Figure 2. KC and DAn PPL1 neurons axoaxonic reciprocal synapses are functional.

(A) Cyclic AMP responses in DAn (α3, α’3, α2α’2 and γ2α’1) to artificial stimulation of KC using ATP/P2X₂ system. The solid line of each trace represents the mean and shaded area represents ± SEM at various concentrations of ATP used in the experiment. Cyclic AMP accumulation was observed in the axons of the DAn that innervate all of the MB lobe compartments that were recorded. N = 9–14. (B) Calcium responses in DAn (α3, α’3, α2α’2 and γ2α’1) to artificial stimulation of KC using ATP/P2X₂ system. The solid line of each trace represents mean and shaded area represents ± SEM. Calcium responses were observed in the axons of the DAn that innervate all of the MB lobe compartments that were recorded. N = 12–16. (C) Diagram of the experimental setup. A micropipette was used to focally apply ACh to the calyx of the MB while imaging in both the calyx and MB lobes. (D) Calcium responses in both calyx and lobes of the KC to ACh application in the presence (red) or absence (black) of 1 µM TTX. TTX blocked the responses in the lobes but not the calyx, indicating that TTX was functional in blocking action potentials. N = 7–10. (E) Diagram of the experimental setup. A micropipette was used to focally apply 5 mM ATP to the α2α2’ compartment of MB while recording calcium responses in α2α’2 DAn. (F) Calcium responses in α2α’2 DAn in the presence (red) or absence (gray) of 1 µM TTX. TTX was without significant effect, indicating that the artificial activation of KC fibers were capable of evoking responses in DAn axon terminals through local, mono-synaptic transmission. N = 9–11. Data were analyzed using Mann-Whitney U non-parametric test. Bars represent the means ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.23789.005

Figure 2—figure supplement 1. Testing the efficacy of the ATP/P2X₂ system.

(A) Diagram of the experimental setup. Flies co-expressing the P2X₂ channel and GCaMP3 in DAn using TH-gal4 were stimulated by bath application of increasing concentrations of ATP. (B) Calcium responses obtained by stimulation with different concentrations of ATP and measured in all compartments shaded green in panel A. N = 12. The solid lines represent the means and the shaded area represents ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.23789.006

Figure 2—figure supplement 2. DAn are presynaptic to KC.

(A) Diagram of experiment setup. DAn were stimulated with increasing concentrations of ATP while recording cAMP responses in KC. (B) Cyclic AMP dose-response curve after artificial stimulation of DAn using the ATP/P2X₂ system. N = 8. The solid lines represent the means and shaded areas represent ± SEM. Responses were recorded in all compartments shaded red in panel A.

DOI: http://dx.doi.org/10.7554/eLife.23789.007

Figure 2—figure supplement 3. KC and PPL1 DAn axoaxonic reciprocal synapses are functional.

(A) Calcium responses in DAn (α3, α’3, α2α’2 and γ2α’1) to artificial stimulation of KC using the ATP/P2X₂ system along with genetic controls. The uas-alone control (blue bars) showed significant responses to P2X₂ stimulation, suggesting leaky expression of the channel. Bars represent the mean ± SEM at 2.5 mM ATP. Calcium responses were observed in the axons of the DAn that innervate all MB lobe compartments that were recorded. N = 7–12. Data were analyzed using Kruskal-Wallis one-way ANOVA and Dunn’s multiple comparison test. Bars represent the means ± SEM. (B) Testing the efficacy of the light/Chrimson system. Left panel shows a diagram of the experimental setup. Flies co-expressing the Chrimson channel and GCaMP6f in KC using R13F02-gal4 were stimulated by red light and calcium responses recorded. N = 12. The solid lines represent the means with the shaded area ± SEM. (C) Calcium responses in DAn (α3, α’3, α2α’2 and γ2α’1) to artificial stimulation of KC using the light/Chrimson system. The solid line of each trace represents the mean and shaded area represents ± SEM. Calcium responses were observed in the axons of the DAn that innervate all MB lobe compartments that were recorded. N = 6–9. (D) Calcium responses in α2α’2 DAn in the presence (red) or absence (gray) of 1 µM TTX. TTX was without significant effect, indicating that the artificial activation of KC fibers were capable of evoking responses in DAn axon terminals through local, monosynaptic transmission. N = 14. Data were analyzed using a Mann-Whitney U non-parametric test. Bars represent the means ± SEM. (E) Connections between KC and DAn are cholinergic. Calcium responses in α2α’2 DAn to stimulation of KC using the light/Chrimson system were strongly inhibited in presence of nAChR antagonist mecamylamide (500 µM) (left). Vehicle was without effect (right). N = 16–18. Data were analyzed using a Mann-Whitney U non-parametric test. Bars represent the means for calcium response ± SEM.

DOI: http://dx.doi.org/10.7554/eLife.23789.008