Article Figures & Data
Figures
- Figure 1.
Pairing of odor and electric shock induces suppression to CS+ odors in the α′3 compartment. A, Left, Schematic diagram of MB showing α′β′ lobe neuropil and its five compartments from a frontal perspective. The line across α′3 indicates the plane that was selected for imaging. Middle: morphology of MB α′β′ lobe neuropil visualized using c305a-gal4 > mCD8::GFP. Right, Representative in vivo image of GCaMP6f expression in the α′3 region driven by c305a-gal4 (scale bar, 10 μm; brightness and contrast were adjusted for better visualization). B, Schematic diagram of the in vivo training setup. The fly head is glued to a thin metal plate. There is a small hole in the plate through which the head cuticle is dissected and the brain can be imaged. The electric shock pulses are delivered to the fly through an electric grid contacting the fly's legs. Odor is delivered to the fly via glass pipette (∼3 mm diameter) whose tip is close to the antenna of the fly (arrows indicate odor flow direction). C, Paired and unpaired training protocol. In paired training, 1 min of 12 pulses electric shock and odor A (CS+) were presented simultaneously followed by odor B (CS−) that was unpaired with shock. In unpaired training, 1 min of electric shock pulses was presented 3 min before odor A onset. Before training, two pulses of each odor were presented to the fly in alternating fashion for 5 s each with a 30 s interstimulus interval (Pre). After training, an identical set of odor pulses were presented to the fly (Post). D, Behavior performance of flies receiving paired and unpaired training protocols. Paired training induced robust memory performance at both 3 min and 1 h after training. However, unpaired training induced no memory at 3 min or 1 h after training. Mean ± SEM, ***p < 0.0001, ns, not significant, p ≥ 0.4976, one sample t test against a theoretical mean of “0,” n = 6. E, Pseudocolored peak responses of α′3 axon segments to CS+ and CS− before (Pre) and 3 min after (Post) paired training. F, Left top, Time course of GCaMP6f response in α′3 to CS+ (MCH) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during the 5 s odor presentation. Right, Responses to the CS− (OCT). Data are shown as mean ± SEM. ***p = 0.0003; ns, not significant, p > 0.9999; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 8. G, Left top, Time course of GCaMP6f response in α′3 to OCT (CS+) during a 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during 5 s odor presentation. Right, Responses to the CS− (MCH). Data are shown as mean ± SEM. **p = 0.0022; ns, not significant, p ≥ 0.4230; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 8. H, Top, Mean odor-evoked Pre and Post responses during 5 s odor presentation to MCH (CS+) with 6 pulses of 30 V electric shock training protocol. Bottom, Responses to the CS− (OCT). Mean ± SEM, **p = 0.0077; ns, not significant, p ≥ 0.6267; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 6.
- Figure 2.
Conditioning-induced suppression to CS+ in the α′3 compartment persists <30 min. A, Change of odor response in α′3 axon segments between Post and Pre odor stimuli at 3, 15, 30, and 45 min after paired or unpaired training with MCH as CS+ and OCT as CS−. The change of odor response within each fly was calculated as (Post-Pre)/Pre. The paired training-induced suppression of the CS+ persisted for at least 15 min and became nonsignificant at 30 and 45 min. No significant difference was observed in CS− between paired and unpaired groups at any time point. Data are shown as mean ± SEM. ***p = 0.0002, **p = 0.0047; ns, not significant, p ≥ 0.2345; Mann–Whitney U test, n = 8. B, Change of odor response in the α′3 compartment between Post and Pre at 3, 15, 30, and 45 min after paired or unpaired training with OCT as CS+ and MCH as CS−. The paired training-induced suppression of the CS+ persisted for at least 15 min and became nonsignificant at 30 and 45 min. No significant difference was observed in CS− between paired and unpaired groups at any time point. Data are shown as mean ± SEM. ***p = 0.0006, *p = 0.0379; ns, not significant, p ≥ 0.1304; Mann–Whitney U test, n = 8.
- Figure 3.
Pairing of odor and electric shock induces suppression to CS+ odors in the α′2 compartment. A, Left, Schematic diagram of MB showing α′β′ lobe neuropil and its five compartments. The line across α′2 indicates the plane that was selected for imaging. Middle: morphology of MB α′β′ lobe visualized using c305a-gal4 > mCD8::GFP. Right, Representative in vivo image of GCaMP6f expression in the α′2 region driven by c305a-gal4 (scale bar, 10 μm; brightness and contrast were adjusted for better visualization). B, Pseudocolored peak responses of α′2 to CS+ and CS− before (Pre) and 3 min after (Post) paired training. C, Left top, Time course of GCaMP6f response in α′2 to CS+ (OCT) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during 5 s odor presentation. Right, Responses to the CS− (MCH). Data are shown as mean ± SEM. **p = 0.0085; ns, not significant, p ≥ 0.3129; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 9. D, Left top, Time course of GCaMP6f response in α′2 to MCH (CS+) during a 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during 5 s odor presentation. Right, Responses to the CS− (OCT). Data are shown as mean ± SEM. *p = 0.0192; ns, not significant, p > 0.9999; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 7.
- Figure 4.
Pairing of odor and electric shock induces suppression in α′3 MBOn. A, Morphology of postsynaptic MBOn that innervates α′3 compartment visualized using MB027B split gal4 > mCD8::GFP. The line across α′3 MBOn indicates the plane that was selected for imaging. B, Left, Mean odor-evoked GCaMP6f responses in α′3 MBOn to MCH (CS+) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training with 12 pulses of 90 V electric shock. Right, Responses to OCT (CS−). Data are shown as mean ± SEM. ***p = 0.0008, **p ≤ 0.0021; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 7. C, Pseudocolored peak responses of α′3 MBOn to CS+ (MCH) and CS− (OCT) before (Pre) and 3 min after (Post) paired training with 6 pulses of 30 V electric shock. D, Left top, Time course of GCaMP6f response in α′3 MBOn to CS+ (MCH) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training with 6 pulses of 30 V electric shock. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during the 5 s odor presentation. Right, Responses to the CS− (OCT). Data are shown as mean ± SEM. ***p = 0.0009, *p ≤ 0.0119; ns, not significant, p = 0.2951; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 6. E, Left, Mean odor-evoked GCaMP6f responses in α′3 MBOn to OCT (CS+) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training with 6 pulses of 30 V electric shock. Right, Responses to MCH (CS−). Data are shown as mean ± SEM. **p = 0.0029; ns, not significant, p ≥ 0.1236; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 8. F, Change of odor response in α′3 MBOn between Post and Pre at 3, 15, and 30 min after paired or unpaired training (CS+:MCH, CS−:OCT). The paired training-induced suppression of CS+ persisted for at least 15 min and became nonsignificant at 30 min. No significant difference was observed in CS− between paired and unpaired groups at any time point. Data are shown as mean ± SEM. **p = 0.0087, *p = 0.0411; ns, not significant, p ≥ 0.3095; Mann–Whitney U test, n = 6. G, Mean odor-evoked GCaMP6f response at 30 min after training in α′3 MBOn during 5 s odor presentation with 12 pulses of 90 V electric shock (CS+:MCH, CS−:OCT). Data are shown as mean ± SEM. ns, not significant, p ≥ 0.1372; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 7. H, Blocking synaptic output of α′3 MBOn immediately after training through testing impaired 15 min memory. Flies were trained at 23°C, transferred to 30°C immediately after training for 15 min, and tested at 30°C. Data are shown as mean ± SEM. *p = 0.0186; ns, not significant, p > 0.9999; two-way ANOVA with Bonferroni post hoc tests, n = 6.
- Figure 5.
Pairing of odor and electric shock reduces ACh release in the α′3 compartment. A, Morphology of postsynaptic MBOn that innervates α′3 compartment visualized using MB027B split gal4 > mCD8::GFP. The line across α′3 MBOn indicates the plane that was selected for imaging. B, Left top, Time course of GACh (ACh sensor) response in the dendrites of α′3 MBOn to CS+ (MCH) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training with 6 pulses of 30 V electric shock. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during the 5 s odor presentation. Right, Responses to the CS− (OCT). Data are shown as mean ± SEM. *p = 0.0457; ns, not significant, p > 0.9999 for MCH in unpaired group, p = 0.3003 for OCT in paired group and p = 0.0856 for OCT in unpaired group; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 6. C, Mean odor-evoked GACh response at 30 min after training in the dendrites of α′3 MBOn during 5 s odor presentation with 6 pulses of 30 V electric shock (CS+:MCH, CS−:OCT). Data are shown as mean ± SEM. ns, not significant, p ≥ 0.6660; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 6.
- Figure 6.
Suppression in α′3 is not induced by GABAergic inhibition through Rdl GABAA receptor. A, No change in 3 min memory with 12 pulses of 90 V electric shock when Rdl (GABAA receptor) was knocked down in α′β′ MBn. Data are shown as mean ± SEM. ns, Not significant; one-way ANOVA with Tukey's post hoc tests, n = 6. B, There was significant enhancement of 3 min memory expression with 12 pulses of 30 V electric shock when Rdl was knocked down in α′β′ MBn. Data are shown as mean ± SEM. *p ≤ 0.0391; one-way ANOVA with Tukey's post hoc tests, n = 6. C, Knocking down Rdl did not impair the odor suppression to the CS+ in the α′3 compartment. Left top, Time course of GCaMP6f response in the α′3 compartment to the CS+ (MCH) during a 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during 5 s odor presentation. Right, Responses to the CS− (OCT). Data are shown as mean ± SEM. ***p = 0.0009; ns, not significant, p ≥ 0.1515; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 8. D, Control for Rdl knockdown in α′β′ MBn. Left top, Time course of GCaMP6f response in the α′3 compartment to the CS+ (MCH) during 5 s odor presentation before (Pre, blue) and 3 min after (Post, red) paired or unpaired training. Traces show the average response (±SEM) across all flies tested. Bottom, Mean odor-evoked Pre and Post responses during 5 s odor presentation. Right, Responses to the CS− (OCT). Data are shown as mean ± SEM. ***p = 0.0006; ns, not significant, p ≥ 0.5049; repeated-measures two-way ANOVA with Bonferroni post hoc tests, n = 6.
