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...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:1033px;" width="1032">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Tungsten rod</td>
			<td style="width:327px;">A-M systems, Sequim, WA, USA</td>
			<td style="width:165px;">717000</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blu Tack</td>
			<td>Bostik, Paris, France</td>
			<td>3049100</td>
			<td>putty-like reusable adhesives</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Watch glass, square, 1 5/8 in</td>
			<td>Carolina Biological Supply Company, Burlington, NC, USA</td>
			<td>742300</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>TAKARA Bio Inc., Kusatsu, Shiga, Japan</td>
			<td>T900</td>
			<td>Phosphated buffered salts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CCHa2</td>
			<td>SCRUM Inc., Tokyo, Japan</td>
			<td></td>
			<td>Custum-synthesized peptide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ghrerin</td>
			<td>Peptide institute Inc., Osaka, Japan</td>
			<td>4372-s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nociceptin</td>
			<td>Peptide institute Inc., Osaka, Japan</td>
			<td>4313-v</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Axio Imager A2</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td>Axio Imager A2</td>
			<td>fluorescence microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">P-725 PIFOC objective scanner with long travel range</td>
			<td>Physik Instrumente GmbH &#38; Co. KG, Karlsruhe, Germany</td>
			<td>N/A</td>
			<td>piezoelectric-activated lens mover</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">ImagEM C9100-13</td>
			<td>Hamamatsu Photonics, Sizuoka, Japan</td>
			<td>C9100-13</td>
			<td>EM-CCD camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OBIS 488 nm LS 60 mW</td>
			<td>Coherent, Santa Clara, CA, USA</td>
			<td>1178770</td>
			<td>488-nm laser</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">&#181;Manager</td>
			<td>Open Imaging, Inc.</td>
			<td>N/A</td>
			<td>https://micro-manager.org</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ImageJ</td>
			<td>U. S. National Institutes of Health</td>
			<td>N/A</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TurboReg</td>
			<td>Philippe Th&#233;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 ...

This method can help answer key questions in the endocrinology field, as well as the neuroscience field, such as our research to review brains responses to endocrine signals. The main advantage of this technique is that you can examine direct effects of endocrine signals on the brain separated from other tissues. To begin this procedure, use forceps to scratch the bottom center of a plastic culture dish to create a dent for easier mounting of the ventral ganglion of the larval brain after dissection.With a toothpick, place a small drop of superglue on either side of the dent, and attach a tungsten rod to the glue. Then, using a small piece of putty-like reusable adhesive, make a circular wall surrounding the dent. To dissect the larval brain, 90 to 120 hours after egg laying collect larvae and wash them at least three times with distilled water to remove adherent food debris.Next, place a larva on a square one and a half inch watch glass filled with ice cold PBS. Use a pair of forceps to gently grab the middle part of the larva. Use another pair of forceps to grab and pull the mouth hooks gently to separate the anterior part of the larva containing the brain from the rest of the body.Then, hold the anterior tip with the forceps and turn the larva inside out. Remove the extraneous tissues attached to the brain, such as imaginal discs, fat bodies, and ring gland. Gently separate the brain from the mouth parts.Afterwards, apply 200 microliters of PBS to the inside of the adhesive ring prepared earlier in the imaging chamber. Gently suck the dissected brain with PBS into a Pasteur pipette, and transfer it to the imaging chamber. In the imaging chamber, grab the muscle fibers extending from the ventral ganglia and insert the brain gently into the dent underneath the tungsten wire.Then, pull the tungsten wire slightly up to place the brain at the correct position for imaging. To acquire calcium fluorescence images, place the imaging chamber containing the brain explant under the microscope. Lower the objective lens until it touches the PBS, and under bright field illumination position the brain and bring it into focus.Switch to fluorescent light and adjust the focus on the GCaMP success labeled cells. Start the acquisition at 250 ms/frame, at a resolution of 512 x 512 pixels in water cooled mode. Then, adjust the exposure time to obtain the fluorescence values within the CCD camera dynamic range, but not lower than 1000 arbitrary units with 16 bit images.Once the imaging parameters are determined, take the images for one minute before peptide administration to detect baseline signal intensities. Subsequently, apply the test peptide directly, by pipetting 100 microliters of the prepared peptide solution into the larval bath. Record the GCaMP success emission for a few minutes.To analyze the data, open the analysis software and use the first frame which was before peptide application as a reference image. Then, select Plugins, TurboReg. Choose the serial image file as the Source, and the reference image as the Target.Next, check Rigid Body and Accurate for the processing method and quality, respectively. Subsequently, click Batch to start image processing. Select multiple regions of interest by using Analyze, Tools, ROI Manager, in image tray.Then, measure the pixel intensity by clicking More, Multi Measure, OK, in the ROI Manager window. In this experiment, Wild type brain was exposed to CCHa2, Ghrelin, and Nociceptin, whereas CCHa2 Receptor mutant brain was exposed to CCHa2. GCaMP6 signals in insulin producing cells were detected by confocal microscopy at 250 ms/frame, and here are the still images of selected time points.The ratio of the change of ROI intensity to baseline signal intensity at each time point was plotted. ROIs where set on cell bodies that were detected in the same focal plane. The solid lines indicate the mean of five to 10 samples, and the dotted lines mark upper and lower limits of the standard error of the mean.The shaded areas indicate the variation of the signals in the experiments. Once mastered, this technique can be done in one hour, if it is preformed properly. While attempting this procedure, it's important to remember to prepare the brain sample quickly, to avoid damage to it.This procedure can be combined with genetics in order to answer additional questions like on receptor specificity. The system used in this protocol is easy to prepare and reusable. Thus, this protocol will be useful for, like in screening.After its development, this technique paved the way for researchers in the field of endocrinology and neuroscience to explore hormonal networks that control brain function in Drosophila.

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9.1K visualizações.  Nagoya University. This method can help answer key questions in the endocrinology field, as well as the neuroscience field, such as our research to review brains responses to endocrine signals. The main advantage of this technique is that you can examine direct effects of endocrine signals on the brain separated from other tissues. To begin this procedure, use forceps to scratch the bottom center of a plastic culture dish to create a dent for easier mounting of the ventral ganglion of the larval brain after dis...