culturing drosophila third instar larvae for brain metabolite studies

Monitorización del transporte de metabolitos cerebrales en Drosophila mediante sensores FRET

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.

Autor destacado: Avances en la investigación del metabolismo energético cerebral utilizando el modelo de Drosophila

Visualización de monocarboxilatos y otros metabolitos relevantes en el cerebro de larvas de Drosophila ex vivo utilizando sensores codificados genéticamente

Watch this Scientific Journal Video about Culturing Drosophila Third Instar Larvae for Brain Metabolite Studies at JoVE.com

Cultivo de larvas de Drosophila en el tercer estadio para estudios de metabolitos cerebrales

Para comenzar, coloque una placa de Petri de 60 milímetros cubierta con 1% de agarosa y PBS en una cámara de puesta de huevos. Coloque una gota de levadura líquida en el centro del plato para estimular la puesta de huevos. A continuación, introduce 300 moscas gypsophila en la cámara.Mantenga las moscas a 25 grados centígrados durante tres días. Vuelva a colocar la placa de Petri en levadura recién disuelta dos veces al día. Al cuarto día, deje que las moscas pongan huevos durante cuatro horas.Después de reemplazar el plato, deje que las moscas pongan huevos durante tres horas más en el plato fresco. Después de tres horas, coloque la placa con los huevos a 25 grados centígrados durante 24 horas. De esta placa, recoja de 50 a 100 larvas recién eclosionadas y colóquelas en un frasco de plástico que contenga alimento estándar.

culturing-drosophila-third-instar-larvae-for-brain-metabolite-studies

Research

JoVE Journal

Neuroscience

Culturing Drosophila Third Instar Larvae for Brain Metabolite Studies

79 vistas. Para comenzar, coloque una placa de Petri de 60 milímetros cubierta con 1% de agarosa y PBS en una cámara de puesta de huevos. Coloque una gota de levadura líquida en el centro del plato para estimular la puesta de huevos. A continuación, introduce 300 moscas gypsophila en la cámara.Mantenga las moscas a 25 grados centígrados durante tres días. Vuelva a colocar la placa de Petri en levadura recién disuelta dos veces al día. Al cuarto día, deje que las moscas pongan huevos dur...

Video: Cultivo de larvas de Drosophila en el tercer estadio para estudios de metabolitos cerebrales

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.