期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 136, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/57701
关键词
Neuroscience; Issue 136; Calcium imaging; GCaMP; brain explant; peptide hormone; G-protein-coupled receptor; Drosophila
资金
- JSPS [15K07147, 17K07419]
- Inamori Foundation Research Grant
- Program of the Joint Usage/Research Center for Developmental Medicine, at the Institute of Molecular Embryology and Genetics, Kumamoto University
- Grants-in-Aid for Scientific Research [17K07419, 15K07147] Funding Source: KAKEN
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 Ca(2+)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.
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