We thank the other Broadie Lab members for their valuable input. Figures were created using BioRender.com. This work was supported by National Institute of Health grants MH084989 and NS131557 to K.B.

1. Odorant exposure
<ol>
	<li>Using a fine paintbrush, sort 40-50 developmentally-staged animals as pharate dark pupae (90+ h post-pupariation at 25 &#176;C) into 25 mm x 95 mm polystyrene Drosophila vials containing standard cornmeal molasses food (Figure 1A).</li>
	<li>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...

Olfactory Experience-Induced Synaptic Remodeling in &lt;i&gt;Drosophila&#039;s&lt;/i&gt; Juvenile Brain

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M., Vest, J., Vita, D. J., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+remodeling+of+Drosophila+olfactory+sensory+neuron+brain+innervation+during+an+early-life+critical+period.">Activity-dependent remodeling of Drosophila olfactory sensory neuron brain innervation during an early-life critical period.</a> J Neurosci. 39 (16), 2995-3012 (2019).</li><li>Golovin, R. 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V., Ramaswami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerulus-selective+regulation+of+a+critical+period+for+interneuron+plasticity+in+the+drosophila+antennal+lobe.">Glomerulus-selective regulation of a critical period for interneuron plasticity in the drosophila antennal lobe.</a> J Neurosci. 40 (29), 5549-5560 (2020).</li><li>Stephan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Psidin+regulates+olfactory+neuron+number+and+axon+targeting+through+two+distinct+molecular+mechanisms.">Drosophila Psidin regulates olfactory neuron number and axon targeting through two distinct molecular mechanisms.</a> J Neurosci. 32 (46), 16080 (2012).</li><li>Doll, C. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+FMRP+requirements+in+development+of+the+neural+circuitry+of+learning+and+memory.">Activity-dependent FMRP requirements in development of the neural circuitry of learning and memory.</a> Development. 142 (7), 1346-1356 (2015).</li><li>Tito, A. J., Cheema, S., Jiang, M., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+one-step+dissection+protocol+for+whole-mount+preparation+of+adult+Drosophila+brains.">A simple one-step dissection protocol for whole-mount preparation of adult Drosophila brains.</a> J Vis Exp. (118), e55128 (2016).</li><li>Okumura, M., Kato, T., Miura, M., Chihara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+axon+targeting+of+Drosophila+olfactory+receptor+neurons+specified+by+the+proneural+transcription+factors+Atonal+and+Amos.">Hierarchical axon targeting of Drosophila olfactory receptor neurons specified by the proneural transcription factors Atonal and Amos.</a> Genes to Cells. 21 (1), 53-64 (2016).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Vita, D. J., Meier, C. J., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+fragile+X+mental+retardation+protein+activates+glial+insulin+receptor+mediated+PDF-Tri+neuron+developmental+clearance.">Neuronal fragile X mental retardation protein activates glial insulin receptor mediated PDF-Tri neuron developmental clearance.</a> Nat Comm. 12 (1), 1160 (2021).</li><li>Gugel, Z. V., Maurais, E. G., Hong, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+odors+at+naturally+occurring+concentrations+triggers+limited+plasticity+in+early+stages+of+Drosophila+olfactory+processing.">Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing.</a> eLife. 12, 85443 (2023).</li></ol>

The authors declare no competing interests.

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 2nd day after eclosion3,4,5. In contrast, we begin odorant exposure in pharate pupa...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>For Odor Exposure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila vials</td>
			<td>Genesee Scientific</td>
			<td>32-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl butyrate</td>
			<td>Sigma Aldrich</td>
			<td>E15701</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Odor chambers</td>
			<td>Glasslock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brushes</td>
			<td>Winsor &#38; Newton</td>
			<td>Series 233</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Thermofisher</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire mesh</td>
			<td>Scienceware</td>
			<td>378460000</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 190 proof</td>
			<td>Decon Labs</td>
			<td>2801</td>
			<td>Diluted to 70%</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Electron Microscope Sciences</td>
			<td>157-8</td>
			<td>Diluted to 4%</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific&#160;</td>
			<td>08-757-100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011-044</td>
			<td>Diluted to 1x</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Electron Microscope Sciences</td>
			<td>24236-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Immunocytochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>488 goat anti-chicken</td>
			<td>Invitrogen</td>
			<td>A11039</td>
			<td></td>
		</tr>
		<tr>
			<td>546 goat anti-rat</td>
			<td>Invitrogen</td>
			<td>A11081</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>Abcam</td>
			<td>13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Avantor</td>
			<td>48366-067</td>
			<td>25 x 25 mm</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>34-8724-5228-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G&#160;</td>
			<td>Electron Microscope Sciences</td>
			<td>17984-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-2</td>
			<td>75 x 25 mm</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Sally Hansen</td>
			<td>109</td>
			<td>Xtreme Wear, Invisible</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-CadN</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AB_528121</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal/Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Any computer/laptop</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Zeiss 510 META&#160;</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Fiji</td>
			<td></td>
			<td>Version 2.14.0/1.54f</td>
		</tr>
	</tbody>
</table>

experience-dependent remodeling of juvenile brain olfactory sensory neuron synaptic connectivity in an early-life critical period

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.

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.

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...

Read this Scientific Article Protocol about Author Spotlight: Exploring Glial Influence in Experience-Dependent Synaptic Pruning During Critical Periods at JoVE.com

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

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially ...

experience-dependent-remodeling-juvenile-brain-olfactory-sensory

Research

JoVE Journal

Neuroscience

Inducing Ethyl Butyrate (EB) Experience-Dependent Synaptic Pruning of Or42a Receptor

To begin, take the vial containing the drosophila larvae. Using a fine paintbrush, sort 40 to 50 pharate dark pupae in polystyrene drosophila vials containing standard cornmeal molasses food. Place fine stainless steel wire mesh over the end of the drosophila vials to contain the flies while allowing good airflow.Secure the wire mesh caps with a tape transparent film onto the side of the vials. Then, add one milliliter of 100%mineral oil, or dissolve EB in mineral oil in 1.5 milliliter tubes. Place these tubes upright within a 3, 700 milliliter glass lock container.Using tape, anchor the tubes in the center of the odorant chambers, along with the drosophila vials. Place the sealed vehicle control and EB odorant chambers containing the pupae vials in a humidified incubator on a 12 hour light and 12 hour dark cycle. After four hours of odorant chamber exposure, rapidly transfer 20 to 25 newly eclosed flies to fresh drosophila vials in chambers with freshly made odorant.Keep the flies in their sealed odorant chambers in the incubators for a total of 24 hours.

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.

Confocal Imaging and Synaptic Measurements to Analyze Experience-Dependent Critical Period Synaptic Glomeruli Pruning

To begin, take a slide and add two thin strips of double-sided adhesive tape to it to prepare for imaging. Pipette approximately 10 to 15 microliters of mounting medium between the two strips of tape for brain mounting. transfer the label drosophila brains onto a microscope slide with a P20 pipette tip prerinsed with PBS and 0.2%Triton X-100.Once the brains are transferred, use a fine paintbrush to align them, ensuring the antennal lobes face upwards. After orienting the brains, cover them with a glass coverslip. Then, fill in the sides of the coverslip with additional mounting medium.Seal the edges of the cover slip with clear nail polish. After drying the slide thoroughly, store it in the refrigerator for subsequent imaging. Use a laser scanning confocal microscope equipped with a 63X oil immersion objective for imaging.Employ an argon 488 and helium neon 543 laser for imaging the an antennal lobe's synaptic glomeruli and Or42a olfactory sensory neurons. Determine the optical gain and offset for both channels. Position the imaging to the center of the brain and focus on the VM7 glomeruli.Locate approximately to the hole in the middle of the brain. Select the imaging resolution of 1024 by 1024. Capture an entire confocal Z stack projection through the antennal lobe to ensure the full Or42a neuron innervation of the VM7 glomeruli is captured.Load the genotype or condition blinded image into Fiji. To split the laser line channels, go to image, click color, and select split channels. In the Or42a olfactory sensory channel, scroll through the Z stack to determine which slices contain the Or42a neuron innervation, identifying the beginning and end of fluorescence.To create a sum slices projection that includes only slices with Or42a neuron innervation, click image and go to stacks. Then, click Z project, select sum slices, and enter the desired range. Use the lasso tool in Fiji to trace the outline of the Or42a neuron innervation in the VM7 glomerulus.Multiply the circumference of the traced area by the number of Z stack slices and the thickness of each slice to calculate the VM7 synaptic glomerulus innervation volume. After 24 hour exposure to a control odorant vehicle, dense Or42a MCD:GFP innervation persists in VM7 glomeruli of both antennal lobes. In contrast, a 24 hour exposure to 25%EB odorant causes significant pruning and loss of synaptic glomerular volume.Increasing EB odorant concentration causes progressively greater synaptic glomeruli pruning. Representative quantifications for this range of EB odorant concentrations showed similar results for fluorescence intensity and innervation volume.

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

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

Summary