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Detection and Quantification of Spreading Depolarization in Drosophila melanogaster Using High-Throughput, Wide-Field Brain Imaging.

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Τίτλος: Detection and Quantification of Spreading Depolarization in Drosophila melanogaster Using High-Throughput, Wide-Field Brain Imaging.
Συγγραφείς: Stein W; School of Biological Sciences, Illinois State University, Normal, Illinois.; These authors contributed equally to this work., Harris AL; Department of Physics, Illinois State University, Normal, Illinois.; These authors contributed equally to this work.
Πηγή: Current protocols [Curr Protoc] 2026 Oct; Vol. 6 (10), pp. e70475.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: John Wiley & Sons Country of Publication: United States NLM ID: 101773894 Publication Model: Print Cited Medium: Internet ISSN: 2691-1299 (Electronic) Linking ISSN: 26911299 NLM ISO Abbreviation: Curr Protoc Subsets: MEDLINE
Imprint Name(s): Original Publication: Hoboken, NJ : John Wiley & Sons, [2021]-
Ιατρικοί όροι (MeSH): Drosophila melanogaster*/physiology , Brain*/diagnostic imaging , Brain*/physiology , Optical Imaging*/methods , Cortical Spreading Depression*/physiology , High-Throughput Screening Assays*/methods, Animals ; Larva
Περίληψη: Spreading depolarization (SD) is a ubiquitous neural phenomenon implicated in mammalian neurological disorders including cerebral ischemia, brain death, stroke, brain injury, and migraine, as well as in insect chill coma. During SD, a breakdown of ion homeostasis and a slowly spreading wave of neural depolarization leads to processing deficits, such as sensory disturbances and paralysis. Our protocols introduce high-throughput techniques using wide-field fluorescence brain imaging in Drosophila melanogaster larvae and adults to detect and quantify the spatial and temporal dynamics of SD and its initiation points. Two SD initiation techniques, rapid cooling and pharmacological manipulation, are detailed. The larval protocols describe how to elicit, detect, and quantify SD in up to 16 animals simultaneously in either intact or dissected preparations. The adult SD protocols describe the detailed steps to elicit, detect, and quantify SD in either fully intact animals or after microdissection that enables high resolution imaging of the brain. Detection, quantification, and analysis techniques are described in a separate protocol. Our protocols establish the fly as a high-throughput model for studying conserved cellular and circuit-level mechanisms of SD initiation and propagation. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: High-throughput SD detection in intact Drosophila larvae with fluorescent calcium reporters Alternate Protocol 1: High-resolution SD imaging in fillet larvae with fluorescent calcium reporters Support Protocol 1: SD imaging in larvae with fluorescent membrane voltage reporters Basic Protocol 2: High-throughput SD detection in adult Drosophila with fluorescent calcium reporters Alternate Protocol 2: High-resolution SD imaging in adult Drosophila with fluorescent calcium reporters Support Protocol 2: SD imaging in adults with fluorescent membrane voltage reporters Basic Protocol 3: High-throughput SD detection analysis in Drosophila with fluorescent calcium reporters Alternate Protocol 3: High-resolution SD imaging analysis in Drosophila with fluorescent calcium reporters Support Protocol 3: SD detection and spread analysis in Drosophila with fluorescent membrane voltage reporters.
(© 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.)
References: J Exp Biol. 2023 Sep 15;226(18):. (PMID: 37665251)
J Insect Physiol. 2008 Apr;54(4):708-18. (PMID: 18342328)
Sci Rep. 2017 Aug 31;7(1):10297. (PMID: 28860653)
Nat Commun. 2023 Nov 25;14(1):7729. (PMID: 38007508)
iScience. 2026 Jun 18;29(7):116389. (PMID: 42375524)
J Neurophysiol. 2016 Sep 1;116(3):1117-27. (PMID: 27334953)
J Cereb Blood Flow Metab. 2004 Oct;24(10):1167-71. (PMID: 15529017)
PLoS Biol. 2019 Feb 15;17(2):e2006732. (PMID: 30768592)
Nat Commun. 2021 Apr 13;12(1):2206. (PMID: 33850125)
Cold Spring Harb Protoc. 2010 Apr;2010(4):pdb.prot5405. (PMID: 20360359)
Stroke. 2025 Apr;56(4):974-986. (PMID: 40013372)
Nat Methods. 2012 Jun 28;9(7):676-82. (PMID: 22743772)
J Insect Physiol. 2010 Aug;56(8):980-90. (PMID: 20361971)
Physiol Rev. 2015 Jul;95(3):953-93. (PMID: 26133935)
Ann Neurol. 2018 Feb;83(2):295-310. (PMID: 29331091)
J Insect Physiol. 2012 Nov;58(11):1511-6. (PMID: 23017334)
Curr Opin Insect Sci. 2023 Aug;58:101055. (PMID: 37201631)
J Cereb Blood Flow Metab. 2026 Jul;46(7):1759-1781. (PMID: 41863251)
J Neurophysiol. 2016 Sep 1;116(3):1152-60. (PMID: 27358319)
Physiol Rev. 2001 Jul;81(3):1065-96. (PMID: 11427692)
Grant Information: Illinois State University
Contributed Indexing: Keywords: calcium imaging; chill coma; migraine; spreading depolarization; spreading depression
Entry Date(s): Date Created: 20261007 Date Completed: 20261007 Latest Revision: 20261008
Update Code: 20261008
DOI: 10.1002/cpz1.70475
PMID: 42841759
Βάση Δεδομένων: MEDLINE
FullText Text:
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PubType: Academic Journal
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  Data: Detection and Quantification of Spreading Depolarization in Drosophila melanogaster Using High-Throughput, Wide-Field Brain Imaging.
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  Data: <searchLink fieldCode="AU" term="%22Stein+W%22">Stein W</searchLink>; School of Biological Sciences, Illinois State University, Normal, Illinois.; These authors contributed equally to this work.<br /><searchLink fieldCode="AU" term="%22Harris+AL%22">Harris AL</searchLink>; Department of Physics, Illinois State University, Normal, Illinois.; These authors contributed equally to this work.
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  Data: <searchLink fieldCode="JN" term="%22101773894%22">Current protocols</searchLink> [Curr Protoc] 2026 Oct; Vol. 6 (10), pp. e70475.
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  Data: <i>Original Publication</i>: Hoboken, NJ : John Wiley & Sons, [2021]-
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  Data: <searchLink fieldCode="MM" term="%22Drosophila+melanogaster%22">Drosophila melanogaster*</searchLink>/<searchLink fieldCode="MM" term="%22Drosophila+melanogaster+physiology%22">physiology</searchLink> <br /><searchLink fieldCode="MM" term="%22Brain%22">Brain*</searchLink>/<searchLink fieldCode="MM" term="%22Brain+diagnostic+imaging%22">diagnostic imaging</searchLink> <br /><searchLink fieldCode="MM" term="%22Brain%22">Brain*</searchLink>/<searchLink fieldCode="MM" term="%22Brain+physiology%22">physiology</searchLink> <br /><searchLink fieldCode="MM" term="%22Optical+Imaging%22">Optical Imaging*</searchLink>/<searchLink fieldCode="MM" term="%22Optical+Imaging+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Cortical+Spreading+Depression%22">Cortical Spreading Depression*</searchLink>/<searchLink fieldCode="MM" term="%22Cortical+Spreading+Depression+physiology%22">physiology</searchLink> <br /><searchLink fieldCode="MM" term="%22High-Throughput+Screening+Assays%22">High-Throughput Screening Assays*</searchLink>/<searchLink fieldCode="MM" term="%22High-Throughput+Screening+Assays+methods%22">methods</searchLink><br /><searchLink fieldCode="MH" term="%22Animals%22">Animals</searchLink> ; <searchLink fieldCode="MH" term="%22Larva%22">Larva</searchLink>
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  Data: Spreading depolarization (SD) is a ubiquitous neural phenomenon implicated in mammalian neurological disorders including cerebral ischemia, brain death, stroke, brain injury, and migraine, as well as in insect chill coma. During SD, a breakdown of ion homeostasis and a slowly spreading wave of neural depolarization leads to processing deficits, such as sensory disturbances and paralysis. Our protocols introduce high-throughput techniques using wide-field fluorescence brain imaging in Drosophila melanogaster larvae and adults to detect and quantify the spatial and temporal dynamics of SD and its initiation points. Two SD initiation techniques, rapid cooling and pharmacological manipulation, are detailed. The larval protocols describe how to elicit, detect, and quantify SD in up to 16 animals simultaneously in either intact or dissected preparations. The adult SD protocols describe the detailed steps to elicit, detect, and quantify SD in either fully intact animals or after microdissection that enables high resolution imaging of the brain. Detection, quantification, and analysis techniques are described in a separate protocol. Our protocols establish the fly as a high-throughput model for studying conserved cellular and circuit-level mechanisms of SD initiation and propagation. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: High-throughput SD detection in intact Drosophila larvae with fluorescent calcium reporters Alternate Protocol 1: High-resolution SD imaging in fillet larvae with fluorescent calcium reporters Support Protocol 1: SD imaging in larvae with fluorescent membrane voltage reporters Basic Protocol 2: High-throughput SD detection in adult Drosophila with fluorescent calcium reporters Alternate Protocol 2: High-resolution SD imaging in adult Drosophila with fluorescent calcium reporters Support Protocol 2: SD imaging in adults with fluorescent membrane voltage reporters Basic Protocol 3: High-throughput SD detection analysis in Drosophila with fluorescent calcium reporters Alternate Protocol 3: High-resolution SD imaging analysis in Drosophila with fluorescent calcium reporters Support Protocol 3: SD detection and spread analysis in Drosophila with fluorescent membrane voltage reporters.<br /> (© 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.)
– Name: Ref
  Label: References
  Group: RefInfo
  Data: J Exp Biol. 2023 Sep 15;226(18):. (PMID: <searchLink fieldCode="PM" term="%2237665251%22">37665251)</searchLink><br />J Insect Physiol. 2008 Apr;54(4):708-18. (PMID: <searchLink fieldCode="PM" term="%2218342328%22">18342328)</searchLink><br />Sci Rep. 2017 Aug 31;7(1):10297. (PMID: <searchLink fieldCode="PM" term="%2228860653%22">28860653)</searchLink><br />Nat Commun. 2023 Nov 25;14(1):7729. (PMID: <searchLink fieldCode="PM" term="%2238007508%22">38007508)</searchLink><br />iScience. 2026 Jun 18;29(7):116389. (PMID: <searchLink fieldCode="PM" term="%2242375524%22">42375524)</searchLink><br />J Neurophysiol. 2016 Sep 1;116(3):1117-27. (PMID: <searchLink fieldCode="PM" term="%2227334953%22">27334953)</searchLink><br />J Cereb Blood Flow Metab. 2004 Oct;24(10):1167-71. (PMID: <searchLink fieldCode="PM" term="%2215529017%22">15529017)</searchLink><br />PLoS Biol. 2019 Feb 15;17(2):e2006732. (PMID: <searchLink fieldCode="PM" term="%2230768592%22">30768592)</searchLink><br />Nat Commun. 2021 Apr 13;12(1):2206. (PMID: <searchLink fieldCode="PM" term="%2233850125%22">33850125)</searchLink><br />Cold Spring Harb Protoc. 2010 Apr;2010(4):pdb.prot5405. (PMID: <searchLink fieldCode="PM" term="%2220360359%22">20360359)</searchLink><br />Stroke. 2025 Apr;56(4):974-986. (PMID: <searchLink fieldCode="PM" term="%2240013372%22">40013372)</searchLink><br />Nat Methods. 2012 Jun 28;9(7):676-82. (PMID: <searchLink fieldCode="PM" term="%2222743772%22">22743772)</searchLink><br />J Insect Physiol. 2010 Aug;56(8):980-90. (PMID: <searchLink fieldCode="PM" term="%2220361971%22">20361971)</searchLink><br />Physiol Rev. 2015 Jul;95(3):953-93. (PMID: <searchLink fieldCode="PM" term="%2226133935%22">26133935)</searchLink><br />Ann Neurol. 2018 Feb;83(2):295-310. (PMID: <searchLink fieldCode="PM" term="%2229331091%22">29331091)</searchLink><br />J Insect Physiol. 2012 Nov;58(11):1511-6. (PMID: <searchLink fieldCode="PM" term="%2223017334%22">23017334)</searchLink><br />Curr Opin Insect Sci. 2023 Aug;58:101055. (PMID: <searchLink fieldCode="PM" term="%2237201631%22">37201631)</searchLink><br />J Cereb Blood Flow Metab. 2026 Jul;46(7):1759-1781. (PMID: <searchLink fieldCode="PM" term="%2241863251%22">41863251)</searchLink><br />J Neurophysiol. 2016 Sep 1;116(3):1152-60. (PMID: <searchLink fieldCode="PM" term="%2227358319%22">27358319)</searchLink><br />Physiol Rev. 2001 Jul;81(3):1065-96. (PMID: <searchLink fieldCode="PM" term="%2211427692%22">11427692)</searchLink>
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