This work was supported by Grants-in-Aid for Scientific Research (15K07147, 17K07419) from JSPS (to Hiroshi Ishimoto, Hiroko Sano), an Inamori Foundation Research Grant (to HI), and the Program of the Joint Usage/Research Center for Developmental Medicine, at the Institute of Molecular Embryology and Genetics, Kumamoto University (to HS). We thank Dr. Azusa Kamikouchi and Mr. Daichi Yamada for their help in using the imaging system.

1. Preparation of Larval Brain Explants
<ol>
	<li>Make an imaging chamber. 
	NOTE: Stable recording requires that the brain explants be tethered. Here, a low-cost, hand-made imaging chamber is used in the Ca2+ imaging system.
	<ol>
		<li>Using forceps, scratch the bottom center of a plastic culture dish (35 mm x 10 mm) to create a dent for the ventral ganglion of the larval brain making mounting easier (step 1.3, Figure 1).</li>
		<li>With a toothpick, place a small drop of superglue on either side of the dent and attach a tungsten rod (0.125 mm diameter, 6 to 7 mm length) to the glue.</li>
		<li>Using a small piece of putty-like reusable adhesive, make a circular wall (10 mm in diameter and 3 mm high) surrounding the dent (Figure 1). 
		NOTE: The space inside the wall will be later filled with phosphate buffered saline (PBS; 0.14 M NaCl, 0.0027 M KCl, 0.01 M PO43-, pH 7.4 &#177; 0.05 at 25 &#176;C).</li>
	</ol>
	</li>
	<li>Animal preparation 
	NOTE: For calcium imaging, a calcium indicator, GCaMP6s, is expressed in the cell type of interest, which is commonly achieved using combinations of tissue-specific GAL4 and UAS-GCaMP6s. To ensure that the signal is mediated through the candidate receptor, GCaMP6s is also expressed in a mutant defective in the receptor.
	<ol>
		<li>When experimental comparisons for different genotypes such as wild type and mutants are required, age match test larvae to avoid GCaMP6 expression level variations and physiological differences due to developmental stages.</li>
		<li>Keep parental flies (30 flies for each sex) in a culture vial containing standard fly food for 8 h to allow egg-laying on the surface of the food. Culture larvae in mass under a 12-h light-dark cycle at 25 &#176;C and 60-70 % relative humidity.</li>
	</ol>
	</li>
	<li>Dissection of the larval brain
	<ol>
		<li>At 90-120 h after egg laying (AEL), collect larvae and wash them at least 3 times with distilled water to remove adherent food debris.</li>
		<li>Place a larva on a square 1.5-inch watch glass filled with ice cold PBS. 
		NOTE: The next two steps are carried out in this watch glass under the dissecting microscope.</li>
		<li>Use forceps to gently grab the middle part of the larva. Use another forceps to grab the mouth hooks and pull the mouth hooks gently to separate the anterior part of the larva containing the brain from the rest of the body. 
		NOTE: Alternatively, surgical scissors can be used.</li>
		<li>Hold the anterior tip by forceps and turn the larva inside out. Remove extraneous tissue attached to the brain, such as imaginal disks, fat bodies, and ring gland. Gently separate the brain from the mouthparts.</li>
		<li>Apply 200 &#181;L of PBS to the inside of the ring made of the putty-like reusable adhesive in the imaging chamber. Gently suck the dissected brain with PBS into a Pasteur pipette, and then transfer it into the imaging chamber.</li>
	</ol>
	</li>
	<li>Setting the brain into the imaging chamber
	<ol>
		<li>Use forceps to grab muscle fibers extending from the ventral ganglia. Insert the brain gently into the dent underneath the tungsten wire.</li>
		<li>Using another forceps, pull the tungsten wire slightly up to place the brain at the correct position for imaging (Figure 1).</li>
	</ol>
	</li>
</ol>
2. Acquisition of Ca2+ Fluorescence Images
NOTE: Brain explants immersed in PBS are imaged using a fluorescence microscope equipped with a 20X water-immersion objective lens (N.A. = 0.5). PBS does not activate cells in the brain. If Z-axis images are needed, the objective lens is mounted with a piezoelectric-activated lens mover. A spinning disc confocal head is used to enhance the time resolution. For low-light imaging, an electron multiplying charge-coupled device camera is mounted on the microscope. The imaging of GCaMP6s fluorescence (excitation/emission: 488/509 nm) requires a 488-nm laser for excitation with a dichroic beam splitter and an emission filter (e.g., 528 &#177; 38 nm band bandpass filter).
<ol>
	<li>Place the imaging chamber containing the brain explant under the microscope.</li>
	<li>Lower the objective lens until it touches the PBS. Under bright-field illumination, position the brain and bring it into focus. Switch to fluorescent light and adjust the focus on the GCaMP6s-labeled cells. 
	NOTE: The basal green fluorescence of the GCaMP6s should be visible.</li>
	<li>Start acquisition at 250 ms/frame at a resolution of 512 &#215; 512 pixels in water-cooled mode using the appropriate acquisition software (e.g., &#181;Manager). Adjust the exposure time to obtain fluorescence values within the dynamic range of the CCD camera, but not lower than 1000 (arbitrary units), with 16-bit images (dynamic range 0-65,535). 
	NOTE: A low exposure time should be used to minimize photo-bleaching of the GCaMP6s. Exposure time should be optimized in each experiment as it depends on the expression levels of GCaMP6 and the sensitivity of the detection system. It was 100 ms in our experiment using dilp2&#62;GCaMP6s. When z-sections are required for a given imaging depth, several z-section images can be acquired depending on the exposure time and frame intervals. If the camera has enough spatial resolution, the binning size (number of registers on the chip that will be binned into one digital pixel) should be increased to reduce the exposure time.</li>
	<li>Once imaging parameters are determined, take images for 1 min before peptide administration to detect baseline signal intensities (F0).</li>
	<li>Apply the test peptide; for this, dissolve 100 &#181;L of peptide solution in PBS and pipette it directly into the larval bath to yield an optimal concentration. 
	NOTE: The peptide solution should be injected slowly (e.g., flow rate 20 &#181;L/s) into the bath solution using a pipette. Unrelated synthetic peptide dissolved in PBS is used as a negative control.</li>
	<li>Record the GCaMP6s emission for a few minutes.</li>
</ol>
3. Data Analysis
Note: Imaging data are analyzed offline with the ImageJ. During the time-lapse imaging, pipetting causes the larval brain to move. Therefore, positional aberrations of the serial images should be corrected before analysis. The correction can be performed using an ImageJ plug-in, TurboReg as follows.
<ol>
	<li>Open the analysis software and use the first frame (before peptide application) as a reference image.</li>
	<li>Select Plugins | Registration | TurboReg.</li>
	<li>Choose the serial image file as the Source and the reference image as the Target.</li>
	<li>Check Rigid Body (parallel displacement and rotation of images) and Accurate (&#34;Fast&#34; is coarse but fast analysis instead) for the processing method and quality, respectively.</li>
	<li>Click Batch to start the image processing.</li>
	<li>Select multiple regions of interest (ROIs) by using Analyze | Tools | ROI Manager in ImageJ.</li>
	<li>Measure the pixel intensity by clicking More | Multi Measure | OK in the ROI Manager window.</li>
	<li>Calculate &#916;F/F0 = (F - F0)/F0, where F is the ROI intensity at the time of the stimulus, and F0 is the intensity at a time window immediately before a particular experimental event (e.g., 30 frames preceding the stimulus onset).</li>
	<li>Repeat the experiment using multiple brain samples, conducting statistical processing to obtain the mean and standard error of the mean (SEM) at each time point.</li>
</ol>

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<li>Berridge, M. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/8381210">Inositol trisphosphate and calcium signalling.</a> Nature. 361 (6410), 315-325 (1993).</li>
<li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high+signal-to-noise+Ca%282%2B%29+probe+composed+of+a+single+green+fluorescent+protein%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nature Biotechnology. 19 (2), 137-141 (2001).</li>
<li>Rulifson, E. J., Kim, S. K., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ablation+of+insulin-producing+neurons+in+flies%3A+growth+and+diabetic+phenotypes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes.</a> Science. 296 (5570), 1118-1120 (2002).</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>Nassel, D. R., Kubrak, O. I., Liu, Y., Luo, J., Lushchak, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+that+regulate+insulin+producing+cells+and+their+output+in+Drosophila%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">Factors that regulate insulin producing cells and their output in Drosophila.</a> Frontiers in Physiology. 4, 252(2013).</li>
<li>Sano, H., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/26020940">The Nutrient-Responsive Hormone CCHamide-2 Controls Growth by Regulating Insulin-like Peptides in the Brain of Drosophila melanogaster.</a> PLoS Genetics. 11 (5), 1005209(2015).</li>
<li>Tsao, C. K., Ku, H. Y., Lee, Y. M., Huang, Y. F., Sun, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long+Term+Ex+Vivo+Culture+and+Live+Imaging+of+Drosophila+Larval+Imaginal+Discs%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Long Term Ex Vivo Culture and Live Imaging of Drosophila Larval Imaginal Discs.</a> PLoS One. 11 (9), 0163744(2016).</li>
<li>Sabado, V. aN., Nagoshi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+Resolution+Fluorescence+Live+Imaging+of+Drosophila+Circadian+Clocks+in+Larval+Brain+Culture%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture.</a> Journal of Visualized Experiments. 131, e57015(2018).</li>
<li>Apostolopoulou, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caffeine+Taste+Signaling+in+Drosophila+Larvae%5BTitle%5D)+AND+%22Frontiers+in+Cellular+Neuroscience%22%5BJournal%5D)">Caffeine Taste Signaling in Drosophila Larvae.</a> Frontiers in Cellular Neuroscience. 10, 193(2016).</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>Nikolaev, V. O., Bunemann, M., Hein, L., Hannawacker, A., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+single+chain+cAMP+sensors+for+receptor-induced+signal+propagation%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Novel single chain cAMP sensors for receptor-induced signal propagation.</a> Journal of Biological Chemistry. 279 (36), 37215-37218 (2004).</li>
<li>Sankaranarayanan, S., De Angelis, D., Rothman, J. E., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+pHluorins+for+optical+measurements+of+presynaptic+activity%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">The use of pHluorins for optical measurements of presynaptic activity.</a> Biophysical Journal. 79 (4), 2199-2208 (2000).</li>
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The authors have nothing to disclose.

The Ca2+ imaging method described here is a useful system for testing the function of hormones in the brain. In vivo, the brain receives hormones from various endocrine organs. In addition, the brain constantly perceives ascending sensory information, which causes spontaneous neuronal activation. This ex vivo system eliminates such noise and yields a better signal/noise ratio than in vivo imaging. In this system, molecules can be tested singly or in combination. In addition to peptid...

Organ-to-organ communication is an evolutionarily conserved strategy for maintaining homeostasis to cope with environmental changes. In humans, a variety of hormones arereleased from the endocrine glands into the circulation. Many of these hormones target the hypothalamus of the brain, which regulates metabolic processes and fundamental behaviors such as feeding1,2. Many hormones have been discovered using mammalian models. However, the mechanisms of their action, especially the interorgan networks in which they participate, remain largely unclear.
Drosophila melanogaster has emerged as a useful model for studying organ-to-organ communication. In insects, many physiological processes are controlled by hormones. Early studies focusing on growth and metamorphosis used large insects. In these studies, the removal or transplantation of specific organs predicted the existence of inter-organ signaling molecules; later, Juvenile hormone (JH), Prothoracicotropic hormone (PTTH), and Ecdysone were biochemically purified3,4,5. A large family of intercellular signaling peptides is thought to be involved in various physiological events during the insect life cycle6,7. Most of these peptides act on G-protein-coupled receptors (GPCRs), although the specific GPCRs were initially difficult to identify using conventional approaches. The publication of the Drosophila whole genome sequence8&#160;was the breakthrough that enabled the identification of Drosophila bioactive peptides based on their homology to those found in other insects. In addition, the receptors for several peptides were identified from GPCRs predicted in the genome using cell-culture-based GPCR-ligand binding assays. Next, expression analyses predicted the organ-to-organ pathways elicited by these peptides and receptors. Notably, many of the putative peptide receptors are expressed in the brain, suggesting that the brain is a major target of peptide hormones9. Moreover, the advanced genetic tools in Drosophila have contributed to the identification of physiological roles of peptide-GPCR combinations. For instance, the GAL4 and LexA-based binary transcriptional systems enable gene knockdown or overexpression in a spatially and temporally controlled manner. GAL4, a transcription factor identified in yeast, binds to a specific cis-regulatory sequence called Upstream Activation Sequence (UAS). In the GAL4/UAS system, the driver line provides tissue-specific or stage-specific GAL4 expression and the responder line carries UAS upstream of the gene of interest or the construct to drive shRNA expression. LexA/LexAop system is based on a similar mechanism. Phenotypic analyses of tissue-specific peptide-knockdown and tissue-specific GPCR-knockdown animals can reveal information about the peptide-GPCR signaling&#39;s mode and site of action. However, the conclusions that can be reached solely by genetic data are limited. On the other hand, once the putative target of a particular peptide hormone is narrowed down to a tissue or cell type, ex vivo calcium imaging in organ explants can be used to elucidate the organ-to-organ communication mediated by peptide-GPCR signaling. Upon the activation of a Gq-coupled GPCR, the intracellular Ca2+ concentration is increased due to the release of Ca2+ from the ER10. In the brain, neural activation also elevates the intracellular Ca2+ levels. Such Ca2+ increases can be detected by a calcium sensor, GCaMP, which undergoes conformational changes in the presence of Ca2+ resulting in fluorescence emission11.
In this article, a calcium imaging method using Drosophila brain explants is described. To test the ability of a peptide to activate specific neurons, a test peptide is applied to the GCaMP-expressing brain explant, and fluorescence changes are monitored by confocal microscopy. The involvement of the GPCR is then confirmed by performing the same assay using a mutant brain lacking the GPCR. This combination of imaging and genetics provides precise information about organ-to-organ communication by peptides-GPCRs. Possible modifications and applications of this protocol are also discussed.

<table><tbody><tr><td>Tungsten rod</td><td>A-M systems, Sequim, WA, USA</td><td>717000</td><td></td></tr><tr><td>Blu Tack</td><td>Bostik, Paris, France</td><td>3049100</td><td>putty-like reusable adhesives</td></tr><tr><td>Watch glass, square, 1 5/8 in</td><td>Carolina Biological Supply Company, Burlington, NC, USA</td><td>742300</td><td></td></tr><tr><td>PBS</td><td>TAKARA Bio Inc., Kusatsu, Shiga, Japan</td><td>T900</td><td>Phosphated buffered salts</td></tr><tr><td>CCHa2</td><td>SCRUM Inc., Tokyo, Japan</td><td></td><td>Custum-synthesized peptide</td></tr><tr><td>Ghrerin</td><td>Peptide institute Inc., Osaka, Japan</td><td>4372-s</td><td></td></tr><tr><td>Nociceptin</td><td>Peptide institute Inc., Osaka, Japan</td><td>4313-v</td><td></td></tr><tr><td>Axio Imager A2</td><td>Carl Zeiss, Oberkochen, Germany</td><td>Axio Imager A2</td><td>fluorescence microscope</td></tr><tr><td>Objective W N-Achroplan 20x/0.5 M27</td><td>Carl Zeiss, Oberkochen, Germany</td><td>420957-9900-000</td><td>water-immersion objective lens</td></tr><tr><td>P-725 PIFOC objective scanner with long travel range</td><td>Physik Instrumente GmbH &amp;amp; Co. KG, Karlsruhe, Germany</td><td>N/A</td><td>piezoelectric-activated lens mover</td></tr><tr><td>Confocal Scanner Unit CSU-W1</td><td>Yokogawa Electric Corporation, Tokyo, Japan</td><td>CSU-W1</td><td>spinning disc confocal head</td></tr><tr><td>ImagEM C9100-13</td><td>Hamamatsu Photonics, Sizuoka, Japan</td><td>C9100-13</td><td>EM-CCD camera</td></tr><tr><td>OBIS 488 nm LS 60 mW</td><td>Coherent, Santa Clara, CA, USA</td><td>1178770</td><td>488-nm laser</td></tr><tr><td>488/568/647 nm Yokogawa dichroic beamsplitter</td><td>Semrock, Rochester, NY, USA</td><td>Di01-T405/488/568/647-13x15x0.5</td><td>dichroic beam splitter</td></tr><tr><td>528/38 nm BrightLine single-band bandpass filter</td><td>Semrock, Rochester, NY, USA</td><td>FF01-528/38-25</td><td>emission filter</td></tr><tr><td>&amp;micro;Manager</td><td>Open Imaging, Inc.</td><td>N/A</td><td>https://micro-manager.org</td></tr><tr><td>ImageJ</td><td>U. S. National Institutes of Health</td><td>N/A</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>TurboReg</td><td>Philippe Th&amp;eacute;venaz, Biomediccal Imaging Group, Swiss Federal Institute of Technology Lausanne</td><td>N/A</td><td>http://bigwww.epfl.ch/thevenaz/turboreg/</td></tr></tbody></table>

ex vivo calcium imaging for visualizing brain responses to endocrine signaling in drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Using this protocol, the activation of brain insulin-producing cells (IPCs) by the peptide hormone, CCHa2, was examined. IPCs of the wild-type brain were labeled with GCaMP6s using a combination of dilp2-GAL412 (a gift from Dr. Rulifson) and UAS-GCaMP6s13 (a gift from Dr. Kim). Larval brain explants were treated with a synthetic CCHa2 peptide, and the fluorescence from GCaMP6s was recorded in real time. In this experiment, ...

Watch this Scientific Journal Video about Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila at JoVE.com

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

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

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

ex-vivo-calcium-imaging-for-visualizing-brain-responses-to-endocrine

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Neuroscience

9.2K Views.  Nagoya University. Dit witboek beschrijft een protocol voor ex vivo calcium beeldvorming van de hersenen van Drosophila . Bij deze methode, kunnen natuurlijke of synthetische verbindingen worden toegepast op de buffer voor het testen van hun vermogen om bepaalde neuronen in de hersenen activeren.

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

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Developmental Biology

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

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

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

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

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

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

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, 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.

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

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.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Immunology and Infection

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

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

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.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

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.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

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

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

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.

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

Ex Vivo Calcium Imaging to Study Drosophila Brain Responses to Neuropeptides

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/57701/ex-vivo-calcium-imaging-for-visualizing-brain-responses-to-endocrine-signaling-in-drosophila'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Ishimoto, H.&#160;et. al.,<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2018)</a> &#160;This video demonstrates calcium imaging to study&#160;Drosophila brain responses to neuropeptides. Neurons expressing a fluorescent calcium indicator reveal intracellular calcium dynamics and functional responses to signaling molecules.

Source:&#160;Ishimoto, H.&#160;et. al., Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

Bioluminescence Imaging for Studying Calcium Transients in Drosophila Brain Structures

Source:&#160;Lark, A. R.,&#160;et. al.,&#160; In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.&#160;J. ...

Synaptic & Cellular Imaging

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

Source: Hancock, C. E. et al., <a href="https://app.jove.com/v/60288">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster.</a>&#160;J. Vis. Exp. (2019).
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.

1. Transgenic fruit flies,&#160;Drosophila melanogaster

	Cross female virgin and male flies (raised at 25 &#176;C in 60% relative humidity on a 12 h ...

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

Samenvatting

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