IPC activity is modulated by the nutritional state and increases in response to feeding on nutritive sugars.

A) Schematic of the setup for in vivo IPC whole cell patch-clamp recordings. B) IPCs in the Drosophila brain. UAS-myr-GFP was expressed under a Dilp2-GAL4 driver to label all 14 IPCs. The GFP signal was enhanced with anti-GFP (green), brain neuropils were stained with anti-nc82 (magenta). C) Representative examples of the membrane potential of two IPCs recorded in fed (magenta) and starved (cyan) flies. D) Average baseline spike rate and E) membrane potential of IPCs in fed (magenta) and starved (cyan) flies. F) Schematic of the experimental starvation and refeeding protocol. HG: High glucose, HG+SD: High glucose with standard diet, HF: High fructose, HA+SD: High arabinose with standard diet, SD: Standard diet, HP: High protein. G) Comparison of IPC spike rates in fed flies (magenta), increasingly starved flies (cyan), and flies refed on HG for different durations (green). Right axis shows fraction of active IPCs (number of IPCs with spike rate > 0 Hz, gray lines and circles). H) Comparison of IPC spike rate in flies refed on different diets. Reference lines represent median IPC activity in flies refed with HG for 18-24 h (green), and in flies starved for 24 h (cyan). Each circle represents an individual IPC, N = number of IPCs (see table S5 for number of flies), error bars indicate median (circle) and interquartile range (IQR, bars). For D), E) and G), p-values are reported from Wilcoxon rank-sum test. For H), a Kruskal-Wallis test followed by post hoc Wilcoxon rank-sum tests were used for pairwise comparisons. p-values are reported after Holm-Bonferroni correction.

Walking activity is modulated by the nutritional state, IPCs and OANs.

A) Schematic showing the UFO setup. B) Average forward velocity (FV) of flies in different feeding states. Median and IQR are shown. p-values from Wilcoxon rank-sum test. C) Average FV of flies representing one replicate during optogenetic activation of IPCs in fed flies (magenta). Empty split-GAL4 was used as control for all experiments (black). Red shading, optogenetic activation (300 s). D) Average FV across all trials from two replicates for IPC activation in fed flies: 300 s before stimulus onset, during stimulus, and after stimulus offset. N = number of flies, n = number of activation trials. Thin lines represent individual trials, thick lines represent median of all trials. E) Average FV of all flies during each stimulus trial (1-5) and poststimulus trial (300 s window immediately after activation ceased, P1-P5) for IPC activation in fed flies. F) Average FV of flies representing one replicate during optogenetic activation of IPCs in starved flies (cyan). G) Average FV across all trials from two replicates for IPC activation in starved flies (plot details as in D). H) Average FV during individual trials for IPC activation in staved flies (plot details as in E). I) Average FV of flies representing one replicate during optogenetic activation of OANs in fed flies (lilac). J) Average of FV across all trials from two replicates for OAN activation in fed flies (plot details as in D). K) Average FV during individual trials for OAN activation in fed flies (plot details as in E). L) and M) Average FV pooled across all stimulus (1-5) and post-stimulus trials (P1-P5), respectively. Median and IQR are shown. p-values are reported from Wilcoxon rank-sum test. Where no detailed p-value is stated, asterisks represent a significant difference. See also tables S1 and S2.

IPCs are not sensitive to glucose perfusion but DH44PINs are.

A) IPC spike rate and delta spike before and after perfusing extracellular saline with high glucose concentration, in starved flies. Baseline spike rates were recorded in glucose-free saline, followed by recordings in glucose-rich saline. Spike rates were averaged within a 5 min window. Delta spike rate was calculated by subtracting the baseline (Pre) from each trial for each IPC. Pre: 5 min recording in glucose-free saline. Glucose-rich saline was allowed to perfuse for about 8 min before analyzing IPC activity. Glu1 and Glu2: Two subsequent, 5 min long recordings in glucose-rich saline, starting eight minutes after onset of saline perfusion. Each circle represents an individual IPC from a different fly, the thick line represents the grand mean of all recordings. p-values are reported from Wilcoxon signed-rank test. B) Same experiment as in A), but with perfusion of trehalose-rich saline after recording the baseline. Note that our glucose-free saline was also devoid of trehalose (see methods). Other details same as A). C) Comparison of IPC spike rate between starved, glucose-refed, glucose-perfused and trehalose-perfused flies, highlighting the ‘incretin effect’. Median and IQR are indicated. D) Staining showing Drosophila brain with IPCs (magenta) and DH44Ns (green). UAS-myr-GFP was expressed under a DH44-GAL4 driver to label DH44 neurons. GFP was enhanced with anti-GFP (green), brain neuropils were stained with anti-nc82 (cyan), and IPCs were labelled using a Dilp2 antibody (magenta). White arrow indicates Dilp2 and DH44-GAL4 positive neuron. The other white regions in the image result from an overlap in z-projections between the two channels, rather than from antibody colocalization. E) Example membrane potential of a DH44PIN recorded in a fed (black) and a starved fly (orange). F) Baseline DH44PIN spike rate in fed (black) and starved flies (orange). Each circle represents an individual neuron (see table S6 for number of flies). Median and IQR are indicated. p-values are reported from Wilcoxon rank-sum test. G) Spike rate and delta spike rate of DH44PINs before and during glucose perfusion in starved flies. Plot details same as A). H) Schematic showing the location of cell bodies (left), and the regulation of IPCs, DH44PINs and DH44Ns outside the PI.

DH44Ns outside the PI inhibit IPCs and drive increases in locomotor activity

A) Immunolabelling showing DH44 expression in the brain and the VNC in the broad DH44-GAL4 driver line. GFP was enhanced with anti-GFP (green), brain and VNC neuropils were stained with anti-nc82 (magenta). B) Example recording of an IPC during optogenetic activation of the DH44Ns (red shading). Upper panel shows individual trials, lower panel shows ten trials overlaid and the median of all trials (purple trace). C) Upper panel shows the spike density of individual IPCs across 10 DH44N activations. Lower panel shows the baseline-subtracted, median filtered Vm traces for each IPC. Thick lines represent the grand mean. D) Effect of DH44N activation on IPCs. Delta Vm is plotted by calculating the median baseline subtracted Vm from C) 500 ms before (Pre) and 200 ms after DH44N activation (Post). Each circle represents one IPC recording. p-values from Wilcoxon signed-rank test. E) Immunolabelling showing GFP expression in the brain and the VNC in the sparse DH44PI-GAL4 driver line. F) Example recording of an IPC during optogenetic activation of DH44 neurons using the sparse DH44PIN driver line. Plot details as in B). G) Spike density and baseline subtracted median Vm of individual IPCs during activation of the DH44PIN driver line. Plot details as in C). H) Pre and post delta Vm of IPCs before and after optogenetic activation of the DH44PIN line. Plot details as in in D). See table S5 for number of flies. I) Average FV of 20 flies during optogenetic activation of the DH44Ns using the broad driver line (see A). Empty split-GAL4 was used as a control for all experiments (black). Red shading indicates 300s optogenetic activation windows. J) Average FV across all DH44N activation trials based on two independent replications of the experiment in I. Note that the peak in average FV lies within the first frame of the stimulation window. K) Average FV of all flies during each stimulus trial (1-5) and post-stimulus trial (300 s window immediately after activation ceased, P1-P5). Circles and bars show median and IQR, respectively. Asterisks represent a significant difference according to a Wilcoxon rank-sum test. L-N) Behavioral effects of optogenetic DH44PIN activation (see E). Plot details as in I, J and K, respectively. See Table S3 and S4 for p-values.