Figure 2. Connections between olfactory receptor neurons and antennal lobe neurons revealed by TRACT.
(a) Olfactory receptor neurons (ORNs) labeled in red, have their cell bodies located into two peripheral sensory organs, antennae (illustrated here) and maxillary palps, and have axons that project into the antennal lobe (AL) in the brain. All axons from ORNs expressing the same olfactory receptor converge in two glomeruli (one in each hemisphere) within the AL (red circles). There are two main types of neurons (green) in the antennal lobe, projection neurons (PNs) and local neurons (LNs). PNs have dendrites that branch in glomeruli and axons that project towards the mushroom body (MB) and/or the lateral horn (LH). (b, c) There are two types of PNs, uniglomerular PNs (uniPNs) and multiglomerular PNs (multiPNs). (b) The dendrites of uniPNs branch in a single glomerulus, and their axons project into the MB and LH via the medial antennal lobe tract (mALT). (c) The dendrites of multiPNS branch into multiple glomeruli, and their axons project into the LH via the mediolateral ALT (mlALT). (d) LNs are axonless neurons and their dendrites branch into many (or most) of the glomeruli. (e) Detection of synaptic contacts between olfactory receptor neurons and antennal lobe neurons in the adult Drosophila antennal lobe with the nlgSNTG4 receptor driven by nSybE enhancer (top panels) and GH146 enhancer (bottom panels). Induction of GFP expression in neurons (arrows) surrounding the antennal lobe when the ligand (nSyb::CD19) was driven by the orco driver in ORNs. Arrowheads indicate the axons of PNs in mALT and mlALT. Left: nSyb::CD19+ axons from ORNs (red); middle: GFP+ neurons in the antennal lobe (green); right: merged images of nSyb::CD19 and GFP. In the brains with the nlgSNTG4 receptor driven by the nSybE enhancer (top panels) GFP expression was induced in different neuronal types, including uniPNs, LNs and and several multiPNs at the ventral part. In the brain with the nlgSNTG4 receptor driven by the PN-specific driver GH146 (bottom panels), only PNs were GFP+. Most of these cells are uniPNs, but there was also one GFP+ multiPN in each antennal lobe (yellow arrow). Maximum projection of z-stack confocal images. Scale bar = 50 µm. See Supplementary file 2 for additional information.
Figure 2—figure supplement 1. TRACT reveals antennal lobe neurons that have cell-cell contacts with olfactory receptor neurons.
(a) and (b) Detection of neuron-neuron contacts in the adult Drosophila antennal lobe (stippled circle) with nlgSNTG4 driven by elav promoter. Top panels show maximum projection of z-stack confocal images; the bottom panels show single optical sections from the z-stack on the top panels. (a) Control samples without orco-lexA driver (carrying elav-nlgSNTG4, 5xUAS-CD4::tdGFP and LexAop-CD19mch) show no induction in antennal lobe neurons. The stars mark the ligand-independent background in the mushroom body (top left) and axons from Johnson’s organ neurons (bottom right). (b) Top panels show induction of CD8:GFP expression in neurons (arrows) surrounding the antennal lobe (stippled circle) when the ligand (CD19mch) was driven by the orco driver in ORNs. Left: CD8:GFP+ neurons; middle: CD19mch+ axons from ORNs; right: merged images of CD19mch (red), CD8:GFP (green). Bottom panels show a single optical section from the stack on top with GABA immunostaining (magenta), confirming that some of the GFP+ neurons (green) induced by CD19mch+ ORNs are GABAergic LNs (arrows). The arrowheads indicate the GFP+, GABA- neurons. left: distribution of GFP+ neurons; middle: GABA immunostaining; right: merged images of GABA (magenta), GFP (green). Scale bar = 20 μm. (c, d) Detection of neuron-neuron contacts in the adult Drosophila antennal lobe with nlgSNTG4 driven by nSyb promoter. (c) Control brains without orco-lexA driver (carrying nSyb-nlgSNTG4, 5xUAS-CD4::tdGFP and LexAop-CD19mch) had low levels of background GFP expression inside of the antennal lobe (arrowheads) and in the antennal mechanosensory and motor center (star in the bottom panel). (d) When CD19mch was driven by the orco-lexA driver in ORNs, it triggered induction of GFP expression in neurons whose dendrites branched throughout the whole antennal lobe. Left: GFP+ cell bodies and neurites from the antennal lobe neurons; middle: ChAT immunostaining (blue); right: merged images of CD19mch (red), CD4::tdGFP (green), and CD19mch+ axons from ORNs (red). The images of two single optical section (top and bottom panels) at different depths show ChAT immunostaining (blue), indicating that some of the GFP+ neurons (green) induced by CD19mch+ ORNs are cholinergic PNs (arrows in the top panels). Bottom panels, some of the GFP+ neurons (green) are non-cholinergic cells, suggesting that they are LNs (arrows in the bottom panels). Scale bar = 50 µm.
Figure 2—figure supplement 2. Expression of the nlgSNTG4 receptor under the SybE enhancer.
The receptor nlgSNTG4 was tagged with the V5 epitope in its C terminus. Immunostaining with an anti-V5 antibody shows that the nlgSNTG4 receptor is highly expressed in the central brain, in regions such as antennal lobe and subesophageal ganglion, but its expression was much weaker in the optic lobe (bottom left and top right corners). The expression level of the receptor protein in the homozygote SybE-nlgSNTG4 (right) is higher than in the heterozygote (middle). Notice that the expression of the receptor is enriched in the neuropil regions. Scale bar = 50 µm.
Figure 2—figure supplement 3. Expression of CD19 fused with different presynaptic proteins in ORNs.
The ligand, CD19, was fused with domains from the following pre-synaptic markers: nSyb (a), syx (b), Nrx1 (c), Sdc (d), Dip (e) and Dpr (f). The distribution of the different ligands expressed in ORN axons driven by the orco driver is shown in single optic sections (a–f) or maximum projections (a’–f’). nSyb::CD19 (a and a’) was selectively accumulated at the presynaptic terminals and could not be detected along axons, thus allowing for the visualization of individual glomeruli. The other ligands (syx (b), Nrx1 (c), Sdc (d), Dip (e) and Dpr (f)) are homogeneously distributed throughout the surface of the axons, including axon shafts, thus allowing to detect the ligand on the antennal commissure (arrowheads in b-f and b’-f’). Scale bar = 50 μm.
Figure 2—figure supplement 4. Comparison of localization of the CD19::Nrx, nSyb::CD19 and CD19::sdc ligands into presynaptic sites in the ORNs targeting DA1.
Different intracellular and transmembrane domains (from Nrx, nSyb, and sdc) were fused to CD19 and expressed into ORNs targeting the DA1 glomerulus using the CD19::Nrx (top panels), nSyb::CD19 (middle panels) and CD19::Sdc (bottom panels), with the R17H02 driver (in green). The brain samples were co-immunostained with antibodies against the OLLAS tag (present in the ligand) and against the pre-synaptic protein, BRP (in magenta). The ligand proteins are co-localized with or adjacent to BRP (arrows), demonstrating that all ligands are enriched at the presynaptic terminals of the ORNs, but CD19::Nrx was also expressed at strong levels in the axon shaft outside of the glomerulus (arrowheads in top panels). Scale bar = 20 μm.
Figure 2—figure supplement 5. Identity of the postsynaptic targets of olfactory receptor neurons in the antennal lobe detected by TRACT.
Cholinergic (ChAT+)and GABAergic (GABA+) neurons were labeled with GFP when the nSyb::CD19 ligand was expressed in most of the ORNs by the orco-lexA driver. The position of the cell bodies of the cholinergic and GABAergic GFP+ neurons was consistent with them being PNs and LNs, respectively. The images of two single optical section from two brain samples show ChAT immunostaining (top row, third column in blue) and GABA immunostaining (bottom row, third column in blue), indicating that some of the GFP+ neurons (top and bottom rows, second column in green) induced by nSyb::CD19+ ORNs (top and bottom rows, first column in red) are cholinergic (not GABAergic) PNs (arrows in (top row) and arrowhead in (bottom row)). Some GFP+ neurons on the lateral part of the antennal lobe are GABA+ and ChAT- (arrowheads in (top row) and arrows in (bottom row)), which indicates they are probably GABAergic LNs. First column: NSYB::CD19+ axons from ORNs (red); Second column: distribution of GFP+ neurons in the antennal lobe; Third column: GABA or ChAT immunostaining; Fourth column: merged images of GABA or ChAT (blue), GFP (green) and nSyb::CD19 (red). Scale bar = 20 μm.
Figure 2—figure supplement 6. TRACT with non-synaptically localized ligand reveals neurons that are not exclusively connected by synapses.
To selectively detect the connections between ORNs and PNs, the GH146 enhancer was used to drive expression of nlgSNTG4 selectively into PNs, and the ligand was expressed in subsets of ORNs that project to identified glomeruli. (a) Control brain without lexA driver (carrying GH146-nlgSNTG4, 5xUAS-CD4::tdGFP and LexAop-CD19mch) reveals low levels of background GFP expression that can be barely observed in several brain regions, including DA1 glomerulus (star in the right). (b, c, and d) Expression of the ligand CD19mch in most glomeruli with the orco driver (b), or in identified glomeruli using the R17H02 (c) and R28H10 (d) drivers. (b) Left - CD19mch+ axons from ORNs (red) driven by orco branch in the antennal lobe. Middle - Induction of CD4::tdGFP expression in PNs triggered by CD19mch+ ORNs (red, left image). Axon bundles of the CD4::tdGFP+ uniPNs in the iACT (arrows) branch in the mushroom body. (c–d) CD4::tdGFP+ PNs (middle panels) induced by CD19mch+ ORN axons (red, left images) that innervate one (VC1) to three (DA1/VA6/VA1lm) identified glomeruli (stippled circles in the left panels). Although the ligand proteins were only present in specific glomeruli (stippled circles, the expression level in VA6 was low and undetectable after immunostaining with anti-RFP antibody), strong CD4::tdGFP induction (middle panels) was detected in PNs with dendrites outside the CD19mch+ glomeruli. In particular, there are GFP+ neurites within DA1 (stars) in all the cases. The arrows indicate the axons of uniPNs, which run through the mALT. In (b–d), in addition to the uniPNs, few multiPNs were labeled as well (arrowheads). Their dendrite arborizations cover the entire antennal lobes. The right panels show the merged images of CD4::tdGFP (green) and CD19mch (red). Scale bar = 50 μm.
Figure 2—figure supplement 7. Control brains without the lexA driver to assess the levels of ligand-independent background.
(a) The confocal images show that the negative controls of Figure 2e without lexA drivers (carrying nlgSNTG4 driven by the nSybE- (top panels) or GH146 enhancers (bottom panels), 5xUAS-CD4::tdGFP and LexAop-nSyb::CD19) have no induction in antennal lobe neurons. (b) The images show that control brains of Figure 3 without LexA driver (carrying GH146-nlgSNTG4, 5xUAS-CD4::tdGFP and LexAop-nSyb::CD19 (top panels) or CD19::Sdc (bottom panels) have very low levels of ligand-independent background GFP expression in the antennal lobe (stippled circle). Scale bar = 50 μm.