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 Drosophila's Juvenile Brain

<ol>
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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. M., Vest, 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=Neuron-specific+FMRP+roles+in+experience-dependent+remodeling+of+olfactory+brain+innervation+during+an+early-life+critical+period.">Neuron-specific FMRP roles in experience-dependent remodeling of olfactory brain innervation during an early-life critical period.</a> J Neurosci. 41 (6), 1218-1241 (2021).</li><li>Chodankar, A., Sadanandappa, M. K., Raghavan, K. 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 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.