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 Sylgard plate in the following text).</li>
	<li>To prepare micro pins for immobilizing explant on this plate, use a pair of forceps to stick multiple micro pins on a tape with the sharp ends aligned on one side (Figure 2B). Use a pair of scissors to cut ~2 mm from the sharp ends of the micro pins (Figure 2B&#39;). Use forceps to hold the cut micro pins and insert into the Sylgard layer of a pre-made Sylgard plate (Figure 2C). Two micro pins are used to immobilize one explant.</li>
	<li>Use a brush to collect white pupae, which form puparium within 1 h, of hsFLP, pebbled-GAL4/+; UAS-FRT100-stop-FRT100-mCD8-GFP8 genotype and transfer them to new vials. Heat shock in 37 &#176;C water bath for 40 min to induce sparse ORN clones from random types. After heat shock, put the vials at 25 &#176;C for 30 h, resulting in pupae aged at 30 h after puparium formation (APF).</li>
	<li>To prepare the culture medium for explant, add 5 mL of Penicillin-Streptomycin (10,000 U/mL) to 500 mL Schneider&#39;s Drosophila Medium. Filter the medium and make 45 mL aliquots in 50 mL conical tubes. The medium can be stored at 4 &#176;C for 1-2 months.</li>
	<li>On the day of imaging, take one tube of 45 mL of Schneider&#39;s Drosophila medium and add 5 mL of Fetal Bovine Serum (10% v/v), 125 &#181;L of 4 mg/mL human insulin stock solution (10 &#181;g/mL final concentration), 50 &#181;L of 1 mg/mL 20-hydroxyecdysone stock solution dissolved in ethanol (1 &#181;g/mL final concentration). Mix well and transfer 15 mL of full medium into a new 50 mL conical tube. The rest of the full medium can be stored at 4 &#176;C for a week. Fetal Bovine Serum, human insulin stock solution and 20-hydroxyecdysone stock solution are aliquoted and stored at -20 &#176;C.</li>
	<li>Oxygenate the 15 mL full medium by pumping oxygen bubbles from an oxygen cylinder under the liquid surface through a sterile 5 mL pipette tip at the rate of one bubble/s for 20-30 min. Use a paraffin film to cover the opening of the tube during this process.</li>
	<li>Sterilize the dissection well surface and the Sylgard plate (with micro pins inserted on the Sylgard layer, prepared in steps A1 and A2) with 70% ethanol. Let them dry before use.</li>
</ol>
2. Explant dissection
<ol>
	<li>Use a brush to transfer 30 h APF (30 h after puparium formation) pupae to a paper tissue and dry the external surface of the pupae for 5 min.</li>
	<li>Put a piece of double-sided tape on a glass slide. Carefully attach the dried pupae on the sticky surface of the tape with the dorsal side facing upward. Gently press the pupae with a brush to help the ventral side of the pupae attach well to the tape (Figure 3A). Do not damage the pupa.</li>
	<li>Use a pair of forceps to remove brown pupal case covering the dorsal side of the head (Figure 3A,B). Insert one sharp tip of the forceps between the brown pupal case and the pupa from the lateral side and carefully break the brown pupal case through a line to the posterior end of the pupa (Figure 3B,C). Open the brown pupal case. Use a pair of forceps to gently hold the pupa and transfer to the dissection well with 1 mL of oxygenated full medium. Submerge floating pupa on the medium surface to help it sink to the bottom of the well (Figure 3D). 
	NOTE: Do not insert the forceps tip too deeply inside the brown pupal case to prevent injuring the pupa with the forceps.</li>
	<li>To dissect the antennae-brain explant from the pupa, use forceps to gently hold the pupa with one hand and use a pair of microscissors to cut a small hole from the posterior side of the pupa with the other hand (Figure 3E). This small hole releases the high pressure inside the pupa.</li>
	<li>Cut through the ventral midline of the pupa from the hole until the neck (the narrow structure that connects the head and the thorax) with the microscissors (Figure 3F). Then, cut through the circumference of the neck to detach the head from the body of the pupa (Figure 3G). Remove the body and place it in a different well. 
	NOTE: Do not cut the neck directly from the dorsal/ventral side of the pupa, which may squeeze the brain.</li>
	<li>Cut the transparent cuticle that covers the dorsal side of the brain (Figure 3H). This will expose the fat body on top of the brain. Keep some cuticle to which the retina and antennae attach. Repeat the same procedure to the ventral side of the brain. 
	NOTE: Do not insert the blade of the scissors too deeply under the cuticle as this will cause severing of the antennal nerves connecting the antennae and brain (Figure 3H&#39;).</li>
	<li>Use a P10 pipette to gently wash out the fat body that covers the brain and antennae by pipetting the medium toward the open regions on the dorsal and ventral sides of the head (Figure 3I). 
	NOTE: Be very gentle when pipetting the medium as the brain can easily be detached from the cuticle. Make sure all fat body is removed during this step. Arrested development of ORN axons were observed when fat body was not cleaned well, probably due to poor oxygen access from the medium.</li>
	<li>To study the interaction of bilateral ORN axons or ORN axons to PN dendrite targeting, sever one or two antennal nerves with the microscissors during this stage8 (Figure 3J). Carefully place the blades of the scissors between the cuticle and the brain and sever interested antennal nerves.</li>
	<li>To transfer the dissected explant to the Sylgard plate, place a droplet of oxygenated full medium (~200 &#181;L) on the Sylgard surface. Coat the inner surface of a 200 &#181;L wide tip pipette tip with the fat body from the dissected trunk (step 1.6) by pipetting the fat body several times, which prevents the explants from sticking the pipette tip during transfer. Then, use this wide tip pipette tip to transfer the explant from the dissection well to the medium droplet on the culture plate (Figure 3K).</li>
	<li>Use forceps to pin the explant on the Sylgard layer in the two optic lobes (Figure 3L). Carefully position the Sylgard plate on the imaging station and immobilize the plate with tapes. Slowly add 10 mL of oxygenated full medium to the Sylgard plate using P1000 pipette. 
	&#8203;NOTE: Avoid disrupting the explant when adding the medium to the Sylgard plate.</li>
</ol>
3. Two-photon microscopy-based live imaging
<ol>
	<li>To perform time-lapse imaging, use a two-photon microscope, a Ti:Sapphire laser, a 20x water-immersion objective (1.0 NA) and an imaging software. Use the excitation wavelength at 920 nm for imaging GFP proteins. Adjust the pixel dwell time to 10 &#181;s.</li>
	<li>Adjust the imaging station position so that the explants are roughly under the objective. Use 70% ethanol to sterilize the lens before imaging. Slowly lower the objective under the medium close to the explants. Check whether there is any bubble on the lens of objective.
	<ol>
		<li>If so, lift the objective above the medium and repeat this until the bubble is gone. Find the explants using the eyepiece and center one explant in the field.</li>
	</ol>
	</li>
	<li>To ensure an explant with a few ORNs sparsely labeled for time lapse imaging, dissect ~10 explants each time and align on y axis on the culture plate. Screen all explants by moving the objective along y axis and choose an explant in which a few single ORN axons have just reached the antennal lobe for imaging (Figure 4A).
	<ol>
		<li>Recognize the antennal lobe by its oval shape and the ORN axons that are beginning to circumnavigate it. Image a ~150 &#181;m x 150 &#181;m area in the xy plane (3x zoom with the 20x objective). Estimate the boundary of the two antennal lobes and center them in the imaging area.</li>
	</ol>
	</li>
	<li>Select an initial imaging region along the z axis by defining the bottom section and top section of scanning. Set up imaging area along the z axis. Set the deepest section with ORN axon signals as the first imaging session and the session 100 &#181;m above (more superficial side) as the last imaging session (Figure 4B). 
	NOTE: This leaves some sections on top (superficial side) of the ORN axons and avoids shifting of ORN axons upward outside the imaging area due to the growth of the brain during culture.
	<ol>
		<li>Image at 2 &#181;m intervals. Set automatic imaging scanning at the frequency of every 20 min using imaging software.</li>
	</ol>
	</li>
	<li>Shift the imaging region 20 &#181;m upward along the z axis after the first 4 h imaging and another 20 &#181;m upward along the z axis after 16 h imaging. This can be achieved by setting a script and different z stacks in the imaging software.</li>
	<li>Culture the explant for an additional period post imaging (up to 24 h ex vivo) before fixation and staining with N-cadherin, a neuropil marker, to reveal the genetic identity of each single ORN by the glomerulus it targets to.</li>
</ol>
4. Image processing
<ol>
	<li>To process z stack images from section series taken at each time point using the Fiji software, open image section series, click on Image | Stacks | Z project [/].</li>
	<li>To correct lateral drift of the sample during culture, install TurboReg Plugin in Fiji.
	<ol>
		<li>Open a z stack image series and a single z stack image from the series. Open Plugins | Registration | TurboReg.</li>
		<li>Select the z stack image series in Source and the single z stack image in Target | Translation. Click on the Batch button to register all the images from the opened z stack image series.</li>
	</ol>
	</li>
	<li>To maximize the utility of imaged samples, separate sparsely labeled single axons in the vicinity to each other from the z stack images following 3D image sections.
	<ol>
		<li>To extract single ORN axons from a few axons in the same image, open the image section series, click on Plugins | Segmentation | Segmentation Editor [/]. Select the brush tool and mask interested ORN axon in the Segmentation Editor working window using Select &#34;+&#34; or &#34;-&#34; buttons on each image section.</li>
		<li>Click on Process | Image Calculator [/]. Select &#34;Image X&#34; in Image 1, &#34;Multiply&#34; in Operation, &#34;Image X. labels&#34; in Image 2, [/]. This generates a new image series file with the interested axon only. Perform step 4.1 to process the z stack image. Repeat this step for all time points to generate a time series image file.</li>
	</ol>
	</li>
	<li>To pseudocolor different axons from the same image, first perform step 4.3 to generate the time series image file of each axon separately. Open the time series image files for different single axons from the same raw data image. Click on Image | Color | Merge Channels. Select different time series image files in different color channels and click OK.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+smell+and+taste+in+Drosophila.">Molecular architecture of smell and taste in Drosophila.</a> Annual Review Neuroscience. 30, 505-533 (2007).</li><li>Hong, W., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+wiring+specificity+in+the+fly+olfactory+system.">Genetic control of wiring specificity in the fly olfactory system.</a> Genetics. 196 (1), 17-29 (2014).</li><li>Bentley, D., Caudy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pioneer+axons+lose+directed+growth+after+selective+killing+of+guidepost+cells.">Pioneer axons lose directed growth after selective killing of guidepost cells.</a> Nature. 304 (5921), 62-65 (1983).</li><li>Godement, P., Wang, L. 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The authors have nothing to disclose.

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 of the olfactory circuit. Therefore, thorough sterilization of the culture dish and pins before imaging and keeping the imaging room clean and isolated is important.
This explant supports long-term two-photon microscopy based time-lapse imaging. Combined with a newly developed reporter for sparse labeling of single ORNs, the explant system allows high-resolution imaging from single axon terminus. This system is powerful to study the cell biological mechanisms underpinning the dynamic process of olfactory circuit assembly8. Although olfactory circuit development was shown here as an example, this system can potentially be expanded to studies of other circuits or other developmental processes in the developing central brain.
The explant maintains normal development in culture for at least 24 h, which can capture the entire process of ORN targeting. It enables researchers to reveal the genetic identity of single ORN axons through counter-staining with a neuropil marker post fixation, as the antennal lobe already develops obvious glomerular structure by the end of the culture. This strategy circumvents the problem of lacking specific genetic drivers for many ORN types at an early developmental stage to achieve imaging of specific types of ORNs using a pan-ORN driver.
To achieve higher spatiotemporal resolution, this explant system can be imaged using more advanced microscopy, the adaptive optics-lattice light-sheet microscopy (AO-LLSM). It has been shown that the AO-LLSM enables visualization of fine structures of axon terminals and scanning frequency at every 30 second per volume8,9,10,11. One advantage of the explant is its compatibility with Janelia Fluorophore dye12,13,14 labeling by incubating the explant expressing Halo-tag in specific neurons with dyes in the medium before imaging. It was noticed that incubating the explant with dye-containing medium results in much stronger labeling than feeding the larvae with the dye. This unique advantage allowed us to image axons at an early developmental stage that was hardly visualized by GFP labeling8.
In addition to time-lapse imaging, the explant system has other advantages. For example, it is possible to sever antennal nerves from the dissected explant unilaterally or bilaterally at specific developmental time points (step 2.8). This allows researchers to probe the requirement of ORN axons in the targeting of any neuron types in the olfactory circuit at distinct developmental steps. In particular unilateral antennal nerve severing assays, which cannot be achieved by traditional genetic manipulation, led to an interesting discovery that interaction between bilateral ORN axons is required for correct contralateral targeting of ORN axons8. Furthermore, the explant is cultured directly in medium instead of being embedded in agarose, therefore allowing fast delivery and wash out of some small molecules or drugs. Compared with genetic manipulation, drug treatment has the advantages of rapid effect and reversibility of the manipulation. It enables researchers to assess some essential processes for cells at later developmental stages by bypassing cell lethality or unhealthy issues due to constitutive disruption through genetic manipulations. It also helps visualization of subtle changes by comparing before and after drug treatment, and after drug washout.

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><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&amp;rsquo;s &lt;em&gt;Drosophila &lt;/em&gt;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).
To separate a few ORN axons from the same explant, one example is shown in Figure 5. Following the procedure in step 4.3, VA1d and VA1v axons from explant shown in Figure 5A were extracted to generate a new z stack image with only these two axons (Figure 5B). Similarly, VM2 and VM3 axons (Figure 5B&#39;) and DL2 axon (Figure 5B&#39;&#39;) were extracted. Figure 5C shows a merge of images in Figure 5B-B&#39;&#39; with pseudocolors. The genetic identities of each ORN axons were revealed by immunostaining of a neuropil marker N-cadherin of the fixed explant (Figure 5D,E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62983/62983fig01.jpg" /> 
Figure 1: Structure of fly olfactory circuit. (A) An adult fly head is shown with one ORN from the right antennae (green) sending its axon to both antennal lobes (ALs) in the brain and forming synaptic connection in a specific glomerulus with dendrites of PNs (red) in the ipsilateral and contralateral ALs. Dashed vertical line indicates midline in this and subsequent diagrams and images. (B) Diagram showing the olfactory circuit development. (1) PN dendrites first innervate a region in the antennal lobe (red). ORN axons reach the antennal lobes in the brain. (2) ORN axons take either a dorsolateral (green) or ventromedial (blue) trajectory to circumnavigate the antennal lobe. (3) ORN axons cross the midline. (4) ORN axons innervate glomeruli in the antennal lobe. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62983/62983fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62983/62983fig02.jpg" /> 
Figure 2: Preparation of the imaging chamber for the explant. (A) Lay a layer of silicone elastomer (~0.5 cm) at the bottom of a 60 mm Petri dish. (B-B&#39;) Align pins on a tape and cut to ~2 mm long using a pair of scissors. (C) Pin the micro pins on the silicone elastomer layer of the culture plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62983/62983fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62983/62983fig03.jpg" /> 
Figure 3: The dissection procedure for the antennae-brain explant. (A) Attach the ventral side of a paper tissue-dried pupa on a double-sided tape on a glass slide. (B-C) Use forceps to cut the external brown cuticle to expose the pupa inside. (D) Transfer the pupa to a dissection well with oxygenated full medium. (E-G) Carefully separate the pupal trunk from the head using microscissors. (H) Cut the pieces of semitransparent cuticle covering the dorsal and ventral sides of the brain. Keep some cuticle on the anterior and lateral sides of the brain to retain connections between the retina, antennae, and brain. (H&#39;) Avoid severing the antennal nerve during this step. (I) Clean the fat body covering the brain by gently pipetting. (J) Sever one or two antennal nerve(s) using microscissors in certain experiments. (K) Place a droplet of oxygenated full medium on the surface of the culture plate. Transfer the dissected explant using a wide tip pipette tip. (L) Use forceps to pin the two optic lobes of the explant on the silicone elastomer layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62983/62983fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62983/62983fig04.jpg" /> 
Figure 4: Time-lapse imaging of single ORN axon targeting from an explant. (A) Select an explant with a few ORN axons just reaching the antennal lobe. Estimate the shape of two antennal lobes by the curvature of the axons and center the antennal lobes in the imaging field. (B) Set the imaging region along the z axis. Consider that the antennal lobe will shift upward as the brain grows and develops. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62983/62983fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62983/62983fig05.jpg" /> 
Figure 5: Extract single ORNs and reveal their glomerular identities. (A) A maximum projection image of an explant with 5-10 single ORN axons using two-photon microscopy with 20x objective and 3x zoom in. (B-B&#39;&#39;) 1-2 single axons are extracted from (A) by manually creating masks in image sections from the raw image data. (C) Merge the images of (B-B&#39;&#39;) with each axon pseudo-colored differently. (D-D&#39;) Maximum projection confocal images taken with 40x objective and 1.5x zoom. Explant shown in (A) was fixed followed by staining with anti-GFP and anti-N-cadherin (neuropil marker). Anterior and posterior halves of the antennal lobes are stacks separately in (D) and (D&#39;). (E) Antennal lobe map shows extracted ORN axons in (B,C). Some images shown in this figure are modified from a prior study8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62983/62983fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Two-photon microscopy based time-lapse images show targeting of two ORN axons, before and after image registration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62983/video 1.mp4" target="_blank">Please click here to download this Video.</a>

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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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.1K Views.  Stanford University. 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.

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

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Neuroscience

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

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

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

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

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

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Rozdziały w tym wideo

Live-imaging of the Drosophila Pupal Eye

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

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