1. Volume 28, Issue 6, Pages 847-858.e3 (March 2018)
Identification of a Single Pair of Interneurons for Bitter Taste Processing in the Drosophila Brain 
Ali Asgar Bohra, Benjamin R. Kallman, Heinrich Reichert, K. VijayRaghavan 
Current Biology 
Volume 28, Issue 6, Pages e3 (March 2018)
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2. Figure 1
VGN 6341 Labels Neurons Involved in Inhibition of Appetitive Behavior
(A) Schematic for behavioral screening by activating neurons in selected GAL4 lines.
(B) Quantification of PER phenotypes upon activation of VGN 6341 GAL4 neurons and stimulation with 10% sucrose.
(C) Quantification of PER phenotypes upon activation of VGN 6341 GAL4 neurons and stimulation with water.
∗∗∗p < 0.001; Student’s t test was used for all statistical comparisons. For each bar, n = 4 trials of 15 flies each. n.s., not significant. See also Table S1 and Movie S1.
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3. Figure 2
VGN 6341 Labels Neurons Involved in the Bitter Taste Neural Pathway
(A) Quantification of PER phenotypes upon inactivation of VGN 6341 GAL4 neurons and stimulation with 10% sucrose. ts, temperature sensitive.
(B) Quantification of PER phenotypes upon silencing of VGN 6341 GAL4 neurons and stimulation with 10% sucrose + 1 mM quinine mixture.
(C) Quantification of PER phenotypes upon silencing of VGN 6341 GAL4 neurons and stimulation with 10% sucrose + 100 mM caffeine.
∗∗∗p < 0.001; Student’s t test was used for all statistical comparisons. For each bar, n = 3–4 trials of 10–15 flies each.
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4. Figure 3 Expression Pattern of VGN 6341 GAL4 in the CNS
(A) VGN 6341-GAL4-driven expression of UAS-mCD8::GFP in the whole brain. The SEZ is delineated by dotted lines.
(B) Zoomed-in image of the SEZ as delineated in (A), showing a medial cluster of labeled cells.
(C) Zoomed-in image of the SEZ as delineated in (A), showing a lateral cluster of labeled cells of the SEZ.
In (B) and (C), co-labeling showing VGN6341-labeled neurons and motor neurons labeled by dye-filling from the proboscis is shown; arrows indicate motor neurons.
(D) Schematic showing the cell body position of neurons labeled by VGN 6341 GAL4 in the SEZ region of the brain. Interneurons are indicated in green, while red indicates neurons.
(E and E’) Shown here: (E) visualization of presynaptic terminal using VGN 6341 Gal4/+; UAS syt-HA/+ flies shows the projection of VGN 6341 neurons in the whole brain and (E’) zoomed-in SEZ.
(F) VGN 6341-GAL4-driven expression of UAS-mCD8::GFP in ventral nerve cord shows expression in thoracic and abdominal ganglia.
(G) Motor neurons innervating proboscis muscles 4 and 5.
(H) Sagittal view of proboscis showing motor neuron innervation of proboscis muscles 6, 7, and 8 labeled by VGN 6341 GAL4. Schematic showing sagittal view of proboscis muscles is adapted from [33]. Motor neurons are numbered according to the muscles they innervate. Nomenclature is according to [34].
Scale bars, 20 μm. See also Figure S1.
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5. Figure 4
Interneurons Labeled by VGN 6341 in Central Brain Are Responsible for Inhibition of Appetitive PER
(A) Expression of mCD8::GFP driven by VGN 6341 GAL4; tsh-GAL80 in central brain remains unchanged.
(B) Tsh-GAL80 removes the labeling of neurons by VGN-6341-GAL4 in ventral nerve cord.
(C) Quantification of PER phenotypes upon activation of VGN 6341 GAL4 neurons in background of tsh-Gal80 and stimulation with 10% sucrose. Left side, control without VGN 6341 Gal4 driver; right side, experimental with VGN 6341 Gal4 driver.
(D) Expression of mCD8::GFP driven by VGN 6341 GAL4;Cha-GAL80 in central brain does not remove labeling of motor neurons in SEZ and labeling of interneurons in pars intercerebralis.
(E and E’) Cha-GAL80 does not remove labeling of motor neurons via VGN 6341 GAL4 driver; hence, labeled motor neuron innervation to proboscis muscles remains unchanged. Motor neurons are numbered according to the muscles they innervate. Nomenclature is according to [34].
(F) Quantification of PER phenotype upon activation of VGN 6341 GAL4 neurons in the presence of Cha-Gal80 and stimulation with 10% sucrose. Left side, control without VGN 6341 Gal4 driver; right side, experimental with VGN 6341 Gal4 driver.
∗∗∗p < For each bar, n = 3 trials of 10 flies each. Scale bar, 20 μm.
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6. Figure 5 VGN 6341 Neurons Synapse with Bitter Sensory Neurons
(A) Axonal termini of the bitter sensory neurons in SEZ marked by flies of genotype Gr66a LexA; LexAop mCD8GFP.
(B) Expression pattern of VGN 6341-labeled neurons in SEZ, showing dendritic arbors of neurons in proximity of axonal termini of bitter sensory neurons.
(C) Image of the SEZ stained with anti-GFP of control flies with genotype VGN 6341>LexAop-CD4::spGFP11; UAS-CD4::SpGFP1-10.
(D) Representative image of the SEZ showing the GRASP signal in flies with the genotype Gr66a LexA/LexA-op-CD4::SpGFP11;VGN 6341/UAS-CD4::SpGFP1-10 (n = 10).
Scale bars, 20 μm. See also Figure S2.
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7. Figure 6
Clonal Analysis Reveals a Pair of Interneurons in SEZ Region Sufficient to Mediate Inhibition of PER
(A–C) Representative images of GFP+ clones of pair of interneurons (arrowheads) in the SEZ.
(A’–C’) Higher magnifications of the same interneurons mediating inhibition of proboscis extension response. Clones are derived from hsflp, UAS-mCD8::GFP;Tub > Gal80 >, UAS-TrpA1;VGN 6341 flies. n = 41.
(D–F) Flies showing that normal proboscis extension response does not contain the interneurons mediating inhibition of PER. n = 86.
(G) Schematic of SEZ showing approximate position of bGLNs.
(H) Single-cell clone of the bGLNs eliciting inhibition of appetitive PER; GFP (green) and nc82 (red); superposition of all labeled optical section revealing the overall extent of the cell’s arbors in the SEZ.
(I) Fewer optical sections of the labeled single-cell clone of the bGLNs, revealing the short axon-like process of the cell.
Scale bars, 20 μm. See also Figure S3.
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8. Figure 7 VGN 6341 bGLNs Are Activated by Bitter Tastants
(A–A”) Color-coded images and mean fluorescence change of GCaMP in VGN 6341 bGLNs in flies of the genotype VGN 6341>GCaMP6s.
(A) Pre-stimulus change in GCaMP fluorescence.
(A’) Post-stimulus showing change in fluorescence (ΔF/F) to bitter stimulant.
(A”) Representative trace showing fluorescence changes of VGN 6341 bGLNs in response to stimulation with bitter tastant; time of stimulation marked by an arrow. Schematic shows approximate region of calcium imaging (rectangular box).
(B–B”) Color-coded images and mean fluorescence changes of GCaMP in VGN 6341 bGLNs in flies of the genotypes VGN 6341 > GCaMP6s and Gr66a > dTrpA1.
(B) Image taken before heat activation of Gr66a+ neurons by IR laser; (B’) following heat activation by IR laser showing change in fluorescence (ΔF/F).
(B”) Representative traces showing fluorescence changes of VGN 6341 bGLNs in response to heat activation of bitter sensory neurons. Time of IR laser activation is marked by an arrow. Fluorescence intensity measurements were taken from areas within circles: region of interest (white) and background (red). Schematic shows approximate region of calcium imaging (rectangular box).
(C) Maximum fluorescence changes of GCaMP in VGN 6341 bGLNs upon stimulation of the proboscis with bitter and sugar tastants (n = 6–8 flies for each tastants). ∗∗p < 0.01.
(D) Schematic of CaMPARI experimental setup.
(E) Flies of the genotype VGN6341 > UAS CaMPARI show no conversion of green to red upon stimulation with 10% sucrose in the medial SEZ cluster where bGLNs present.
(F) Bitter stimulation (1 mM quinine and 100 mM caffeine mixture) converts green to red fluorescence in bGLNs in the medial SEZ cluster (arrowheads and dashed circles).
In (E) and (F), the left panels show neurons labeled by VGN6341 before stimulation, the middle panels show neurons activated upon stimulation, and the right panels show an overlay of the two.
Scale bar, 20 μm. See also Figure S4 and Movies S2 and S3.
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