Long-range projection neurons in the taste circuit of Drosophila
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Identification of taste projection neurons.
(A) Projection pattern of bitter (red) and sugar (green) GRNs into the SEZ (top) and VNC (bottom). For A-D, magenta indicates neuropil; arrows indicate cell bodies; and scale bar, 50 μm. (B) Full …
Figure 1—figure supplement 1
Detailed anatomy of TPNs.
(A) Single cell example of TPN1 (R30A08-Gal4 stochastically labels a single TPN1 neuron). TPN1 cell bodies are on the ventral surface of the third neuromere in the VNC. Each TPN1 cell (arrow) sends …
Figure 2 with 1 supplement
TPNs are in close proximity to gustatory projections.
(A) Double labeling of TPNs (green) with sugar, bitter, pheromone, or water GRNs (magenta). Shown are the projections of TPN1 and TPN2 in the first leg ganglia of the VNC and the projections of TPN3 …
Figure 2—figure supplement 1
Single plane examples of TPNs and gustatory projections.
Single plane images of TPNs (green) and sugar or bitter GRNs (magenta). Single planes (4.6 µm optical sections) are from the same specimens shown in Figure 2A.
Figure 3 with 1 supplement
TPNs respond to taste compounds.
(A) Schematic brains showing anatomy of TPNs (green) and approximate region of calcium imaging (dashed box). (B) UAS-cd8-tdTomato signal used to locate axonal arbors of each neuron type. (C) △F …
Figure 3—figure supplement 1
Calcium Imaging of TPNs with ectopic activation of GRNs.
Calcium responses in each TPN class were observed using GCaMP5 while a heat-activated channel (LexAop-dTRPA1) was expressed in either sugar GRNs (Gr5a-LexA) or bitter GRNs (Gr66a-LexA). In all …
Figure 4
TPNs arborize near olfactory projection neurons.
(A) Co-labeling of TPN2 (green) and mushroom bodies (magenta) shows no overlap. (B) Co-labeling of TPN2 (green) and olfactory projection neurons (magenta) shows overlap in lateral horn (LH). Blue …
Figure 5 with 1 supplement
Taste projection neurons influence proboscis extension.
(A) PER to activation with CsChrimson, in absence of a taste stimulus. Activation of sugar GRNs (Gr64f), TPN1, and TPN2 increase PER, compared to no retinal controls. For Gal4 control and …
Figure 5—figure supplement 1
Genetic controls for PER experiments.
(A) Genetic controls for Figure 5a: PER responses to red light. No significant effect on PER was observed. Fisher’s exact tests. n = 51–55. (B) Genetic controls for Figure 5b: PER rate to …
Figure 6 with 1 supplement
Taste projection neurons are essential for conditioned taste aversion.
(A) Schematic of aversive taste memory protocol experiments with flies expressing Shits in TPN1, TPN2 or TPN3. Flies were initially presented with 500 mM sucrose (pre-test) and only flies that …
Figure 6—figure supplement 1
Memory experiment controls.
(A–B) Shibire and CsChrimson aversive memory experiments for second TPN3 Gal4 line (C220) shows qualitatively similar results to first Gal4 line (Figure 6D and F). Error bars are 95% CI; Fisher’s …
Figure 7
TPN2 carries the CS signal, while TPN3 carries the US signal.
A and B. Aversive taste memory is impaired upon conditional silencing of TPN2 during either the pairing (A) or the testing/retrieval (B) phases, suggesting TPN2 carries the CS signal. (C) …
Figure 8 with 1 supplement
TPNs act upstream of mushroom bodies to influence learned behaviors.
(A) Schematic for imaging calcium responses of PPL1-MV1 neurons to CsChrimson activation of TPN2 and TPN3. (B) Example single-animal light-triggered averages of PPL1-MV1 calcium responses to red …
Figure 8—figure supplement 1
Controls for PPL1 imaging experiments.
(A–B) Imaging calcium responses of PPL1-MV neurons to CsChrimson activation of second TPN3 Gal4 line (C220) shows qualitatively similar results to first Gal4 line (Figure 8B and D). n = 9–11. Error …
Figure 9
TPNs action on all PPL1 lobes.
(A) Schematic specifying PPL1 region focused on for calcium imaging, with CsChrimson activation of TPN2 or TPN3. (B) Average response (black line) and individual traces (gray, blue, and red lines) …
Figure 10
TPN3 is not the only relay for bitter information to mushroom bodies .
(A and B) Schematic of imaging and nerve-cut experiment. Bitter GRNs are activated via CsChrimson and calcium responses of TPN3 and the PPL1-MV1 cluster are imaged. Dashed boxes indicate approximate …
Tables
Table 1
Genotypes of Experimental Flies
| Figure | Description | Genotype |
|---|---|---|
| 1A | GRN anatomy | Gr66a-Gal4/CyO; UAS-cd8-tdTomato/Gr64fLexA, LexAop-cd2-GFP |
| 1B | TPN1 anatomy | UAS-GFP/+; R30A08-Gal4/+ |
| 1C | TPN2 anatomy | UAS-GFP/+; VT57358-Gal4/+ |
| 1D | TPN3 anatomy | tub>Gal80>/X; UAS-GFP/+; C220-Gal4/hsFLP, MKRS |
| 1 – fs1A | TPN1 single | UAS-GFP/+; R30A08-Gal4/+ |
| 1 - fs1B | TPN1 dendrite/axon | +/CyO; R30A08-Gal4/UAS-DenMark, UAS-synaptotagmin-eGFP |
| 1 - fs1C & D | TPN2 mosaics | tub>Gal80>/X; UAS-GFP/+; VT57358-Gal4/ hsFLP, MKRS |
| 1 - fs1E | TPN2 dendrite/axon | +/CyO; VT57358-Gal4/UAS-DenMark, UAS-synaptotagmin-eGFP |
| 1 - fs1F | TPN3 – 2nd Gal4 | UAS-GFP/+; C220-Gal4/+ |
| 1 - fs1G | TPN3 – 1st Gal4 | UAS-GFP/+; R11H09-Gal4/+ |
| 1 - fs1H | TPN3 – 2 cells only | UAS-GFP/UAS-RedStinger; C220-Gal4/R11H09-Gal4 |
| 1 - fs1I | TPN3 dendrite/axon | +/CyO; C220-Gal4/UAS-DenMark, UAS-synaptotagmin-eGFP |
| 2A & 2 – fs1 | Sugar GRN doubles | UAS-cd8tdTomato/CyO; R30A08-Gal4 / Gr64fLexA, LexAop-cd2-GFP UAS-cd8tdTomato/CyO; VT57358-Gal4 / Gr64fLexA, LexAop-cd2-GFP UAS-cd8tdTomato/CyO; C220-Gal4 / Gr64fLexA, LexAop-cd2-GFP |
| 2A & 2 – fs1 | Bitter GRN doubles | UAS-cd8tdTomato/Gr66a-LexA; R30A08-Gal4 / LexAop-cd2-GFP UAS-cd8tdTomato/Gr66a-LexA; VT57358-Gal4 / LexAop-cd2-GFP UAS-cd8tdTomato/Gr66a-LexA; C220-Gal4 / LexAop-cd2-GFP |
| 2A | Pheromone GRN doubles | UAS-cd8tdTomato/ppk23-LexA; R30A08-Gal4 / LexAop-cd2-GFP UAS-cd8tdTomato/ppk23-LexA; VT57358-Gal4 / LexAop-cd2-GFP UAS-cd8tdTomato/ppk23-LexA; C220-Gal4 / LexAop-cd2-GFP |
| 2A | Water GRN doubles | UAS-cd8tdTomato/ppk28-LexA, LexAop-cd2-GFP; R30A08-Gal4 / TM2 or 6b UAS-cd8tdTomato/ppk28-LexA, LexAop-cd2-GFP; VT57358-Gal4 / TM2 or 6b UAS-cd8tdTomato/ppk28-LexA, LexAop-cd2-GFP; C220-Gal4 / TM2 or 6b |
| 2B | Sugar GRN GRASP | Gr5a-LexA/X; UAS-cd8tdTomato/LexAop-CD4::spGFP11; R30A08-Gal4 or VT57358-Gal4 or C220-Gal4/UAS-CD4::spGFP1-10 |
| 2B | Bitter GRN GRASP | Gr66a-LexA/X; UAS-cd8tdTomato/LexAop-CD4::spGFP11; R30A08-Gal4 or VT57358-Gal4 or C220-Gal4/UAS-CD4::spGFP1-10 |
| 3 A-F top | TPN1 GCaMP | UAS-cd8tdTomato/UAS-GCaMP6s; R30A08-Gal4/UAS-GCaMP6s |
| 3 A-F middle | TPN2 GCaMP | UAS-cd8tdTomato/UAS-GCaMP6s; VT57358-Gal4/UAS-GCaMP6s |
| 3 A-E bottom and G | TPN3 GCaMP | UAS-cd8tdTomato/UAS-GCaMP6s; R11H09-Gal4/UAS-GCaMP6s |
| 3 – fs1 A | TPN1-GCaMP sugar-TRP | Gr5a-LexA/X; UAS-GCaMP5/UAS-cd8tdTomato; R30A08-Gal4/LexAop-dTRP |
| TPN1-GCaMP bitter-TRP | Gr66a-LexA/X; UAS-GCaMP5/UAS-cd8tdTomato; R30A08-Gal4 /LexAop-dTRP | |
| 3 – fs1 B | TPN2-GCaMP sugar-TRP | Gr5a-LexA/X; UAS-GCaMP5/UAS-cd8tdTomato; VT57358-Gal4 /LexAop-dTRP |
| TPN2-GCaMP bitter-TRP | Gr66a-LexA/X; UAS-GCaMP5/UAS-cd8tdTomato; VT57358-Gal4 /LexAop-dTRP | |
| 3 – fs1 C | TPN3-GCaMP sugar-TRP | Gr5a-LexA/X; UAS-GCaMP5/UAS-cd8tdTomato; C220-Gal4/LexAop-dTRP |
| TPN3-GCaMP bitter-TRP | Gr66a-LexA/X; UAS-GCaMP5/UAS-cd8tdTomato; C220-Gal4/LexAop-dTRP | |
| 4A | TPN2 & MB | MB-dsRed/UAS-GFP; VT57358-Gal4/ TM2 or 6b |
| 4F | TPN3 & MB | MB-dsRed/UAS-GFP; R11H09-Gal4/TM2 or 6b |
| 4 B-E | TPN2 & olfactory PN | GH146-QUAS, QUAS-mtdTomato / UAS-GFP; VT57358-Gal4/+ |
| 4 G-J | TPN3 & olfactory PN | GH146-QUAS, QUAS-mtdTomato / UAS-GFP; R11H09-Gal4/+ |
| 5 A & B | PER: noGal4 | UAS-CsChRimson/X; +; + |
| 5 A & B | PER: GR Gal4s | UAS-CsChRimson/X; Gr64f-Gal4/+; TM2 or TM6b/+ UAS-CsChRimson/X; Gr66a-Gal4/+; TM2 or TM6b/+ |
| 5 A & B | PER: TPN Gal4s | UAS-CsChRimson/X; +; R30A08-Gal4/+ UAS-CsChRimson/X; +; VT57358-Gal4/+ UAS-CsChRimson/X; +; R11H09-Gal4/+ |
| 5 C & D | Shi PER behavior | + / UAS- Shibire(ts) R30A08-Gal4/UAS-Shibire(ts) VT57358-Gal4/UAS-Shibire(ts) R30A08-Gal4, VT57358-Gal4/UAS-Shibire(ts) R11H09-Gal4/UAS-Shibire(ts) C220-Gal4/UAS-Shibire(ts) |
| 5 – fs1A & B | Gr-Gal4s | Gr64f-Gal4/+; TM2 or TM6b/+ Gr66a-Gal4/+; TM2 or TM6b/+ |
| 5 – fs1A & B | TPN-Gal4s | R30A08-Gal4/+ VT57358-Gal4/+ R11H09-Gal4/+ C220-Gal4/+ |
| 5 – fs1 C & D | TPN-Gal4s (controls for PER with Shi) | R30A08-Gal4/+ VT57358-Gal4/+ R11H09-Gal4/+ C220-Gal4/+ |
| 6B | Memory – TPN1 Shi | R30A08-Gal4/UAS-Shibire(ts) |
| 6C | Memory – TPN2 Shi | VT57358-Gal4/UAS-Shibire(ts) |
| 6D | Memory – TPN3 Shi | R11H09-Gal4/UAS-Shibire(ts) |
| 6F | Memory – TPN3 Chrimson | UAS-CsChRimson/X; +; R11H09-Gal4/+ |
| 6 – fs1A | TPN3 – 2nd Gal4 | X; +; C220-Gal4/UAS-Shibire(ts) |
| 6 – fs1B | TPN3 – 2nd Gal4 | UAS-CsChRimson/X; +; C220-Gal4/+ |
| 7 A & B | Memory – TPN2 Shi | VT57358-Gal4/UAS-Shibire(ts) |
| 7 C & D | Memory – TPN3 Shi | R11H09-Gal4/UAS-Shibire(ts) |
| 8 & 9 | PPL1 - control | UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/TM3-ser |
| 8 & 9 | PPL1 – TPN2 | UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/ VT57358-Gal4 |
| 8 & 9 | PPL1 – TPN3 | UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/ R11H09-Gal4 |
| 8 – fs1A | PPL1 imaging | UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/C220-Gal4 |
| 8 – fs1B | PPL1 imaging | UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/TM3-ser UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/C220-Gal4 |
| 8 – fs1C-E | PPL1 ex vivo imaging | UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/ VT57358-Gal4 UAS-CsChRimson/X; LexAop-GCaMP6s; TH-LexA/ R11H09-Gal4 |
| 10 | Gr66a-LexA, LexAop-CsChrimson / UAS-CD8-tdTomato; UAS-GCaMP6s / MB065B-Gal4 AD; R11H09-Gal4, UAS-GCaMP6s / MB065B-Gal4 DBD |
