drosophila brain dissection to analyze ethyl butyrate (eb) experience-dependent synaptic pruning of or42a receptor

Olfactory Experience-Induced Synaptic Remodeling in Drosophila's Juvenile Brain

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 glomeruli3,4. 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 volume8,9,10. 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 glomeruli3,7.

Author Spotlight: Exploring Glial Influence in Experience-Dependent Synaptic Pruning During Critical Periods

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

My research entails exploring glial roles and experience dependent critical period synapse remodeling. I'm determining the role of glia in synaptic pruning, specifically during critical periods and the neuron to glia communication, enabling pruning driven by experience. Our protocol has the proven power of genetics to define molecular mechanisms.To study gene environment interactions, we can precisely manipulate the odor, concentration, duration, and timing in a short, well-defined critical period. This protocol is incredibly versatile in inducing synapse remodeling. Our results established an excellent model amenable to genetic and pharmaceutical manipulations that could potentially reopen critical period like plasticity during adulthood, which is a topic of enormous interest for the treatment of injury, trauma, and neurological disorders.

Drosophila Brain Dissection to Analyze Ethyl Butyrate (EB) Experience-Dependent Synaptic Pruning of Or42a Receptor

To begin, take drosophila flies exposed to mineral oil and EB for 24 hours. Label vials as a vehicle control or EB exposed, and maintain these designations throughout brain dissection and processing. Using forceps, transfer a single anesthetized fly into a small dish of freshly made PBS.Fully submerge the fly in PBS for full brain dissection. Then, position a fly ventral side up. Take two fine sharpened forceps to grasp the upper thorax with one forcep and the head, under the proboscis with the other.Pull the head and the rest of the body in opposite directions to remove the head. Ensure the head easily detaches from the thorax and leaves the isolated head for dissection. Slide the forceps, previously used to grasp the thorax, under the opposite side of the proboscis.Gently pull the exoskeleton cuticle in opposite directions to tear up between the eyes and reveal the brain. Continue to remove the exoskeleton cuticle from the head, ensuring all parts are removed. Now, rinse a P20 pipette tip with PBS and 0.2%Triton X-100.Transfer the brains into the fixing solution in the cap tube immediately after dissection.