Figures and data
Expression of neuromodulator receptors in IPCs.
A: Expression of biogenic amine and neuropeptide receptors in IPC single-nucleus transcriptomes. Novel receptors are depicted in black, and receptors previously shown to be expressed in IPCs are shown in gray. B: t-SNE plot based on unsupervised clustering of IPC transcriptomes reveals 4 clusters. C: t-SNE plots showing expression of 5-HT1A receptor and Allatostatin-A receptor 2. D: Expression of IPC markers (Ilp2, Ilp3, Ilp5 and Piezo) and select neuromodulatory receptors across different IPC clusters. Note that some receptors are expressed in all clusters while others are only expressed in a subset of clusters. E: Maximum projection of representative confocal images showing receptor-T2A-GAL4 driven mCherry expression (magenta) in IPCs (labelled using DILP2 antibody in green). Asterisks indicate IPCs whose nuclei are stained. Scale bars = 10 µm. F: Fraction of IPCs expressing select receptors based on T2A-GAL4 driven mCherry expression in IPCs. Averages are based on 5 preparations for each receptor. See Table S1 for all abbreviations.
Modulation of individual IPCs by aminergic and peptidergic neurons in patch-clamp recordings.
A: Schematic of setup for patch-clamp recordings and optogenetic activation. B: Anatomy of example driver line labeling AstANs (gray) and antibody staining against DILP2 labeling IPCs (green). C: Example responses of three IPCs to optogenetic activation of AstANs (red bar). IPCs with a membrane potential (Vm) rising above the 90th percentile of the baseline during or after stimulation were defined as ‘excited’ (magenta). IPCs with Vm falling below the 10th percentile were defined as ‘inhibited’ (blue). IPCs remaining between the thresholds were ‘unaffected’ (black). D: Normalized overall trends across all tested lines based on the area under the curve (AUC) average of all recorded cells for the respective line. E: DAN, and OAN populations primarily drove excitation or had no effect on IPCs upon activation. Spike events (dots), spike frequency (spk/s), and low-pass filtered Vm (mV) are shown, color-coded according to the clusters as before. Gray lines indicate individual IPCs, while color-coded lines show the cluster average. Fractions indicate number of IPCs per cluster. F: MS- and 5-HT-expressing neurons had inhibitory or no effects on IPCs. Details as in D. G: TKNs, AKHNs, DH31Ns, AstANs, and LKNs evoked mixed effects in IPCs upon activation. Details as in D. H: Baseline subtracted AUC values (ΔAUC) of all recorded cells (gray), and excited (magenta), unaffected (black), and inhibited (blue) clusters. P-values were calculated using the Wilcoxon signed-rank test.
Latency analysis and singly fly recordings.
A: Threshold-based latency analysis for excited (magenta) and inhibited (blue) IPCs during activation of DANs, OANs, AstANs, and LKNs, which had significant effects on the IPC activity upon activation. Activation onset at 0 s. Boxplots show median and interquartile range, dots represent individual recordings. Three outliers for AstAN between 0.4 and 0.8 not shown for clarity of inspection. B: Patch-clamp recordings of 11 IPCs subsequently patched in the same fly reveal a similar distribution of excited, inhibited, and unaffected IPCs compared to the distribution of IPCs recorded across all flies (C). C: All recordings across 22 flies upon AstAN activation. Plot from Figure 2G. D: Cluster distribution across 11 cells in one fly and in 44 cells in 22 flies.
Modulation of the IPC population by aminergic and peptidergic neurons in calcium imaging experiments.
A: Schematic of the setup for optogenetic activation during calcium imaging. B: example images of the GCaMP6m-expressing IPC cell bodies, delimited as ROIs during a time series before and during a 5 s optogenetic activation of OANs C: Normalized ΔF/F traces for ROIs in B during one activation (red shading). D: Super-imposition of two subsequent activations (thin lines) and the mean of both (thick line) from the example in B. The mean was used for further analysis steps. E: Cluster analysis based on the 20th and 80th percentile of the baseline (see Material and Methods) revealed three cluster within this one example animal. F: Heatmaps of the mean ΔF/F traces of all recorded IPCs per driver line. Traces were baseline subtracted based on the activity during a 1 s window (gray box) before the 5 s activation (red box). G: Mean ΔF/F traces (thick lines) for each cluster (color-coded as before) with respective standard deviation (shaded areas). Data correspond to the respective driver line indicated in F. H: AUC of IPCs before activation (pre) and during activation (act.; from onset to 5 s after activation) for all recorded IPCs (grey, left) and for the three clusters (colors, right). P-values were calculated using the Wilcoxon signed-rank test (see Table 4 for all p-values). I: Overall trends across all tested lines based on the AUC averaged across all IPC responses. J: Simultaneous recording of one IPC via patch-clamp (first row, purple), and the same IPC (second row, purple ΔF/F trace) as well as 12 additional IPCs (black ΔF/F traces) via calcium imaging. K: Superimposition of action potentials from J and corresponding ΔF/F traces (black traces, individual events, lavender trace, mean, gray trace, burst of spikes and respective ΔF/F trace).
Neurons containing classical transmitters provide heterogeneous input to IPCs.
A – D: Presynaptic neurons to IPCs in the FlyWire connectome contain acetylcholine (A), glutamate (B), GABA (C), or unknown transmitters (D). Scale bar = 100 µm. E: Connectivity map of presynaptic neurons containing glutamate (purple), acetylcholine (magenta), or GABA (blue) to individual IPCs (green). Connection strength is indicated with the numbers of synapses by line thickness (upper panel) and circle diameter (lower panel). Individual IPCs differ regarding their synaptic input and input strength. Note that 18 IPCs were identified in FlyWire2.
Summary of neuromodulation of IPCs.
A: Comparison of overall activity shifts for all driver lines and IPCs tested between patch-clamp (black) and calcium imaging (white). B: Comparison of cluster proportions between patch-clamp (outer circles) and calcium imaging (inner circles). C: Comparison of, expression data, G-protein coupling prediction, patch-clamp recording, and calcium imaging. Dots indicate percentage of IPCs for expression data, patch-clamp, and calcium imaging, and the confidence for G-protein prediction. For receptor expression data, black dots indicate the expression has been validated with receptor mapping. D: Simplified model of IPC modulation. Overview of receptor expression and functional connectivity in patch-clamp (blue) and calcium imaging (purple) recordings for all modulatory inputs tested. Line thickness indicates fraction of involved IPCs. Receptor expression for TK from 1. E: Simplified hypothesis of IPC population activity shifts through excitatory or inhibitory modulator input and the effects of consecutive input on insulin output.
Fly genotypes for anatomical mapping
Fly genotypes for activation experiments and expression analysis
Antibodies for anatomical receptor mapping, and brain and VNC anatomy
