Name: an explant system for time-lapse imaging studies of olfactory circuit assembly in drosophila
Uploaded: 2021-10-13T14:45:00
Description: Watch this Scientific Journal Video about An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila at JoVE.com

We thank N. &#214;zel and R. Hiesinger for their advice on the explant culture; M. Wagner for technical help of the two-photon microscopy; D.J. Luginbuhl for generating transgenic flies; D. Friedmann for suggestions of Fiji software analysis; Y. Ge for assistance on fly work; C. McLaughlin and K.K.L. Wong for comments on the manuscript. L.L. is a Howard Hughes Medical Institute investigator. This work was supported by National Institutes of Health grants 1K99DC01883001 (to T.L.) and R01-DC005982 (to L.L.).

1. Preparation of reagents
NOTE: All the steps in this protocol are carried out at room temperature (20-25 &#176;C) unless explained otherwise.
<ol>
	<li>To prepare the culture dish for immobilizing the explant during time lapse imaging, lay 0.5 cm thick Sylgard (thoroughly mix two liquid components at 10:1 ratio before use) on the bottom surface of a 60 mm x 15 mm Petri dish and let it cure for 48 h at room temperature (Figure 2A, referred as S...

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The Drosophila antennae-brain explant retains normal targeting of the olfactory circuit. We did notice that the development is 2 times slower ex vivo compared to in vivo. It is noted that the explant system does not retain maxillary palp, which hosts six types of ORNs. To ensure normal development is recapitulated ex vivo, stretching of the antennal nerves needs to be avoided during explant dissection. During ex vivo culture bacteria growth usually causes arrested development ...

Neurons are precisely interconnected to form circuits essential for the proper function of the brain. For over 100 years, neuroscientists have been trying to understand how neurites extend toward their intermediate and final targets with extreme precision. As a result, they have identified important genes that encode guidance cues for developing neuronal processes1. The Drosophila olfactory system provides an excellent model to investigate this process since olfactory receptor neurons (ORNs, the primary sensory neurons) project to 50 identifiable glomeruli with stereotypical size, shape, and relative position, where they form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs), each of which send dendrites to one of the 50 glomeruli2 (Figure 1A). Therefore, it is relatively easy to identify mutant phenotypes at synaptic (glomerular) resolution in the fly olfactory system. This led to discoveries of important genes that regulate olfactory circuit assembly3.
The assembly of the fly olfactory circuit relies on temporally and spatially coordinated developmental processes3. ORNs and PNs acquire distinct cell fates, which set up the program for their wiring specificities. Next, PN dendrites prepattern the antennal lobe (Figure 1B). The axons of ORNs then circumnavigate the ipsilateral antennal lobe and cross the midline of the brain to reach the contralateral antennal lobe. Subsequently, ORN axons invade both ipsi- and contralateral antennal lobes and form synapses with dendrites of their partner PNs in specific glomeruli. This coarse model for olfactory circuit assembly was proposed based on the characterization of fixed samples from intermediate time points during the development. The poor temporal resolution and inability to follow the same neuronal processes across development from fixed tissue limit the mechanistic understanding of the circuit assembly process.
It is technically challenging to live image ORN and PN processes in vivo since the wiring process occurs in the first half of the pupal stage when the antennal lobe is surrounded by opaque fat body inside the pupal case. It is, therefore, impossible to directly image the developing olfactory circuit from intact pupae. Dissected tissues cultured ex vivo can circumvent tissue opacity and have been successfully used to study neural development4,5,6. The challenge of using a similar ex vivo explant culture strategy to study neuronal wiring in the pupal brain is whether it recapitulates the precise neuron targeting in a culture condition. Based on a previously reported ex vivo culture condition for the fly eye-brain complex7, an explant that contains the whole pupal brain, antennae, and the connecting antennal nerves intact has been recently developed, which retains precise targeting of the olfactory circuit and can be subjected to two-photon microscopy-based live imaging for up to 24 h at the frequency of every 20 min8. Here, a detailed protocol of the explant culture and imaging is described. The explant system provides a powerful method to study the assembly of olfactory circuit and potentially other circuits in the central brain.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20-hydroxyecdysone</td>
			<td>Sigma</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Ti:Sapphire laser</td>
			<td>Coherent</td>
			<td>Coherent MRU X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>12585014</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Prairie</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>World Precision Instruments</td>
			<td>501778</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen cylinder</td>
			<td>Praxair</td>
			<td>OX M-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21720024</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0162601</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscopy</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>water immerse objective (20X)</td>
			<td>Zeiss</td>
			<td>421452-9800-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an explant system for time-lapse imaging studies of olfactory circuit assembly in drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

ORN axons arrive at the antennal lobe between 18 h and 36 h APF. They then navigate the antennal lobe, cross the midline, and innervate the glomeruli. Video 1 is a representative video showing the entire process for several individually identifiable axons, taken at the frequency of every 20 min for 24 h. Before registration using TurboReg, the axons exhibit some lateral drifting as the brain develops (first half of the video). After registration, the drifting is corrected (second half of the video).
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Watch this Scientific Journal Video about An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila at JoVE.com

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

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