24 (10), 2496-2505 (2004).</li>
<li>Larval Dissection. , Howard Hughes Medical Institute. 
 <a target="_blank" href="https://www.youtube.com/watch?v=UVCPmuu2b6o">https://www.youtube.com/watch?v=UVCPmuu2b6o</a> (2019).</li>
<li>Schindelin, J., Rueden, C. T., Hiner, M. C., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ImageJ+ecosystem%3A+an+open+platform+for+biomedical+image+analysis%5BTitle%5D)+AND+%22Mol+Reprod+Dev%22%5BJournal%5D)">The ImageJ ecosystem: an open platform for biomedical image analysis.</a> Mol Reprod Dev. 82 (7-8), 518-529 (2015).</li>
<li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pyramid+approach+to+subpixel+registration+based+on+intensity%5BTitle%5D)+AND+%22IEEE+Trans+Image+Process%22%5BJournal%5D)">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li>
<li>Chen, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li>
<li>Dana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-performance+calcium+sensors+for+imaging+activity+in+neuronal+populations+and+microcompartments%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.</a> Nat Methods. 16 (7), 649-657 (2019).</li>
<li>De Vries, S. E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+large-scale+standardized+physiological+survey+reveals+functional+organization+of+the+mouse+visual+cortex%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.</a> Nat Neurosci. 23 (1), 138-151 (2020).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+sensory+transmission+and+neuronal+plasticity+in+primary+sensory+neurons+with+a+positively+tuned+voltage+indicator%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Imaging sensory transmission and neuronal plasticity in primary sensory neurons with a positively tuned voltage indicator.</a> Nat Commun. 16 (1), 6396(2025).</li>
<li>Root, C. M., Ko, K. I., Jafari, A., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presynaptic+facilitation+by+neuropeptide+signaling+mediates+odor-driven+food+search%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search.</a> Cell. 145 (1), 133-144 (2011).</li>
<li>Slankster, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+underlying+starvation-dependent+modulation+of+olfactory+behavior+in+Drosophila+larva%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Mechanism underlying starvation-dependent modulation of olfactory behavior in Drosophila larva.</a> Sci Rep. 10 (1), 3119(2020).</li>
<li>Lehnert, B. P., Baker, A. E., Gaudry, Q., Chiang, A. S., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+roles+of+TRP+channels+in+auditory+transduction+and+amplification+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Distinct roles of TRP channels in auditory transduction and amplification in Drosophila.</a> Neuron. 77 (1), 115-128 (2013).</li>
<li>Murthy, M., Turner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-cell+in+vivo+patch-clamp+recordings+in+the+Drosophila+brain%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Whole-cell in vivo patch-clamp recordings in the Drosophila brain.</a> Cold Spring Harb Protoc. 2013 (2), 140-148 (2013).</li>
<li>Giovannucci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CaImAn%3A+an+open+source+tool+for+scalable+calcium+imaging+data+analysis%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">CaImAn: an open source tool for scalable calcium imaging data analysis.</a> Elife. 8, (2019).</li>
<li>Cao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+targeted+optical+electrophysiology+in+intact+neural+circuits%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genetically targeted optical electrophysiology in intact neural circuits.</a> Cell. 154 (4), 904-913 (2013).</li>
<li>Simpson, J. H., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+imaging+and+optogenetics+in+Drosophila%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Functional imaging and optogenetics in Drosophila.</a> Genetics. 208 (4), 1291-1309 (2018).</li>
</ol>

The authors have no conflicts of interest to disclose.

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...

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.

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

in-vivo and ex-vivo calcium imaging of an olfactory circuit neuron in the third-instar drosophila melanogaster larva

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

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.

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<...

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.

Calcium imaging is crucial for studying neural dynamics and brain function. However, current methods for imaging calcium in Drosophila larvae are ...

This protocol uses GLUture tissue adhesive to enhance the ability in in-vivo and ex-vivo calcium imaging. This allows researchers to understand the calcium dynamics in an olfactory circuit neuron in Third-Instar Drosophila larvae. Simplicity and cost efficiency are the two main advantages for this technique, making experiments more reliable and accessible in neurophysiological research.Prepare two feeding chambers by placing a small square of chem-wipe in six centimeter Petri dishes. For starved conditions, add 350 microliters of distilled water. For non-starved conditions, add 350 microliters of 0.2 molar sucrose solution.Transfer an equal number of washed larvae into each feeding chamber. Allow the larvae to feed on sucrose, non-starved, or distilled water, starved, for two hours at room temperature On a 24 by 50 millimeters, 1.5 millimeters thick microscope cover glass, create a well, using petroleum jelly dispensed from a syringe. Place a small drop of GLUture topical tissue adhesive in the center of the cover glass.Spread the GLUture evenly into a thin, uniform layer, using a glass rod. Carefully transfer a single larva onto the GLUture using a brush or thin wire. Gently press the ventral side of the larvae onto the adhesive.Allow the GLUture to dry, ensuring the larvae is fully immobilized. Once the larvae is immobilized, immerse the larva in 100 microliters of imaging buffer. Place the cover glass onto a Leica DMi8 inverted microscope, connected to a Yokogawa CSU-W1 spinning disc confocal scanner module, and a CCD camera for calcium imaging.Prepare a microscope cover-glass with GLUture for calcium imaging, as described in steps 3.2 to 3.3. Complete the dissection as described by Ishimoto and Sano, 2018. Using a P-10 micro pipette, carefully aspirate the dissected brain with five microliters of dissection solution from the Petri dish.Slowly expel the brain onto the GLUture. Before the GLUture dries, orient the brain in a dorsal side up position with the two brain lobes facing down and the dorsal ventral cord facing up. Using a P-200 micro pipette, transfer 100 microliters of calcium imaging buffer over the immobilized larvae brain on the cover glass.Open VisiView for calcium imaging, using Leica DMi8 inverted spinning microscope. Capture images using a 10X/1.4 any objective with filters, switching between the GFP laser and RFP laser. Exposure values range from 100 to 1000 milliseconds and the gain is set at 50%of the corresponding exposure value.A bidding factor of two is applied during image capture. Identify the neuron of interest by searching for the tdTomato signal with the RFP laser. Once the region of interest is identified, switch to the GFP laser to capture GCaMP6f signals.Ensure both tdTomato and GCaMP signals colloquialize before capturing stack images of both channels. Capture two separate time series recordings of tdTomato and GCaMP6f success signals simultaneously at a rate of 0.5 frames per second, for a total of two minutes. Import the tdTomato and GCaMP6f signal stacks into ImageJ.To identify the correct region of interest, merge the tdTomato and GCaMP6f signals to confirm colocalization. After verification, split the tdTomato and GCaMP6f channels. Import the custom ROI-based macros into ImageJ.Select the GCaMP6f stack and hit run for calcium analysis. The macro first generates maximum intensity projections, performs motion correction, using stack reg plugin, and applies bleach correction. Motion correction was carried out by defining a broad rectangular region of interest, such as the brain lobe on each frame.The rectangular box must cover the entire ROI in all frames to ensure accurate motion correction, while retaining local, spatial, and ROI shape across all frames. Each ROI, the macros calculated one, the intensity across all frames, two, the baseline fluctuation difference, such as the difference between the maximum and minimum intensity values during the first 10 frames, and three, the maximum frame to frame intensity change, Delta F.ROIs showing intensity fluctuations less than 10 units were eliminated. The final dataset was organized into four columns, one, time and frames, two, sample conditions starved or fed, three replica ID, individual samples, and four, normalized intensity.F norm value scaled from zero to one. Finally, data analysis and visualization were performed in R, using custom R scripts. The required packages, including ggplot2, dplyr, and readr were installed and updated prior to running the analysis.The output included heat maps illustrating the normalized calcium intensity for the first 30 frames, 60 frames, and all frames at the time series recording, and box plots to show median of normalized intensities. The significance of normalized calcium intensity was assessed using the Mann-Whitney U test. Figure one is a schematic for in-vivo and ex-vivo calcium imaging setups.Whole larval sample A, or larval brain sample B, was fixed on a thin layer of GLUture in solid blue, spread within a well of petroleum jelly, and round on a microscope cover glass. Samples were immersed in calcium imaging buffer, light blue, and placed for imaging on an inverted spinning disc confocal microscope. Time series of calcium fluorescence images of the samples were captured.Schematic was prepared using BioRender. Figure two illustrates GCaMP6f fluorescence imaging. A, split images showing fluorescence in the 488 nanometer channel, GCaMP6f and green, and the 525 nanometer channel, tdTomato in red.The merged image is shown in the right panel. B, example raw fluorescence traces over time for GCaMP6f in green and tdTomato in red in keystone LNs. Fluctuation in GCaMP6f fluorescence indicate calcium activity while the stable tdTomato signal is used as a control to identify keystone LNs.C, representative GCaMP6f signal images for in-vivo top panels and ex-vivo bottom panel preparations. Non-starved samples are shown on the left, and starved samples are shown on the right. Figure three depicts GCaMP6f fluorescence measurements.A, the top left panel shows a heat map showcasing the average temporal dynamics of calcium signals from keystone elements from non-starved, N=5, and starved larvae, N=5, in the in-vivo preparation whole larvae. The box plot in the top right panel compares normalized calcium signal intensity between non-starved in gray, and starved in white samples in the in-vivo preparation. Mann-Whitney U test, P is less than 0.001.B, the bottom left panel shows a heat map showcasing the average to portal dynamics of calcium signals from Keystone LN from non-starved, N=5 and starved N=5 in the ex-vivo preparation dissected brain. The box plot in the bottom right panel compares normalized calcium signal intensity between non-starved, gray, and starved, white, samples in the ex-vivo preparation. Mann-Whitney U test B is less than 0.001.To demonstrate our larval calcium imaging approach, we successfully applied it to both in-vivo and ex-vivo preparations of the drosophila larvae. In both preparations, we observed higher keystone LN activity and starved samples compared to non-starved samples. These higher keystone activity was clearly observed in heat maps, showcasing temporal representations of the responses over 60 seconds, and in box plots depicting average signal intensity values.These results are consistent with the recent studies that demonstrate higher olfactory neuron activity in starved animals. As demonstrated in this video, GLUture tissue adhesive significantly reduces motion artifacts in both in-vivo and ex-vivo setups. This method could also be applied to study different kinds of stimuli, for example, external application of odors and internal superfusion of neuromodulators.

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Research

JoVE Journal

Neuroscience

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures  for up to a few days1.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

This video describes in vivo calcium imaging in Drosophila neurons using GCaMP. The featured protocol clip shows how to record changes in GCaMP fluorescence from mushroom body neurons during an olfactory conditioning experiment.

Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

This video demonstrates a method for in vivo calcium imaging using synaptically localized calcium sensors in flies. It outlines the steps for preparing the flies for microscopy, conducting olfactory conditioning, and visualizing odor-induced calcium influx.

In Vivo Calcium Imaging Using Synaptically Localized Calcium Sensors in Drosophila melanogaster

Source: Ishimoto, H. et. al., Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila. J. Vis. Exp. (2018) This video demonstrates calcium imaging to study Drosophila brain responses to neuropeptides. Neurons expressing a fluorescent calcium indicator reveal intracellular calcium dynamics and functional responses to signaling molecules.

Ex Vivo Calcium Imaging to Study Drosophila Brain Responses to Neuropeptides

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

Learn calcium imaging protocols for Drosophila larval olfactory neurons using GLUture tissue adhesive immobilization. This method enables stable in-vivo and ex-vivo experiments with custom R scripts for quantitative analysis.

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

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