Assessing Glucose Uptake in the Motor Neurons of Drosophila Larvae with Upregulated Glucose Metabolism

Abstract

Source: Loganathan, S., et al., Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy. J. Vis. Exp. (2021).

The video demonstrates a technique for measuring glucose uptake in Drosophila larval motor neurons using a fluorescence resonance energy transfer (FRET)-based intracellular glucose sensor. After dissecting the larvae to expose the ventral nerve cord, the motor neurons are visualized through the fluorescence emitted by the sensor's donor fluorophore. When stimulated with glucose, FRET between the sensor's donor and acceptor fluorophores triggers the emission of fluorescence from the acceptor, enabling the comparison of glucose uptake between control larvae and mutant larvae with upregulated glucose metabolism.

Protocol

1. Drosophila Ventral Nerve Cord (VNC) dissections

1. Make silicone elastomer dissection dishes.
   1. Pour silicone elastomer components into a 50 mL tube as indicated in the manufacturer's protocol and stir well.
   2. Fill 35 mm tissue culture dishes with approximately 2 mL of the elastomer mixture. Use a syringe to remove any bubbles. Cover the dishes and place them on a level surface in a 60 °C oven overnight to cure.
2. Dissect Drosophila larvae to expose the VNC while maintaining tissue viability.
   1. Collect a wandering third instar larva, rinse with ddH

Results

Figure 1: Image analysis of glucose sensor in the context of TDP-43 mutant. (A) Representative images of FRET and CFP signals in glucose sensor controls and TDP-43^G298S samples under baseline and stimulation conditions. A repres.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm tissue culture dishes	Sigma Aldrich	CLS430165
40X water immersion lens	Zeiss	440090	dippable, N.A. 0.8
dissection scissors	Roboz	RS-5618
Dumont #5 forceps	VWR	100189-236
Dumont #55 forceps	VWR	100189-244
Minutien pins	Fine Science tools	26002-10	used for dissections
SYLGARD 184 Silicone Elastomer Kit	Dow	1317318
Zeiss LSM880 NLO upright multiphoton/confocal microscope	Zeiss	N/A