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

Published: October 27, 2023

doi:

Andrés González-Gutiérrez, Jorge Gaete, Andrés Esparza, Jorge Toledo, Andrés Köhler-Solis, Jimena Sierralta³

¹Biomedical Neuroscience Institute,Universidad de Chile, ²Advanced Scientific Equipment Network (REDECA), Faculty of Medicine,Universidad de Chile, ³Department of Neuroscience, Faculty of Medicine,Universidad de Chile

Summary

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

Abstract

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 Förster 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.

Introduction

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' 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 Förster resonance energy transfer (FRET) microscopy, such as ATeam (ATP), FLII¹²Pglu700µδ6 (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 GABA_A 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.

Protocol

1. Fly strain maintenance and larval synchronization To perform these experiments, use fly cultures raised at 25 °C on standard Drosophila food composed of 10% yeast, 8% glucose, 5% wheat flour, 1.1% agar, 0.6% propionic acid, and 1.5% methylparaben. To follow this protocol, use the following lines: w1118 (experimental control background), OK6-GAL4 (driver for motor neurons), repo-GAL4 (driver for all glial cells), CG-GAL4 (driver for fat bodies), U…

Representative Results

For up to 1 h, this procedure allows for easy measurement of intracellular changes in the fluorescence of monocarboxylate and glucose sensors. As shown in Figure 4, Laconic sensors in both glial cells and motor neurons respond to 1 mM lactate at a similar rate at the start of the pulse, but motor neurons reach a higher increase over the baseline during the 5 min pulse, as previously demonstrated17. This lactate concentration was chosen because it is comparable to the …

Discussion

The use of the Drosophila model for the study of brain metabolism is relatively new²⁶, and it has been shown to share more characteristics with mammalian metabolism than expected, which has primarily been studied in vitro in primary neuron cultures or brain slices. Drosophila excels at in vivo experiments thanks to the battery of genetic tools and genetically encoded sensors available that allows researchers to visualize in real time the metabolic changes caused…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

We thank all the members of the Sierralta Lab. This work was supported by FONDECYT-Iniciación 11200477 (to AGG) and FONDECYT Regular 1210586 (to JS). UAS-FLII¹²Pglu700µδ6 (glucose sensor) was kindly donated by Pierre-Yves Plaçais and Thomas Preat, CNRS-Paris.

Materials

Agarose	Sigma	A9539
CaCl₂	Sigma	C3881
CCD Camera ORCA-R2	Hamamatsu	–
Cell-R Software	Olympus	–
CG-GAL4	Bloomington Drosophila Stock Center	7011	Fat body driver
Dumont # 5 Forceps	Fine Science Tools	11252-30
DV2-emission splitting system	Photometrics	–
Glass coverslips (25 mm diameter)	Marienfeld	111650	Germany
Glucose	Sigma	G8270
GraphPad Prism	GraphPad Software	Version 8,0,2
HEPES	Sigma	H3375
ImageJ software	National Institues of Health	Version 1,53t
KCl	Sigma	P9541
LUMPlanFl 40x/0.8 water immersion objective	Olympus	–
Methylparaben	Sigma	H5501
MgCl₂	Sigma	M1028
NaCl	Sigma	S7653
OK6-GAL4	Bloomington Drosophila Stock Center		Motor neuron driver
Picrotoxin	Sigma	P1675S	CAUTION-Fatal if swallowed
Poly-L-lysine	Sigma	P4707
Propionic Acid	Sigma	P1386
Repo-GAL4	Bloomington Drosophila Stock Center	7415	Glial cell driver (all)
Sodium Lactate	Sigma	71718
Sodium pyruvate	Sigma	P2256
Spinning Disk fluorescence Microscope BX61WI	Olympus	–
Sucrose	Sigma	S0389
Trehalose	US Biological	T8270
UAS-AT1.03NL	Kyoto Drosophila Stock Center	117012	ATP sensor
UAS-Chk RNAi GD1829	Vienna Drosophila Resource Center	v37139	Chk RNAi line
UAS-FLII¹²Pglu700md6	Bloomington Drosophila Stock Center	93452	Glucose sensor
UAS-GCaMP6f	Bloomington Drosophila Stock Center	42747	Calcium sensor
UAS-Laconic	Sierralta Lab	–	Lactate sensor
UAS-Pyronic	Pierre Yves Placais/Thomas Preat	–	CNRS-Paris
UMPlanFl 20x/0.5 water immersion objective	Olympus	–

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Citazione di questo articolo

González-Gutiérrez, A., Gaete, J., Esparza, A., Toledo, J., Köhler-Solis, A., Sierralta, J. Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors. J. Vis. Exp. (200), e65846, doi:10.3791/65846 (2023).