Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Summary

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Abstract

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

Introduction

The remodeling of juvenile brain circuits during early life represents the last chance for large-scale synaptic connectivity changes to match the highly variable, unpredictable environment into which an animal is born. As the most abundant group of animals, insects share this evolutionarily conserved, foundational critical period remodeling mechanism¹. Critical periods open with the onset of sensory input, exhibit reversible circuit changes to optimize connectivity, and then close when stabilization forces resist further remodeling². Insects are particularly reliant on olfactory sensory information and show a well-defined olfactory critical period. Drosophila provides an excellent genetic model to investigate this experience-dependent critical period in the juvenile brain. Odorant experience during the first few days following eclosion drives striking circuit connectivity changes in individually identified synaptic glomeruli^3,4. The direction of remodeling is dependent on the specific input odorant experience. Some odorants cause an increase in the synaptic glomerulus volume for a couple of days post-eclosion (dpe)^3,5,6,7, whereas other odorants cause a rapid elimination of synapses during the 0-2 dpe critical period, resulting in decreased innervation volume^8,9,10. Specifically, ethyl butyrate (EB) odorant experience drives dose-dependent synaptic pruning of the Or42a olfactory receptor neurons only during this early-life critical period⁸. The synapse elimination is completely reversible by modulating EB odorant input within the critical period but becomes permanent following the closure of the critical period. This olfactory experience-dependent synaptic pruning provides a valuable experimental system to elucidate the temporally restricted mechanisms underlying juvenile brain circuit remodeling.

Here, we present a detailed protocol used to induce and analyze EB experience-dependent synaptic pruning of Or42a receptor olfactory sensory neurons during the early-life critical period. We show that Or42a synaptic terminals in the antennal lobe VM7 glomerulus can be specifically labeled by transgenically driving a membrane-tethered mCD8::GFP marker, either directly fused to the Or42a promoter (Or42a-mCD8::GFP)¹¹ or using the Gal4/UAS binary expression system (Or42a-Gal4 driving UAS-mCD8::GFP)¹². Individual Or42a neuron synapses can be similarly labeled using targeted transgenic expression of presynaptic active zone markers fused to an array of fluorescent tags (e.g., Bruchpilot::RFP)⁸ or an electron-dense signal for ultrastructural synapse analyses (e.g., miniSOG-mCherry)⁸. Or42a synaptic terminals can be imaged with a combination of laser-scanning confocal microscopy and transmission electron microscopy. We show that Or42a synaptic glomeruli pruning is EB dose-dependent, scaling to the concentration of the timed odorant experience. The percentage of EB odorant dissolved in mineral oil used as a vehicle can be varied, as can the timed duration of the odorant exposure in developmentally staged animals. Finally, we show the methods used to analyze the extent of synaptic glomeruli pruning by measuring the VM7 innervation fluorescence intensity and volume. Synapse number can also be quantified by counting labeled synaptic puncta and by measuring synaptic ultrastructure parameters using transmission electron microscopy⁸. Overall, the protocol shown here is a powerful approach that enables the systematic dissection of both cellular and molecular mechanisms mediating Drosophila olfactory circuit synaptic connectivity pruning during a juvenile critical period. The general odor exposure setup described in this study has been utilized in previous studies using other odors and assaying other glomeruli^3,7.

Protocol

1. Odorant exposure

1. Using a fine paintbrush, sort 40-50 developmentally-staged animals as pharate dark pupae (90+ h post-pupariation at 25 °C) into 25 mm x 95 mm polystyrene Drosophila vials containing standard cornmeal molasses food (Figure 1A).
2. Place fine stainless-steel wire mesh over the end of the Drosophila vials to contain the flies while also allowing good airflow. Secure the wire mesh caps with taped transparent film onto the side of the Drosophila vials.
3. In a 1.5 mL microcentrifuge tube, place 1 mL of 100% mineral oil (vehicle control) or dissolve the ethyl butyrate (EB) odorant in mineral oil in another tube to produce the desired concentration (e.g., 15% (150 µL) or 25% (250 µL)).
4. Place vehicle control or odorant microcentrifuge tubes upright within an air-tight 3700 mL Glasslock container. Using tape, anchor the tubes securely in the center of odorant chambers together with the Drosophila vials (Figure 1B).
5. Place the sealed vehicle control and EB odorant chambers containing the staged Drosophila pharate pupae vials into a humidified (70%), temperature-controlled (25 °C) incubator on a 12 h/12 h light/dark cycle.
6. Remove the newly-eclosed flies after 4 h of odorant chamber exposure and rapidly transfer 20-25 flies to fresh Drosophila vials in chambers with freshly made odorant (Figure 1A,B). Discard the uneclosed pupae.
7. Keep the flies in their sealed odorant chambers in the incubators for a total of 24 h. For longer odorant dosing, rapidly transfer flies to new Drosophila vials in freshly made odorant chambers every 24 h.
8. Anesthetize the developmentally-staged flies by submerging them in a dish of 70% ethanol for 1 min in preparation for immediate brain dissection and immunocytochemistry processing.

2. Brain dissection

1. Mark vials as vehicle control (100% mineral oil only) or EB exposed (% EB) by labeling and maintain these designations for the entire duration of brain dissection and subsequent processing. Process 10-20 animals for each genotype/odorant condition.
2. Using forceps, transfer a single anesthetized fly into a small dish of freshly made phosphate-buffered saline (PBS)¹³. Immerse the fly in PBS and continue full brain dissection with the fly fully submerged.
3. With fine (#5) sharpened forceps in both hands, place the fly ventral side up and grasp the upper thorax with one forceps and the head under the proboscis with the other forceps. Take care not to penetrate the brain.
4. Remove the head from the rest of the body by pulling gently in opposite directions with both hands. The head should easily detach from the thorax; leave the isolated head for dissection (Figure 1C).
5. Slide the forceps previously used to grasp the thorax under the opposite side of the proboscis. Begin to gently pull the exoskeleton cuticle in opposite directions so that it tears between the eyes to reveal the brain.
6. When pulling, the brain optic lobes may separate along with the exoskeleton. To prevent this, use a slow, steady pull with the two forceps while removing the exoskeleton cuticle (Figure 1C, middle).
7. Continue to remove the exoskeleton cuticle from the head. Ensure that all parts of the exoskeleton are removed. Remove any tissues attached to the brain, including the fat body and any projecting trachea.
8. To prevent sticking, rinse a P20 pipette tip with PBS + 0.2% Triton-X 100 (PBST). Immediately after dissection, transfer the brains into the fixing solution (4% paraformaldehyde (PFA) + 4% sucrose) in a capped tube (Figure 1C,D).

3. Brain immunocytochemistry

1. Fix the brain for 30 min at room temperature (RT) with end-over-end rotation. While keeping the brains in the tube, pipette off and properly dispose of the hazardous fixative. Quickly wash fixed brains 3x with PBS (Figure 1D).
2. Brains can often be found stuck in the cap of the tubes after rotation. To prevent accidental loss of brains, which is a constant hazard in all transfers, use a dissection microscope while pipetting liquids.
3. In preparation for antibody labeling, block the fixed brains for 1 h at RT in PBST + 1% bovine serum albumin (BSA) + 0.5% normal goat serum (NGS) with constant end-over-end rotation (Figure 1D).
4. Remove the block and incubate brains with the selected primary antibody diluted appropriately in PBST + 0.2% BSA + 0.1% NGS. Incubate the brains overnight (14-16 h) at 4 °C with constant rotation.
   NOTE: Example antibodies used: rat anti-CadN (dilution 1:50)¹⁴ to label synaptic glomeruli and chicken anti-GFP (1:1000)⁸ to label Or42a-mCD8::GFP (Figure 1D).
5. Pipette off the primary antibody. Wash brains 3x for 20 min each with PBST. Incubate brains with secondary antibodies diluted in PBST with 0.2% BSA + 0.1% NGS for 2 h at RT with constant rotation.
   NOTE: Alternatively, incubate brains overnight (14-16 h) at 4 °C. Dilute antibodies in PBST with 0.2% BSA + 0.1% NGS; secondary antibodies used here: 488 goat anti-chicken (1:250) and 546 goat anti-rat (1:250).
6. Pipette off the secondary antibodies. Wash brains 3x for 20 min each with PBST. Finish by washing brains for 20 min with PBS (Figure 1D).
7. Prepare glass microscope slides (75 mm x 25 mm) by adding two thin strips of double-sided adhesive tape to the slide ~25 mm apart from one another. This provides a spacer to avoid crushing the brains.
8. Pipette ~10-15 µL of mounting medium between the two strips of tape to mount the brains. With a P20 pipette tip pre-rinsed with PBST, transfer the labeled brains onto the microscope slide (Figure 1E).
9. Once the brains have been transferred, use a fine paintbrush to align them, ensuring the antennal lobes are facing upwards. Look for the side that has the arched hump, which will contain the antennal lobes.
10. Once the brains are properly oriented, cover them with a glass coverslip (No. 1.5H), ensuring the coverslip is secure on the tape. Then, fill in the sides of the coverslip with additional mounting medium (Figure 1E).
11. Seal the edges of the coverslip with clear nail polish. Allow the slide to dry thoroughly, and then store it in the refrigerator for subsequent imaging.

4. Confocal imaging

1. Blind all brain slides to both genotype and experience conditions prior to imaging by marking the slide with a coded label for later decoding.
2. Use a laser-scanning confocal microscope with a 63x oil-immersion objective. We use a Zeiss 510 META microscope in all images shown here (Figure 1F).
3. Use appropriate laser lines for the fluorophores employed. Here, use an Argon 488 and HeNe 543 laser for the antennal lobe synaptic glomeruli and Or42a olfactory sensory neuron imaging (Figure 2).
4. Determine the optimal gain and offset for both channels, ideally keeping the gain for both channels <750 and offset ≈0. This is done to ensure the signal-to-noise is optimal while limiting the background.
5. Position imaging to the center of the brain. When imaging, the VM7 glomeruli reside proximally to the hole left in the middle of the brain following removal of the esophagus during dissection (Figure 2A).
   NOTE: The VM7 glomeruli will always be close to the opening of the esophagus. However, the exact location and size vary slightly based on brain dissection and mounting differences, so take this into consideration when imaging.
6. Select the imaging resolution and optical slice thickness. Here, 1024 x 1024 resolution with a Z-stack slice thickness of 0.37 µm is used.
7. Take an entire confocal Z-stack projection through the antennal lobe, ensuring the capture of the full Or42a neuron innervation of the VM7 glomeruli.

5. Synaptic measurements

1. Load the genotype/condition-blinded image into Fiji (1.54f). Split the laser line channels by clicking Image > Color > Split Channels.
2. In the Or42a olfactory sensory channel, determine which slices contain the Or42a innervation by scrolling through the entirety of the Z-stack, identifying where the fluorescence begins and ends.
3. Create a sum slices projection including only slices containing Or42a neuron innervation by clicking Image > Stacks > Z Project > Sum Slices and entering the desired range.
4. In Fiji, click the Lasso tool on the top bar to trace the outline of the Or42a neuron innervation in the VM7 glomerulus, treating each brain side glomerulus independently (Figure 3).
5. Multiply the circumference by the number of Z-stack slices and the thickness of each slice to obtain the VM7 synaptic glomerulus innervation volume. To quantify synapse number, the find maxima tool can be used along with the find maxima stacks macro to count synapse puncta in the outlined region^8,15.

Results

Figure 1 shows the workflow for the olfactory experience-dependent critical period odorant exposure and brain imaging methods. The protocol starts with the age-matching of pharate dark pupae immediately prior to eclosion (Figure 1A). The pupae are placed into odorant chambers for 4 h, and then newly-eclosed adults are flipped into fresh vials in either the vehicle control or dosed EB odorant chambers (Figure 1B). We typically expose...

Discussion

The odorant exposure and brain imaging protocol presented here can be used to reliably induce and quantify experience-dependent olfactory sensory neuron synaptic glomeruli pruning during an early-life critical period. Earlier studies utilizing this treatment paradigm to explore olfactory circuit remodeling began odorant exposure on the 2^nd day after eclosion^3,4,5. In contrast, we begin odorant exposure in pharate pupa...

Materials

Name	Company	Catalog Number	Comments
For Odor Exposure
Drosophila vials	Genesee Scientific	32-110
Ethyl butyrate	Sigma Aldrich	E15701
Microcentrifuge tubes	Fisher Scientific	05-408-129
Mineral oil	Sigma Aldrich	M3516
Odor chambers	Glasslock
Paint brushes	Winsor & Newton	Series 233
Parafilm	Thermofisher	S37440
Wire mesh	Scienceware	378460000
Brain Dissection
Ethanol, 190 proof	Decon Labs	2801	Diluted to 70%
Forceps	Fine Science Tools	11251-30	Dumont #5
Paraformaldehyde	Electron Microscope Sciences	157-8	Diluted to 4%
Petri dishes	Fisher Scientific	08-757-100B
Phosphate-buffered saline	Thermo Fisher Scientific	70011-044	Diluted to 1x
Sucrose	Fisher Scientific	BP220-1
Sylgard	Electron Microscope Sciences	24236-10
Triton-X 100	Fisher Scientific	BP151-100
Brain Immunocytochemistry
488 goat anti-chicken	Invitrogen	A11039
546 goat anti-rat	Invitrogen	A11081
Bovine serum albumin	Sigma Aldrich	A9647
Chicken anti-GFP	Abcam	13970
Coverslips	Avantor	48366-067	25 x 25 mm
Double-sided tape	Scotch	34-8724-5228-8
Fluoromount-G	Electron Microscope Sciences	17984-25
Microscope slides	Fisher Scientific	12-544-2	75 x 25 mm
Nail polish	Sally Hansen	109	Xtreme Wear, Invisible
Normal goat serum	Sigma Aldrich	G9023
Rat anti-CadN	Developmental Studies Hybridoma Bank	AB_528121
Confocal/Analysis
Any computer/laptop
Confocal microscope	Carl Zeiss		Zeiss 510 META
Fiji software	Fiji		Version 2.14.0/1.54f