Name: Dissektion af tredje Instar Drosophila larve til hjernemetabolit billeddannelse
Uploaded: 2023-10-27T12:00:00
Description: Watch this Scientific Journal Video about Dissection of Third Instar Drosophila Larva for Brain Metabolite Imaging at JoVE.com

dissection of third instar drosophila larva for brain metabolite imaging

Overvågning af hjernemetabolittransport i Drosophila ved hjælp af FRET-sensorer

The brain has high energy requirements due to the high cost of restoring ion gradients in neurons caused by neuronal electric signal generation and transmission, as well as synaptic transmission1,2. This high energy demand has long been thought to be met by the continuous oxidation of glucose to produce ATP3. Specific transporters at the blood-brain barrier transfer the glucose in the blood to the brain. Constant glycemic levels ensure that the brain receives a steady supply of glucose4. Interestingly, growing experimental evidence suggests that molecules derived from glucose metabolism, such as lactate and pyruvate, play an important role in the brain cells&#39; energy production5,6. However, there is still some debate about how important these molecules are for energy production and which cells in the brain produce or use them7,8. The lack of appropriate molecular tools with the high temporal and spatial resolution required for this task is a significant issue that has prevented this controversy from being completely resolved.
The development and application of several engineered fluorescent metabolic sensors have resulted in a remarkable increase in our understanding of where and how metabolites are produced and used, as well as how the metabolic fluxes occur during basal and high neuronal activity9. Genetically encoded metabolic sensors based on F&#246;rster resonance energy transfer (FRET) microscopy, such as ATeam (ATP), FLII12Pglu700&#181;&#948;6 (glucose), Laconic (lactate), and Pyronic (pyruvate), have contributed to our understanding of brain energy metabolism10,11,12,13. However, due to the high costs and sophisticated equipment required to conduct experiments on live animals or tissues, results in vertebrate models are still primarily limited to cell cultures (glial cells and neurons).
The emerging use of the Drosophila model to express these sensors has revealed that key metabolic features are conserved across species and their function can be easily addressed with this tool. More importantly, the Drosophila model has shed light on how glucose and lactate/pyruvate are transported and metabolized in the fly brain, the link between monocarboxylate consumption and memory formation, and the remarkable demonstration of how increases in neural activity and metabolic flux overlap14,15,16,17. The method presented here for measuring monocarboxylate, glucose, and ATP levels using genetically encoded FRET sensors expressed in the larval brain allows researchers to learn more about how the brain of Drosophila uses energy, which can be applied to the brains of other animals.
We show that this method is effective for detecting lactate and glucose in glial cells and neurons, and that a monocarboxylate transporter (Chaski) is involved in lactate import into glial cells. We also demonstrate a simple method for studying metabolite changes during increased neuronal activity, which can be easily induced by bath application of a GABAA receptor antagonist. Finally, we show that this methodology can be used to measure monocarboxylate and glucose transport in other metabolically significant tissues, such as fat bodies.

Forfatter Spotlight: Fremskridt inden for forskning i hjernens energimetabolisme ved hjælp af Drosophila-modellen

Visualisering af monocarboxylater og andre relevante metabolitter i ex vivo Drosophila-larvehjernen ved hjælp af genetisk kodede sensorer

The brain is a tissue with a high energy consumption that mostly uses glucose as fuel. On the other hand, experimental data suggests that other metabolites like ketone bodies and monocarboxylates might be useful as energy sources. However, there is a still debate on which brain cells are the producers and primary consumers.Lactate has been demonstrated to be a relevant metabolite in vertebrates to drive brain activity in vivo. Our group and others have shown in Drosophila the importance of monocarboxylate molecules to fuel the high energy requirements in neurons, as well as the need of glial-derived lactate and ketone bodies in memory formation. We have described monocarboxylate transporters in the Drosophila brain and determined that the transfer of lactate from glial cells to neurons is necessary to maintain the neural activity during high-demand periods.The main advantage is to have the possibility to determine the intracellular dynamic of glucose and its metabolites, such as lactate, pyruvate, and ATB in glial cells and neurons during basal and high neural activity. Additionally, to measure the transfer of these molecules in other living tissues, such as the fat bodies. The use of the Drosophila as in vivo model for the study of brain energy metabolism using a simple setup.This will allow the modeling of metabolic diseases or to better understand the energy management in the normal brain or during the development of neurodegenerative diseases.

Watch this Scientific Journal Video about Dissection of Third Instar Drosophila Larva for Brain Metabolite Imaging at JoVE.com

Dissektion af tredje Instar Drosophila larve til hjernemetabolit billeddannelse

Til at begynde med skal du placere 25 millimeter glasdæksel glider ind i brøndene på en seks-brønds cellekulturplade. Placer en dråbe polylysin i midten af hver dæksel. Når inkubationen er afsluttet, vaskes hvert dæksel tre gange med destilleret vand.Vask derefter dækslet glider to gange med en calciumfri saltopløsning. Installer derefter dækslerne i et optagekammer og fyld det med calciumfrit saltvand. Saml de vandrende tredje instar larver fra det ønskede genetiske kryds af drosophila.Overfør larverne til en skål godt, vask derefter larverne tre gange med destilleret vand. Placer larverne i en anden glasdissektionsskål indeholdende 750 mikroliter iskold, calciumfri saltopløsning. Placer nu en larver under et stereomikroskop.Med et par tang skal du lave et tværgående snit over bagsiden af maven. Skub kæben med tangen for at vende larverne indefra og ud. Overhold den ventrale nerveledning ved siden af kæben.Fjern forsigtigt de imaginære diske og resterende hjernevæv. Derefter adskilles den ventrale nerveledning med den centrale hjerne og optiske lobes fra resten af vævet. Den ventrale nerveledning overføres til optagekammeret, der indeholder calciumfri saltopløsning.For at undgå interferens skal du fastgøre de resterende nerver til bunden af dækslet med tang. For at udføre eksperimenter på fedtlegemer skal du fortsætte med at isolere fedtvævet fra den udviste larve. Anbring de isolerede fedtlegemer, der udtrykker båndsensorerne, i optagekammeret.For at erhverve billeder af væv, der udtrykker sensorer, skal du tænde mikroskopets belysningssystem. Anbring optagelseskammeret indeholdende ventrale nerveledninger eller fedtlegemer på scenen af et fluorescerende mikroskop. For at se GCaMP6f-fluorescensen skal du indstille excitationsbølgelængden til 488 nanometer.For metabolitter såsom lakoniske, pyroniske, ATP- eller glukosesensorer indstilles excitationen til 440 nanometer. Indstil nu billedoptagelsen med jævne mellemrum for GCaMP6f og metabolitsensoren. Placer målet for nedsænkning i vandet, og sørg for, at det forbliver nedsænket.Tilslut derefter optagekammeret til et perfusionssystem. Brug en peristaltisk pumpe med lav flux til at ekstrahere væsken fra kammeret og opretholde en konstant strøm på tre milliliter pr. Minut. Placer opløsningen indeholdende rør 25 centimeter over mikroskoptrinnet.Før enhver stimulering opnås en stabil fluorescensbaseline ved at lade optageopløsningen flyde i fem til 10 minutter. Udskift nu flowopløsningen med stimuleringsopløsningen indeholdende glucose, pyruvat eller lactat i fem minutter. Tag billederne af den stimulerede ventrale nerveledning, der blev udsat for 80 mikromolær picrotoxin for at vurdere neuronal aktivitet.Lakoniske sensorer i både gliaceller og motorneuroner reagerer på et millimolært lactat med en lignende hastighed ved starten af pulsen. Imidlertid når motorneuronerne en højere stigning over basislinjen i løbet af fem minutters puls. Glukosesensorens signal steg med en lignende hastighed i gliacellerne og neuronerne, når de blev udsat for fem millimolær glukose.Under glukosepulsen øges signalet i neuroner imidlertid mere end i gliaceller. Knocking out Chaski transportør reducerede laktattransport i gliaceller. Picrotoxin-inducerede stigninger i neuronal aktivitet resulterede i et forbigående fald i ATP-niveauer i soma af motorneuroner.Den lakoniske sensor blev observeret at være godt udtrykt i fedtlegemer. Øget fluorescens af glukosesensorerne blev set, når fedtlegemerne blev udsat for stigende glukosekoncentration. Forhøjede koncentrationer af laktat og pyruvat resulterede i en proportional stigning i lakonisk og pyronisk fluorescens.