Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Summary

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Abstract

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

Introduction

Optogenetics has emerged as a powerful technique combining optics and genetics in neuroscience, providing precise, non-invasive control over neural activity through light stimulation¹. In Drosophila melanogaster, a widely used model organism, optogenetic tools enable the activation and inhibition of specific neurons, allowing researchers to modulate neural circuits. Among the tools used, CsChrimson and GtACR (Guillardia theta anion channel rhodopsins) provide complementary approaches for neuronal targeting. CsChrimson channelrhodopsin, a red-light sensitive cation channel from green algae, facilitates neuronal activation throu....

Protocol

1. Strains, fly rearing, and fly aspirator

1. Strains and maintenance
   1. The strains used in the experiments include HC-Gal4¹⁰, Gr5a-Gal4²⁰ (Bloomington Drosophila Stock Center (BDSC): 57592), Gr66a-Gal4²¹ (BDSC: 57670), UAS-GtACR2³, UAS-CsChrimson² (BDSC: 55136). Rear flies at 25 °C on a standard cornmeal medium under a 12-h light:12-h dark cycle.
      NOTE: 1 L cornmeal medium contains 1 L dH₂O, 79 g of dextrose, 7.5 g of agar, 24 g of flaked....

Discussion

Optogenetic manipulation has transformed the field of neuroscience by enabling precise control of neural circuits with spatiotemporal accuracy²⁷. A neural circuit includes populations of neurons interconnected by synapses, performing specific functions upon activation. The Drosophila whole-brain connectome has been completed, offering comprehensive insights into the synaptic pathways within the Drosophila brain^28,29. Whil.......

Materials

Name	Company	Catalog Number	Comments
1000 mA LED driver	Luxeon Star	3021-D-E-1000
5 mL VWR Culture Tubes, Plastic, with Dual-Position Caps	VWR	60818-664
780 Longpass Filter / IR 780 nm 100 mm x 100 mm	Lee Filters	BH #LE8744	Cut to approximately 47 x 100 fit the plastic cover
Agfabric 6.5 ft. x 15 ft. Insect Bug Netting Garden Net for Protecting Plants Vegetables Flowers Fruits	The Home Depot	EIBNW6515
All trans retinal	Sigma-Aldrich	116-31-4
Aluminum Plate (30.5 cm x 30.5 cm x 0.6 cm)	Amazon		purchased from Amazon
Black Plastic Box	LI-COR	929-97101
CALCIUM CHLORIDE ANHYDRO 25GR	Thermo Fisher Scientific	AC297150250
CX405 Handycam with Exmor R CMOS sensor	SONY	HDR-CX405
Elmer’s “School Glue”	Elmer
Ethyl alcohol, Pure (200 Proof)	Sigma-Aldrich	E7023
Fisherbrand Isotemp Hot Plate Stirrer	Fisher Scientific	SP88850200
Fly line: Gr5a-Gal4	Bloomington Drosophila Stock Center	57592
Fly line: Gr66a-Gal4	Bloomington Drosophila Stock Center	57670
Fly line: HC-Gal4 (II)	Dr. Marco Gallio Lab		A kind gift
Fly line: UAS-CsChrimson	Bloomington Drosophila Stock Center	55136
Fly line: UAS-GtACR2/TM6B	Dr. Quentin Gaudry Lab		A kind gift
Flystuff 62-101 Yellow Cornmeal (11.3 Kg), Yellow, 11.3 Kg/Unit	Genesee Scientific	62-101
Flystuff 62-107 Inactive Dry Yeast, 10 Kg, Nutritional Flake, 10 Kg/Unit	Genesee Scientific	62-107
Flystuff 66-103 Nutri-Fly Drosophila Agar, Gelidium, 100 Mesh, 5 Kg (11.02 lbs)/Unit	Genesee Scientific	66-103
FreeMascot OD 8+ 190 nm–420 nm / 600 nm–1100 nm Wavelength Violet/Red/Infrared Laser Safety Glasses	FreeMascot	B08LGMQ65S	purchased from Amazon
GoPro Hero8 Black	GoPro	6365359
LEE Filters 100×100 mm Infra Red #87 Infrared Polyester Filter	B&H Photo	LE8744
Longpass Filter, Colored Glass, 50.8 x 50.8 mm, 830 nm Cut-on, RG830	Newport	FSQ-RG830
Methyl 4-hydroxybenzoate, 99%, Thermo Scientific Chemicals	Thermo Fisher Scientific	126960025
MicroWell Mini Tray 60 Well, Low Profile NS PS	Thermal Scientific	NUNC 439225	The lids are used as the "plastic cover"
Olympus Plastics 24-160RS, 1000 µL Olympus Ergonomic Pipet Tips Low Binding, Racked, Sterile, 8 Racks of 96 Tips/Unit	Eppendorf	24-160RS
Parafilm M Sealing Film	Heathrow Scientific	HS234526B	4 in x 125 feet
Potassium chloride, ACS, 99.0-100.5%,	Thermo Fisher Scientific	AA1159530
Prism	GraphPad	Version 9	data analysis software
Samco Graduated Transfer Pipettes	Thermo Fisher Scientific	225	3 mL
Slides	Fisher Scientific	12-544-2	5 mm x 75 mm x 1.0 mm
Stereo microscope	OLYMPUS	CZ61
Styrofoam box (27 cm height × 22 cm width × 16 cm length)
Sucrose	Fisher Scientific	225911
Surface temperature probe	Fluke	80PK-3A
Syringe	BD Integra	305270
Tate & Lyle 457 Dextrose, Tate & Lyle, Pow, Tate & Lyle 457 Dextrose, Tate & Lyle, Powder, 50 lbs/Unit	Genesee Scientific	62-113
Traceable Calibrated Big-Digit Thermocouple Thermometer	Traceable by cple-parmer	UX-91210-07	Fisherbrand Traceable BigDigit Type K Thermometer
Triple blue LED starboard	LEDSupply	07007-PB000-D	470 nm
Triple red LED starboard	LEDSupply	07007-PD000-F	627 nm
Tygon PVC Clear Tubing 1/4" ID, 3/8" OD, 5 ft. Length	McMaster Carr Supply Company	6516T21
Univivi IR Illuminator, 850nm 12 LEDs Wide Angle IR Illuminator for Night Vision	Univivi	4331910725
Wakefield Thermal 25.4 mm Round Heatsink Star LED Board - 882-100AB	Wakefield-Vette	882-100AB
Wireless Presenter	DinoFire Store	B01410YNAM	purchased from Amazon