Volume 25, Issue 11, Pages 1535-1541 (June 2015) A Dopamine-Modulated Neural Circuit Regulating Aversive Taste Memory in Drosophila  Pavel Masek, Kurtresha Worden, Yoshinori Aso, Gerald M. Rubin, Alex C. Keene  Current Biology  Volume 25, Issue 11, Pages 1535-1541 (June 2015) DOI: 10.1016/j.cub.2015.04.027 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Aversive Taste Conditioning Is Dependent on Neurotransmission from DANs (A) Schematic of aversive taste memory assay. A pretest of stimulation with 100 mM fructose (pink) alone is followed by three training trial triplets, where fructose is immediately followed by the application of 10 mM quinine (green) to the extended proboscis. Only fructose is provided during the test, and PER is measured. (B) A fly brain expressing CD8::GFP under control of TH-GAL4 (green) reveals broad ramifications of DANs throughout the central brain. Background staining is nc82 antibody (blue). The scale bar represents 20 μm. (C) Dopamine clusters that are labeled by TH-GAL4 are innervating vertical lobes (PPL1) and horizontal lobes (PAM) of the MBs as well as other brain neuropiles (modified from [29]). (D) Average of performance index during testing reveals TH-GAL4>UAS-ShiTS1 (pink) flies do not suppress PER after pairing fructose with quinine to the levels of control flies (n = 10–22). (E) Flies expressing ShiTS1 in TH-GAL4 neurons (TH-GAL4>UAS-ShiTS1) show a significant reduction in PER suppression as early as the second training trial when tested at the non-permissive temperature of 32°C, but not at the permissive temperature of 25°C, when TH-GAL4 neurons are active (n = 10–22). All data represent the mean ± SEM; ∗∗p < 0.01, ∗∗∗p < 0.001. Current Biology 2015 25, 1535-1541DOI: (10.1016/j.cub.2015.04.027) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 PPL1 Neurons Are Required for Aversive Taste Memory (A) Mean PER suppression for PPL1-expressing lines during testing shows impaired PER suppression in flies expressing TRPA1 and tested at 32°C (pink) compared to controls harboring split-GAL4 constructs alone (black) or tested at 25°C (cyan). n = 10–56. (B) Aversive taste memory was disrupted when four lines that express in the PPL1 cluster of DANs were silenced: MB060B (n = 10–29), MB065B (n = 12–56), MB502B (n = 10–27), and MB504B (n = 10–36) (p < 0.05). Lower panels: Innervation patterns of PPL1 neurons in MB lobes visualized with pJFRC225-5xUAS-IVS-myr::smGFP-FLAG reporter in VK00005. The scale bar represents 20 μm. (C) Specific expression of identified PPL1 DANs. Only the MB-V1 and MB-α3 clusters are present in all the identified lines. The gray scale represents subjectively determined intensity of terminals in the MB. All data represent the mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Current Biology 2015 25, 1535-1541DOI: (10.1016/j.cub.2015.04.027) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Thermogenetic Activation of PPL1 DANs Substitutes for a Bitter Punishment (A) Schematic of inducible activation of DANs. Infrared laser stimulation (yellow) is substituted for quinine stimulation to activate DANs following the application of fructose (pink). (B) Activation of four lines labeling the PPL1 cluster (black) that are required for memory formation result in significant PER suppression. Flies harboring GAL4 transgenes (red) or UAS-TRPA1 (green) alone showed no reduction (MB502B) or no significant reduction (all other lines) compared to naive response (n = 10–15). (C) Pairing laser stimulation with the presentation of fructose in flies expressing UAS-TRPA1 under the control of MB060B-GAL4 results in PER suppression during training and test (black). Pairing fructose with quinine (without laser stimulation) also leads to PER suppression to a similar level (blue). Unpaired presentation of laser and fructose (gray), where laser onset precedes fructose application, does not lead to PER suppression. Control flies harboring GAL4 transgenes (red) or UAS-TRPA1 (green) alone showed no significant suppression of PER (n = 10–15). All data represent the mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Current Biology 2015 25, 1535-1541DOI: (10.1016/j.cub.2015.04.027) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 The α2 MB Output Neurons Modulate Naive Sugar Response (A) Average PER suppression reveals impaired memory in flies expressing ShiTS1 in α2 MBONs at 32°C (pink) compared to controls harboring Split-GAL4 constructs alone (black) or tested at 25°C (cyan) (n = 10–36). (B) Three independent lines that label the α2 MBONs disrupt taste memory when used to express ShiTS1 and tested at non-permissive temperature of 32°C (red): MB050B (n = 12–17), MB080C (n = 11–36), and MB542B (n = 10–33). Lower panels: Innervation patterns of the identified MBONs lines visualized with pJFRC225-5xUAS-IVS-myr::smGFP-FLAG reporter in VK00005. The scale bar represents 20 μm. (C) Possible pathway regulating aversive taste conditioning. Identified DANs of PPL1 cluster project to regions of vertical lobes of MBs, including α2 and α3. MBONs project from the α2 region of MBs to dorsal region of the lateral horn (MB080C and MB050B) or to superior medial protocerebrum (MB542B). (D) ShiTS1 expressing in the identified output neurons (MB542B) blocks activity of these neurons at a restrictive temperature of 32°C (red), leading to increased PER to low sugar concentrations compared to measurement at permissive temperature of 25°C (blue) (n = 23–31). (E) Flies expressing TRPA1 in the same output neurons (MB542B) reduce their PER response to high concentrations of sugars upon activation of TRPA1 (red) at an elevated temperature of 32°C compared to control measured at a low temperature of 25°C (blue) (n = 34–38). All data represent the mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Current Biology 2015 25, 1535-1541DOI: (10.1016/j.cub.2015.04.027) Copyright © 2015 Elsevier Ltd Terms and Conditions