Coordinated and Compartmentalized Neuromodulation Shapes Sensory Processing in Drosophila

Abstract

Learned and adaptive behaviors rely on neural circuits that flexibly couple the same sensory input to alternative output pathways. Here, we show that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, depending on the state and experience of the fly. Using functional synaptic imaging and electrophysiology, we reveal that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, we show that the dopaminergic neurons function as an interconnected network, encoding information about both an animal's external context and internal state to coordinate synaptic plasticity throughout the mushroom body. Our data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity. PAPERCLIP.

Figure 1. Compartmentalized Architecture of the Mushroom Body

(A) Schematic of mushroom body anatomy focusing on the γ lobe. Each γ Kenyon cell (KC, blue) receives olfactory input in the calyx and projects a single axon into the γ lobe (dashed line). KCs form en passant synapses with mushroom body output neurons (MBONs, green) and receive modulatory input from dopaminergic neurons (DANs, magenta) within discrete anatomic compartments (shown for γ2–γ5). (B) A single γ KC axon photolabeled with PA-GFP projects its axon across the complete length of the lobe (dashed line). (C) Segregated dendritic innervation of MBONs is revealed by expression of GFP in pairs of MBONs in each panel using MBON-specific drivers. (D) Compartmentalized axonal projections of DANs photolabeled with PA-GFP in alternating compartments. PA-GFP is expressed under the TH and DDC promoters. (E) sytGCaMP expressed in all γ KCs with only a single KC functionally activated. Maximum intensity projection shows peak fluorescence from multiple T-series in different Z planes. Magnified view shows individual KC synaptic puncta (right). See also Figure S1.

Figure 2. DAN Network Activity Reflects Both External Sensory Stimuli and Internal Behavioral State

(A) The sytGCaMP was expressed in DANs of all γ-lobe compartments, driven by the combination of TH and DDC promoters and imaged in response to sucrose ingestion and electric shock. (B and C) Sucrose ingestion and electric shock. Schematic of stimulus (top) with representative heatmap (ΔF/F₀) and normalized intensity trace of DAN sytGCaMP response to the stimulus (B, sucrose; C, shock) below are shown. (Bottom) Stimulus-triggered averages ± SEM for DANs of each compartment are shown. (B, n = 10 traces in nine flies; C, n = 21 traces in 11 flies). Fluorescence in other lobes is masked for clarity. Black scale bar indicates 1 s throughout figures unless otherwise noted. (D) Representative normalized fluorescence traces of γ lobe DANs aligned to fly's motion (top). Dashed lines delineate start and end of a single representative bout of flailing. Cross-correlations between motion trace and activity in DANs of each compartment are shown (bottom, n = 12 traces in six flies). (E) Schematic and still image from video showing the fly in flailing (right) and quiescent (left) behavioral states (top). Representative heatmap (ΔF/F₀) of DAN activity in response to start and stop of flailing is shown (middle). Average DAN fluorescence ± SEM in each compartment aligned to the start and stop of flailing is shown (bottom, n = 14 traces in six flies). See also Figure S2, Table S1, and Movies S1 and S2.

Figure 3. Functional Communication between Compartments Coordinates DAN Network Activity

(A–E) DAN sytGCaMP activity patterns evoked by activation of P2X₂ expressed in the (A) 58E02+ DANs innervating γ4-5, (B) γ2 MBON, (C) γ3 MBON, (D) γ4 MBON, and (E) γ5 MBON. The sytGCaMP was expressed in DANs of all γ-lobe compartments using the TH and DDC promoters. Schematic of stimulus (top left), representative heatmap (bottom left, ΔF/F₀), normalized intensity trace for representative experiment shown (top right), and stimulus-triggered averages ± SEM for DANs of each compartment (bottom right) are shown. ATP stimulation is shown as pink bar (58E02, n = 8; γ2, n = 8; γ3, n = 8; γ4, n = 8; γ5, n = 12). See also Table S1.

Figure 4. Compartmentalized Ca²⁺ Domains along KC Axons In Vivo

(A) Schematic (top) and representative basal fluorescence of sytGCaMP expressed in γ KCs labeled with approximate compartmental borders (bottom) are shown. (B) Volumetric two-photon resonant imaging of odor-evoked sytGCaMP reveals asymmetric presynaptic Ca²⁺ in γ KCs in each imaging plane. (C) Maximum-intensity Z-projection of all 15 imaging planes sampled through the γ lobe in the example shown in (B) (top). Average normalized odor-evoked profile of sytGCaMP fluorescence intensity along the γ lobe (gray line, n = 21 flies) and peak intensity for each compartment (black dots, n = 21) with mean ± SEM in red (middle) are shown. Odor-evoked time courses were imaged in each compartment for representative experiment shown above (bottom, blue lines indicate 1-s odor stimulus). (D) Representative image of sytGCaMP signal in γ KCs in response to direct stimulation of KCs by acetylcholine iontophoresis into the mushroom body calyx in a brain explant (top). Normalized intensity profiles for ex vivo stimulation across a range of iontophoretic voltages (1–10 V) with average profile for each voltage in a different colored line are shown (n = 6). Stimulation-evoked time courses were imaged in each compartment for representative experiment shown above (bottom, blue lines indicate stimulation). (E) tdTomato expressed in γ4 and γ5 DANs using 58E02-LexA (top, middle). Compartmentalized KC sytGCaMP responses in the same fly shows synaptic Ca²⁺ domains have sharp boundaries that align to the border between γ3 and γ4 compartments. (F) Odor response in a sparse subset of γ KCs expressing sytGCaMP (heatmap, top) and tdTomato (grayscale, middle). Odor-evoked time courses were measured at individual synaptic boutons (bottom). All KC heatmaps in this figure represent peak fluorescence. Values marked with different lowercase letters represent significant differences (p < 0.05 by t test with correction for multiple comparisons). See also Figures S3 and S4 and Movie S3.

Figure 5. Dopaminergic Signaling Shapes the Distribution of KC Presynaptic Ca²⁺

(A) Representative odor-evoked KC sytGCaMP response before and after sucrose ingestion (bottom left). Normalized intensity profiles pre- and post-sugar ingestion and the change due to sugar feeding (post-pre) for the representative images are shown (top right). Average change in normalized intensity profile induced by sugar ingestion is shown (bottom right, n = 11 flies). (B) Schematic of γ lobe P2X₂ expression under the 58E02 promoter (top left) and representative odor-evoked responses in γ KCs expressing sytGCaMP, pre- and post-activation of 58E02+ DANs with ATP (bottom left). Normalized intensity profiles and change due to DAN activation for the representative images are shown (top right). Average change in normalized intensity profile induced by DAN activation is shown (bottom right, n = 10 flies). (C) As in (B), but all are shown in control flies lacking P2X₂ expression (n = 6 flies). (D) Representative odor-evoked response of γ KCs expressing sytGCaMP in DopR2 mutant and wild-type flies (top). Fluorescence in other lobes is masked for clarity. Average normalized odor-evoked profile across the γ lobe and compartmental averages (bottom) in flies mutant for DopR2 (red, n = 8) and wild-type (black, n = 8) are shown. (E) As in (D), but this compares γ KC-specific knockdown of DopR2 using RNAi (red, n = 14) to wild-type flies (black, n = 5). All KC heatmaps in this figure represent peak fluorescence to odor stimulation (Experimental Procedures). Error bars in all panels are SEM. Significant differences in relative compartment intensity compared to wild-type are indicated as follows: *p < 0.05, **p < 0.005, and ***p < 0.0005. See also Figure S5.

Figure 6. DANs Selectively Potentiate KC-MBON Synaptic Transmission in Individual Compartments

(A) Schematic of experimental setup. Synaptic currents were measured in the γ4 MBON (green) by voltage-clamp recordings in response to direct KC stimulation by acetylcholine iontophoresis in the calyx (Stim). P2X₂-expressing 58E02+ DANs (magenta) were activated by local ATP injection (left). Representative γ4 MBON recordings (center) show overlay of ten KC stimulations pre- (grayscale) and post-(redscale) activation of 58E02+ DANs by ATP injection. Note the potentiation evident in both spontaneous and evoked EPSCs. Vertical line denotes 2-ms KC stimulation. Amplitude of evoked currents in the γ4 MBON pre- and post-ATP injection is shown (right, average of ten stimulations each in n = 5 recordings). (B–E) MBON responses to KC stimulation are potentiated by activation of DANs within the same compartment, but not in other compartments. Schematic (top), time courses (bottom left), and quantification of responses to KC stimulation (bottom right) before and after ATP injection were recorded in (B) the γ4 MBON with activation of the γ4-γ5 (58E02+) DANs (n = 6), (C) the γ2 MBON with activation of the γ2 DAN (n = 6), (D) the γ2 MBON with activation of the γ4-5 DANs (n = 6), and (E) the γ4 MBON with activation of the γ2 DAN (n = 6). All pairwise comparisons plot mean ± SEM. Significance of change after activation is indicated as follows: *p < 0.05 and **p < 0.005. See also Figure S6.

Figure 7. State-Dependent and Bidirectional Modulation of KC-MBON Signaling

(A) Schematic shows pairs of MBONs expressing soluble GCaMP6s used for functional imaging in (B) and (C). (B and C) Representative heatmaps of evoked fluorescence (top left in each panel, ΔF/F₀), time courses (bottom left), and scatterplots (right) of responses to odor stimuli (blue line) in pairs of MBONs in vivo (B, n = 8 for γ2 versus γ4, n = 11 for γ3 versus γ5) and evoked by calycal stimulation in a brain explant (C, n = 8 for each pair). Values marked with different lowercase letters represent significant differences (p < 0.05 by t test with correction for multiple comparisons). (D and E) MBON olfactory responses are potentiated by activation of DANs within the same compartment, but not in other compartments. (D) Schematic (left) and quantification of γ4 MBON odor responses before and after stimulation of the γ4-5 (58E02+) DANs are shown (n = 6, right). (E) As in (D), but γ2 MBON response with activation of the γ4-5 DANs was quantified (n = 6). (F) Ratio between odor-evoked responses in the γ4 MBON and γ2 MBON before and after sugar feeding is shown (n = 10). (G) Schematic (left) and experimental design (right) for (H)–(J). The γ4 MBON responses to direct KC stimulation (in H–I) or odor stimuli (in J) were recorded before and after 58E02+ DAN activation that was either temporally paired or unpaired with KC stimulation. Dashed lines here and below represent >45-s delays. (H) Representative γ4 MBON GCaMP responses to KC stimulation showing bidirectional modulation of KC-MBON signaling by activation of 58E02+ DANs depending upon whether DAN and KC activation were temporally paired or unpaired. Blue lines indicate time of KC stimulation. (I) Changes in γ4 MBON responses to KC stimulation following activation of 58E02+ DANs that was either paired (left, n = 6, starting from a potentiated state) or unpaired (right, n = 12, starting from a depressed state) with KC stimulation are shown (see Figure S7 and Supplemental Experimental Procedures). (J) Change in γ4 MBON response to an odor that was paired with 58E02+ DAN activation using P2X₂ relative to a second odor that was unpaired is shown (n = 10). All pairwise comparisons in this figure represent the mean (±SEM) with significant changes indicated as follows: *p < 0.05, **p < 0.005, and ***p < 0.0005. See also Figure S7.