Name: Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila (Video) | JoVE Journal
Uploaded: 2018-06-02T14:30:00.000+00:00
Duration: 6 min 49 s
Description: Learn ex vivo calcium imaging to visualize brain responses to endocrine signaling in Drosophila. This method enables direct examination of neuronal activation by hormones using GCaMP6 fluorescence.

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5 minutes

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>

<ol><li>Morton, G. J., Schwartz, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leptin+and+the+central+nervous+system+control+of+glucose+metabolism%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Leptin and the central nervous system control of glucose metabolism.</a> Physiological Reviews. 91 (2), 389-411 (2011).</li>
<li>Stanley, S., Wynne, K., McGowan, B., Bloom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormonal+regulation+of+food+intake%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Hormonal regulation of food intake.</a> Physiological Reviews. 85 (4), 1131-1158 (2005).</li>
<li>Goodman, W. G., Cusson, M. The juvenile hormones. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/B9780123847492100081">Insect Endocrinology</a>. Gilbert, L. I. , Academic Press. 310-365 (2012).</li>
<li>Lafont, R., Dauphin-Villemant, C., Warren, J. T., Rees, H. Ecdysteroid chemistry and biochemistry. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/B9780123847492100044">Insect Endocrinology</a>. Gilbert, L. I. , Academic Press. 106-176 (2012).</li>
<li>Smith, W., Rybczynski, R. Protothoracicotropic hormone. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/B9780123847492100019">Insect Endocrinology</a>. Gilbert, L. I. , Academic Press. 1-62 (2012).</li>
<li>Claeys, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insect+neuropeptide+and+peptide+hormone+receptors%3A+current+knowledge+and+future+directions%5BTitle%5D)+AND+%22Vitamins+and+Hormones%22%5BJournal%5D)">Insect neuropeptide and peptide hormone receptors: current knowledge and future directions.</a> Vitamins and Hormones. 73, 217-282 (2005).</li>
<li>Gade, G., Goldsworthy, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insect+peptide+hormones%3A+a+selective+review+of+their+physiology+and+potential+application+for+pest+control%5BTitle%5D)+AND+%22Pest+Management+Science%22%5BJournal%5D)">Insect peptide hormones: a selective review of their physiology and potential application for pest control.</a> Pest Management Science. 59 (10), 1063-1075 (2003).</li>
<li>Adams, M. D., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/10731132">The genome sequence of Drosophila melanogaster.</a> Science. 287 (5461), 2185-2195 (2000).</li>
<li>Park, D., Veenstra, J. A., Park, J. H., Taghert, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+peptidergic+cells+in+Drosophila%3A+where+DIMM+fits+in%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mapping peptidergic cells in Drosophila: where DIMM fits in.</a> PLoS One. 3 (3), 1896(2008).</li>
<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>
<li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+minimal+hemolymph-like+solution%2C+HL3.1%2C+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae%5BTitle%5D)+AND+%22Journal+of+Neurogenetics%22%5BJournal%5D)">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> Journal of Neurogenetics. 18 (2), 377-402 (2004).</li>
</ol>

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

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, ...

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.

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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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 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

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.

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

Learn ex vivo calcium imaging to visualize brain responses to endocrine signaling in Drosophila. This method enables direct examination of neuronal activation by hormones using GCaMP6 fluorescence.

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

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