Optogenetic control of Drosophila neurons: a laboratory practical for undergraduates and outreach

ABSTRACT

Teaching aspects of neuroscience to large undergraduate classes can be difficult in terms of the cost of equipment involved such as microscopes and electrophysiology equipment, the time taken to master techniques such as dissection or intracellular recording, and ethical concerns when using vertebrates. Here, I describe a practical that uses behavioral readouts and optogenetics on Drosophila that can be implemented with minimal cost as well as reduced ethical concerns and uses mostly observational techniques. The practical can be used to teach aspects of genetics and the tools for manipulating neuronal activity for ascribing neuronal function. The practical can be customized to fit different undergraduate levels and learning objectives.

INTRODUCTION

Understanding how behaviors are encoded by the neural circuits that generate them remains a major goal of neuroscience. Connectomes, which aim to be complete neuronal wiring diagrams of an organism, provide a rich and growing resource for defining neural circuits. The first connectome of any organism was published for Caenorhabditis elegans and was achievable due to its low number of neurons (1). Advances in automation in electron microscopy as well as the segmentation tools that allow automatic identification of features means that connectomes for larger and more complex brains are achievable. In the past year alone, the connectome for both the larval and adult brains of Drosophila has been published (2, 3), and the first vertebrate brains are likely to follow soon (4).

One outstanding bottleneck is to assign functions to these newly mapped neural circuits. Optogenetics, a tool fusing optics and genetics, can be used to modulate and ultimately assign functionality to neurons. This technique uses light-sensitive ion channels, most notably Channel Rhodopsin 2 (ChR2) from the green algae Chlamydomonas reinhardtii (5), to activate neurons when artificially expressed in other species (6).

Several papers have described the equipment and protocols needed to use optogenetics to manipulate Drosophila larval and adult behavior and how they can be applied in neurobiology teaching (7–9). In this article, I describe a simple and accessible component list for the mass production of light-emitting diode (LED) light sources and an updated list of fly lines useful for teaching. I describe how they can be used for a simple undergraduate practical, which introduces biology students to behavioral observation, genetic manipulation in model organisms, and molecular neurobiology. The practical can be adapted for different durations, undergraduate levels or be simplified for outreach demonstrations for high school students.

PROCEDURE

Materials

The following Drosophila Gal4-containing strains can be obtained from the Bloomington Drosophila stock center: P{ChAT-GAL4.7.4} (stock 6793); P{GawB}VGlutOK371 (stock 26160); P{GMR15D07-GAL4}attP2 (stock 47467); P{GMR53F07-GAL4}attP2 (stock 50442); P{w[+mC]=Gr5a-GAL4.8.5}6 (stock 57592). We used a variant of ChR2, T159C (10) that gave better response to blue light than the wild type version, PBac{UAS-ChR2.T159C}VK00018 (stock 58373). Another ChR2 variant, H134R, also worked well in our hands (11). We attempted to use ChR2 XXL (10), but this led to lethality suggesting it is activated even under normal daylight with these drivers. Various wild type stocks can be utilized, for example, Oregon-R (stock 5).

The Gal4 lines should be crossed to the UAS line, and resulting larvae and adults can be used for optogenetics. For larger classes, it is recommended to create a stock containing each Gal4 with the UAS ChR2 (through recombination if they are on the same chromosome, or by making a stock with transgenes on the second and third chromosomes) to avoid the need to perform fly crosses for each practical. Such stocks can also be obtained from the author’s lab. A list of the lines used in the practical is given in Table 1.

TABLE 1.

Drosophila lines, their expression patterns, and response to blue light stimulation^a

Fly genotype	ATR added to media	Expression pattern of ChR2 (reference)	Response of larvae	Response of adults	Notes (reference)
Wild type	Yes	Not present	None	None	Demonstrates that wild type larvae and adults do not respond to blue light
UAS ChR2 T159C	Yes	Not expressed	None	None	Demonstrates the need for a Gal4 for ChR2 expression
ChAT Gal4 UAS ChR2 T159C	Yes	Cholinergic neurons (includes all larval peripheral nervous system sensory neurons)	Body contracts, mouth hooks continue. Some attenuation on prolonged stimulation	Fall to bottom of dish, legs twitch, some attenuation on prolonged stimulation	Activation of all sensory neurons causes larvae to activate motor neurons and tense up
VGlut Gal4 UAS ChR2 T159C	Yes	Glutamatergic neurons (mainly motor neurons)	Body contracts, mouth hooks stop. No attenuation on prolonged stimulation	Flies immobilized	Activation of all motor neurons causes larvae to tense up
ChAT Gal4 or VGlut Gal4 UAS ChR2 T159C	No	As above	None	None	Demonstrates that ATR is essential for the ChR2 to be activatable
GMR15D07Gal4 UAS ChR2 T159C	Yes	Poorly defined (12)	Persistent head casting	No obvious response	(13)
GMR53F07Gal4 UAS ChR2 T159C	Yes	Poorly defined (12)	Reverse locomotion	No obvious response	(13)
Gr5a Gal4 UAS ChR2 T159C	Yes		No obvious response	Proboscis extension reflex, grooming (forelimb rubbing)	(14)

^a These are based on stocks, which contain two copies of both the Gal4 and UAS.

Drosophila media (https://flystocks.bio.indiana.edu/information/recipes/bloomfood.html) was supplemented with 50 µm all trans retinal (ATR) (from a 100 mM stock in ethanol) (e.g., Sigma-Aldrich R2500-25MG, $70, approximately), which is necessary for the ChR2 molecule to respond to blue light. ATR is stable in Drosophila media for >3 months at <10°C.

35 mm apple juice agar plates were produced as per the recipe from CSHL (15). Drosophila larvae and adults were given to students in 28 mL polystyrene fly vials (Greiner Bio-One, catalogue 205101) or 35 mm vented petri dishes (Greiner Bio-One, catalogue 627102), respectively, though similar products are available from other laboratory suppliers. The component list for the battery operated LED is given in Table 2, wiring diagram in Fig. S1 and an optional LED holder 3D (Fig. 1B) printer instruction file can be downloaded from https://www.thingiverse.com/thing:6632298. A stereomicroscope for each pair of students is also required, though this does not need to have high specification.

TABLE 2.

Components needed to create the LEDs with manufacturer codes and the part numbers for a major international electronic component supplier available in Europe/Asia and North America (Digikey)^a

Manufacturer and code	Digikey part number	Description	Approx. cost	Image
New Energy XPEBBL-L1-0000-00301-SB01	1672-1053-ND	LED MOD XLAMP BLUE STARBOARD (475 nm)	$6
Carclo Technical Plastics 10193	1066-1012-ND	LENS CLEAR 3-24DEG SPOT SNAP-IN	$2
Carclo Technical Plastics 10750	1066-1074-ND	OPTIC HOLDER 20 mm HEX XP-E BLACK	$0.5
Wakefield-Vette 624-45ABT3	345-1089-ND	HEATSINK CPU 21 mm SQ W/ADH BLK	$2
Recom Power RCD-24-1.00/W/X3	945-1131-ND	LED DRIVER CC BUCK 3-31 V 1 A	$25
MPD BC3AAW	BC3AAW-ND	HOLDER BATT 3-AA CELLS WIRE LDS	$2
Judco Manufacturing Inc. 40-4692-00	563PB-ND	SWITCH PUSH SPST-NO 2 A 14 V	$2.5
Any hardware supplier	1175-10064-22-1-0500-005-1-TS-DS-ND	HOOK-UP STRND 22AWG 30 V BLU FEET (need approx. 40 cm per unit)	$0.50
Any hardware supplier	1175-10064-22-1-0500-006-1-TS-DS-ND	HOOK-UP STRND 22AWG 30 V BRN FEET (need approx. 40 cm per unit)	$0.50
Any hardware supplier	1920-1363-ND	CONN TERM STRIP 12 POLES 10 mm (need five connectors per unit – but not essential)	$7
Any hardware supplier		BATTERY ALKALINE 1.5 V AA (need three per unit)	$2
Any hardware supplier		Soldering iron and wire solder
Amazon/eBay or similar		50 cm Gooseneck Flex Arm with C-Clamp & Spring Clamp	$15
Optional 3D printed LED holder and lid		.stl files for 3D printing
Pipette tip box		Any supplier		Can be used to encase battery and LED driver
Total per unit			$70 (approx.)	Unit price reduces substantially when purchased in bulk

^a MPD, memory protection device.

FIG 1.

LED microscope setup (A) Photo of the LED wiring given in Fig. S2. An empty tip box makes a cheap way to hide the batteries and other components. (B) Optional 3D-printed LED holder with a lid to encase LED and heat sink. (C) Final LED setup as presented to students.

Safety issues

The light from the LED is bright and is uncomfortable if directly shone into the eyes. The gooseneck arm focusses the LED beam downward onto the microscope stage area and therefore eliminates the possibility of direct eye exposure.

Laboratory exercise

In the pre-exercise briefing, the students are introduced to the framework, into which these experiments and tools fall, that is, the recent production of connectomes for small animal central nervous systems and the resulting need to put functional information on such circuits. The students are also introduced to the discovery and use of channel rhodopsin (in this case ChR2) as a tool to activate neurons in response to blue light (temporal specificity) and the use of the UAS/Gal4 (16) system to express ChR2 in particular neurons (spatial specificity).

Students work in pairs at a workstation, in which they have a dissecting microscope with blue light LED and food vials that contain larvae and mini petri dishes that contain adults with ChR2 expressed in various neurons. The students follow a worksheet (Fig. S2) and transfer a few larvae from the food with a thin spatula onto the agar plate to observe their normal crawling behavior. Then, the students shine short (1–3 s) and prolonged (>10 s) bursts of blue light onto the larvae to see how they respond, if the response attenuates and how quickly they recover post stimulation. The students can observe the effect of blue light on the adults in the petri dishes directly.

The worksheet guides the students as to which order to do the experiments and to record their observations and deductions. The students begin with the widely expressed Gal4s (ChAT and VGlut), which exhibit striking and robust responses and once they become accustomed to observing Drosophila behavior, move onto the more subtle behaviors.

At the end of the practical, we have a debriefing session where we gather the class together and go through the answers on the worksheets and the conclusions that we can draw. We also discuss why optogenetics can be used to study larval and adult nervous systems in Drosophila (the light can reach the neurons as they are small, the larvae translucent and adults have only a thin cuticle) but why this approach would be tricky, for example, in mice.

CONCLUSION

The practical described here can be run in 90 min allowing multiple sessions to be carried out in 1 day where class sizes are large. Understanding can be tested by means of post-lab online multiple choice questions (see Fig. S3 for examples). For smaller classes, the practical can be extended to suit the level of the students, time available, and other skills that could be acquired. For example, it would be possible not only to obtain quantitative data on behavior frequency but also to investigate how different transgene copy numbers or the intensity of the light affects the robustness of the response.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jmbe.00086-24.

Figure S1. jmbe.00086-24-s0001.docx.

Wiring diagram to connect the LED to battery.

Figure S2. jmbe.00086-24-s0002.docx.

Student worksheet.

Figure S3. jmbe.00086-24-s0003.docx.

Sample MCQs.

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