We thank all the members of the Sierralta Lab. This work was supported by FONDECYT-Iniciaci&#243;n 11200477 (to AGG) and FONDECYT Regular 1210586 (to JS). UAS-FLII12Pglu700&#181;&#948;6 (glucose sensor) was kindly donated by Pierre-Yves Pla&#231;ais and Thomas Preat, CNRS-Paris.

1. Fly strain maintenance and larval synchronization
<ol>
	<li>To perform these experiments, use fly cultures raised at 25 &#176;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.</li>
	<li>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...

Monitoring Brain Metabolite Transport in &lt;em&gt;Drosophila&lt;/em&gt; Using FRET Sensors

<ol>
	<li>Vergara, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+energy+homeostasis+principle:+neuronal+energy+regulation+drives+local+network+dynamics+generating+behavior.">The energy homeostasis principle: neuronal energy regulation drives local network dynamics generating behavior.</a> Frontiers in Computational Neuroscience. 13 (49), 1-18 (2019).</li><li>Pulido, C., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pools+are+a+major+hidden+resting+metabolic+burden+of+nerve+terminals.">Synaptic vesicle pools are a major hidden resting metabolic burden of nerve terminals.</a> Science Advances. 7 (49), 1-9 (2021).</li><li>Benton, D., Parker, P. Y., Donohoe, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+supply+of+glucose+to+the+brain+and+cognitive+functioning.">The supply of glucose to the brain and cognitive functioning.</a> Journal of Biosocial Science. 28 (4), 463-479 (1996).</li><li>Mergenthaler, P., Lindauer, U., Dienel, G. A., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+for+the+brain:+the+role+of+glucose+in+physiological+and+pathological+brain+function.">Sugar for the brain: the role of glucose in physiological and pathological brain function.</a> Trends in Neurosciences. 36 (10), 587-597 (2013).</li><li>Boumezbeur, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+blood+lactate+to+brain+energy+metabolism+in+humans+measured+by+dynamic+13C+nuclear+magnetic+resonance+spectroscopy.">The contribution of blood lactate to brain energy metabolism in humans measured by dynamic 13C nuclear magnetic resonance spectroscopy.</a> The Journal of Neuroscience. 30 (42), 13983-13991 (2010).</li><li>Baltan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+lactate+serve+as+an+energy+substrate+for+axons+in+good+times+and+in+bad,+in+sickness+and+in+health.">Can lactate serve as an energy substrate for axons in good times and in bad, in sickness and in health.</a> Metabolic Brain Disease. 30 (1), 25-30 (2015).</li><li>Barros, L. F., Weber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrossTalk+proposal:+an+important+astrocyte-to-neuron+lactate+shuttle+couples+neuronal+activity+to+glucose+utilization+in+the+brain.">CrossTalk proposal: an important astrocyte-to-neuron lactate shuttle couples neuronal activity to glucose utilization in the brain.</a> The Journal of Physiology. 596 (3), 347-350 (2018).</li><li>Yellen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fueling+thought:+Management+of+glycolysis+and+oxidative+phosphorylation+in+neuronal+metabolism.">Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism.</a> The Journal of Cell Biology. 217 (7), 2235-2246 (2018).</li><li>Koveal, D., Diaz-Garcia, C. M., Yellen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+biosensors+for+neuronal+metabolism+and+the+challenges+of+quantitation.">Fluorescent biosensors for neuronal metabolism and the challenges of quantitation.</a> Current Opinion in Neurobiology. 63, 111-121 (2020).</li><li>Imamura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+ATP+levels+inside+single+living+cells+with+fluorescence+resonance+energy+transfer-based+genetically+encoded+indicators.">Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (37), 15651-15656 (2009).</li><li>Takanaga, H., Chaudhuri, B., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLUT1+and+GLUT9+as+major+contributors+to+glucose+influx+in+HepG2+cells+identified+by+a+high+sensitivity+intramolecular+FRET+glucose+sensor.">GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor.</a> Biochimica et Biophysica Acta. 1778 (4), 1091-1099 (2008).</li><li>San Martin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+FRET+lactate+sensor+and+its+use+to+detect+the+Warburg+effect+in+single+cancer+cells.">A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells.</a> PloS One. 8 (2), 1-13 (2013).</li><li>San Martin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mitochondrial+flux+in+single+cells+with+a+FRET+sensor+for+pyruvate.">Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate.</a> PloS One. 9 (1), 1-9 (2014).</li><li>Placais, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulated+energy+metabolism+in+the+Drosophila+mushroom+body+is+the+trigger+for+long-term+memory.">Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory.</a> Nature Communications. 8, 1-14 (2017).</li><li>Mann, K., Deny, S., Ganguli, S., Clandinin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+activity,+metabolism+and+behaviour+across+the+Drosophila+brain.">Coupling of activity, metabolism and behaviour across the Drosophila brain.</a> Nature. 593 (7858), 244-248 (2021).</li><li>Volkenhoff, A., Hirrlinger, J., Kappel, J. M., Klambt, C., Schirmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+using+a+FRET+glucose+sensor+reveals+glucose+delivery+to+all+cell+types+in+the+Drosophila+brain.">Live imaging using a FRET glucose sensor reveals glucose delivery to all cell types in the Drosophila brain.</a> Journal of Insect Physiology. 106 (1), 55-64 (2018).</li><li>Gonzalez-Gutierrez, A., Ibacache, A., Esparza, A., Barros, L. F., Sierralta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+lactate+levels+depend+on+glia-derived+lactate+during+high+brain+activity+in+Drosophila.">Neuronal lactate levels depend on glia-derived lactate during high brain activity in Drosophila.</a> Glia. 68 (6), 1213-1227 (2020).</li><li>Geistlinger, K., Schmidt, J. D. R., Beitz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocarboxylate+transporters+accept+and+relay+protons+via+the+bound+substrate+for+selectivity+and+activity+at+physiological+pH.">Human monocarboxylate transporters accept and relay protons via the bound substrate for selectivity and activity at physiological pH.</a> PNAS Nexus. 2 (2), 1-8 (2023).</li><li>Pasco, M. Y., Leopold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sugar-induced+insulin+resistance+in+Drosophila+relies+on+the+lipocalin+Neural+Lazarillo.">High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo.</a> PloS One. 7 (5), 1-8 (2012).</li><li>McMullen, E., Weiler, A., Becker, H. M., Schirmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+carbohydrate+transport+at+the+blood-brain+barrier.">Plasticity of carbohydrate transport at the blood-brain barrier.</a> Frontiers in Behavioral Neuroscience. 14, 1-15 (2020).</li><li>Delgado, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaski,+a+novel+Drosophila+lactate/pyruvate+transporter+required+in+glia+cells+for+survival+under+nutritional+stress.">Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.</a> Scientific Reports. 8 (1), 1-13 (2018).</li><li>Stilwell, G. E., Saraswati, S., Littleton, J. T., Chouinard, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Drosophila+seizure+model+for+in+vivo+high-throughput+drug+screening.">Development of a Drosophila seizure model for in vivo high-throughput drug screening.</a> European Journal of Neuroscience. 24 (8), 2211-2222 (2006).</li><li>Lerchundi, R., Huang, N., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+changes+in+astrocytic+and+neuronal+adenosine+triphosphate+using+two+different+variants+of+Ateam.">Quantitative imaging of changes in astrocytic and neuronal adenosine triphosphate using two different variants of Ateam.</a> Frontiers in Cellular Neuroscience. 14 (80), 1-13 (2020).</li><li>Baeza-Lehnert, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-canonical+control+of+neuronal+Energy+Status+by+the+Na(+)+Pump.">Non-canonical control of neuronal Energy Status by the Na(+) Pump.</a> Cell Metabolism. 29 (3), 668-680 (2019).</li><li>Mattila, J., Hietakangas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+carbohydrate+energy+metabolism+in+Drosophilamelanogaster.">Regulation of carbohydrate energy metabolism in Drosophilamelanogaster.</a> Genetics. 207 (4), 1231-1253 (2017).</li><li>De Backer, J., Grunwald, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+glia+in+cellular+and+systemic+metabolism:+insights+from+the+fly.">A role for glia in cellular and systemic metabolism: insights from the fly.</a> Current Opinion in Insect Science. 53 (100947), 1-8 (2022).</li><li>Loganathan, S., Ball, H., Manzo, E., Zarnescu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+glucose+uptake+in+Drosophila+models+of+TDP-43+proteinopathy.">Measuring glucose uptake in Drosophila models of TDP-43 proteinopathy.</a> Journal of Visualized Experiments. (174), e62936 (2021).</li><li>Dienel, G., Rothman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calibration+of+genetically+encoded+metabolite+biosensors+must+account+for+metabolite+metabolism+during+calibration+and+cellular+volume.">In vivo calibration of genetically encoded metabolite biosensors must account for metabolite metabolism during calibration and cellular volume.</a> Journal of Neurochemistry. , (2023).</li><li>Gandara, L., Durrieu, L., Behrensen, C., Wappner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+toolkit+for+the+analysis+of+metabolic+changes+in+Drosophila+provides+new+insights+into+metabolic+responses+to+stress+and+malignant+transformation.">A genetic toolkit for the analysis of metabolic changes in Drosophila provides new insights into metabolic responses to stress and malignant transformation.</a> Scientific Reports. 9, 1-11 (2019).</li><li>Gandara, L., Durrieu, L., Wappner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+FRET+sensors+in+intact+organs:+Applying+spectral+unmixing+to+acquire+reliable+signals.">Metabolic FRET sensors in intact organs: Applying spectral unmixing to acquire reliable signals.</a> BioRxiv. , (2023).</li></ol>

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera ORCA-R2</td>
			<td>Hamamatsu</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-R Software</td>
			<td>Olympus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CG-GAL4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>7011</td>
			<td>Fat body driver</td>
		</tr>
		<tr>
			<td>Dumont # 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>DV2-emission splitting system</td>
			<td>Photometrics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips (25 mm diameter)</td>
			<td>Marienfeld</td>
			<td>111650</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>Version 8,0,2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institues of Health</td>
			<td>Version 1,53t</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>LUMPlanFl 40x/0.8 water immersion objective</td>
			<td>Olympus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylparaben</td>
			<td>Sigma</td>
			<td>H5501</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>OK6-GAL4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td></td>
			<td>Motor neuron driver</td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675S</td>
			<td>CAUTION-Fatal if swallowed</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic Acid</td>
			<td>Sigma</td>
			<td>P1386</td>
			<td></td>
		</tr>
		<tr>
			<td>Repo-GAL4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>7415</td>
			<td>Glial cell driver (all)</td>
		</tr>
		<tr>
			<td>Sodium Lactate</td>
			<td>Sigma</td>
			<td>71718</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk fluorescence Microscope BX61WI</td>
			<td>Olympus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose</td>
			<td>US Biological</td>
			<td>T8270</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-AT1.03NL&#160;</td>
			<td>Kyoto Drosophila Stock Center</td>
			<td>117012</td>
			<td>ATP sensor</td>
		</tr>
		<tr>
			<td>UAS-Chk RNAi GD1829</td>
			<td>Vienna Drosophila Resource Center</td>
			<td>v37139</td>
			<td>Chk RNAi line</td>
		</tr>
		<tr>
			<td>UAS-FLII12Pglu700md6&#160;</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>93452</td>
			<td>Glucose sensor</td>
		</tr>
		<tr>
			<td>UAS-GCaMP6f&#160;</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>42747</td>
			<td>Calcium sensor</td>
		</tr>
		<tr>
			<td>UAS-Laconic</td>
			<td>Sierralta Lab</td>
			<td>-</td>
			<td>Lactate sensor</td>
		</tr>
		<tr>
			<td>UAS-Pyronic</td>
			<td>Pierre Yves Placais/Thomas Preat</td>
			<td>-</td>
			<td>CNRS-Paris</td>
		</tr>
		<tr>
			<td>UMPlanFl 20x/0.5 water immersion objective</td>
			<td>Olympus</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Advances in Brain Energy Metabolism Research Using the &lt;em&gt;Drosophila&lt;/em&gt; Model

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

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.

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 ...

Watch this Scientific Journal Video about Author Spotlight: Advances in Brain Energy Metabolism Research Using the Drosophila Model at JoVE.com

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.

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the ...

visualizing-monocarboxylates-other-relevant-metabolites-ex-vivo

Research

JoVE Journal

Neuroscience

Culturing Drosophila Third Instar Larvae for Brain Metabolite Studies

To begin, place a 60-millimeter Petri dish covered with 1%agarose and PBS in an egg-laying chamber. Put a drop of liquid yeast in the center of the plate to encourage egg-laying. Next, introduce 300 gypsophila flies into the chamber.Keep the flies at 25 degrees Celsius for three days. Replace the Petri dish in freshly dissolved yeast twice daily. On the fourth day, let the flies lay eggs for four hours.After replacing the dish, allow the flies to lay eggs for three more hours on the fresh plate. After three hours, place the plaque with the eggs at 25 degrees Celsius for 24 hours. From this plaque, collect 50 to 100 newly-hatched larvae and place them in a plastic vial containing standard feed.

Dissection of Third Instar Drosophila Larva for Brain Metabolite Imaging

To begin, place 25 millimeter glass cover slips into the wells of a six-well cell culture plate. Place a drop of polylysine in the center of each cover slip. After incubation is complete, wash each cover slip three times with distilled water.Then wash the cover slips two times with a calcium-free saline solution. Next, install the covers in a recording chamber and fill it with calcium-free saline. Collect the wandering third instar larvae from the desired genetic cross of drosophila.Transfer the larvae to a dish well, then wash the larvae three times with distilled water. Place the larvae in another glass dissection dish well containing 750 microliters of ice-cold, calcium-free saline solution. Now place a larvae under a stereo microscope.With a pair of forceps, make a transverse cut across the back of the abdomen. Push the jaw with the forceps to turn the larvae inside out. Observe the ventral nerve cord next to the jaw.Carefully remove the imaginal discs and remaining brain coddle tissues. Next, separate the ventral nerve cord with the central brain and optic lobes from the rest of the tissue. Transfer the ventral nerve cord to the recording chamber containing calcium-free saline solution.To avoid interference, attach the remaining nerves to the bottom of the cover slip with forceps. To perform experiments on fat bodies, proceed to isolate the fat tissues from the turned-out larva. Place the isolated fat bodies expressing the fret sensors in the recording chamber.To acquire images of tissues expressing sensors, turn on the illumination system of the microscope. Place the recording chamber containing ventral nerve cords or fat bodies on the stage of a fluorescent microscope. To view the GCaMP6f fluorescence, set the excitation wavelength to 488 nanometers.For metabolites such as laconic, pyronic, ATP, or glucose sensors, set the excitation to 440 nanometers. Now set the image acquisition at regular intervals for GCaMP6f and metabolite sensor. Place the water immersion objective, ensuring it remains submerged.Next, connect the recording chamber to a perfusion system. Use a low-flux peristaltic pump to extract the liquid from the chamber and maintain a constant flow of three milliliters per minute. Position the solution containing tubes 25 centimeters above the microscope stage.Before any stimulation, obtain a stable fluorescence baseline by allowing the recording solution to flow for five to 10 minutes. Now, replace the flow solution with the stimulation solution containing glucose, pyruvate, or lactate for five minutes. Capture the images of the stimulated ventral nerve cord that was exposed to 80 micromolar picrotoxin to assess neuronal activity.Laconic sensors in both glial cells and motor neurons respond to one millimolar lactate at a similar rate at the start of the pulse. However, the motor neurons reach a higher increase over the baseline during the five minute pulse. The glucose sensor's signal increased at a similar rate in the glial cells and neurons when exposed to five millimolar glucose.During the glucose pulse, however, the signal in neurons increases more than in glial cells. Knocking out Chaski transporter reduced lactate transport in glial cells. Picrotoxin-induced increases in neuronal activity resulted in a transitory drop in ATP levels in the soma of motor neurons.The laconic sensor was observed to be well-expressed in fat bodies. Increased fluorescence of the glucose sensors was seen when the fat bodies were exposed to increasing glucose concentration. Increased lactate and pyruvate concentrations resulted in a proportional increase in laconic and pyronic fluorescence.

387 Views.  Universidad de Chile.  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.

Monitoring Brain Metabolite Transport in Drosophila Using FRET Sensors

Summary