Figures and data
IPC activity depends on the nutritional state and increases after glucose ingestion.
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 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. Each dot represents an individual IPC, error bars indicate median (circle) and inter-quartile range (IQR, bars). P-values from Wilcoxon rank-sum test. F) Schematic of the experimental starvation and refeeding protocol. HG: High glucose, HP: High protein, SD: Standard diet. G) Comparison of IPC spike rates in fed flies (magenta), 24 h starved flies (cyan), and flies refed on HG for different durations (green). H) Comparison of IPC spike rate in 24 h starved flies (cyan), HP (yellow) and SD (grey). Each circle represents an individual IPC, N = number of IPCs (at least 7 flies were used for each condition).
Walking activity is modulated by the nutritional state, OANs and IPCs.
A) Schematic showing the UFO setup. B) Average forward velocity (FV) of flies in different feeding states. Median and IQR are presented. p-values from Wilcoxon rank-sum test. C) Average FV of flies representing one replicate during optogenetic activation of OANs, in fed flies. Empty-GAL4 was used as control for all experiments (black). Red shading, optogenetic activation (300 s each). D) Average FV across all trials from two replicates for OAN activation: 300s before light onset, during stimulus, and after light 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 while activating OANs. Left: each stimulus trial (1-5) and Right: 300s after light offset (P1-P5). F) Average FV of flies representing one replicate during optogenetic activation of IPCs in fed flies. G) Average FV across all trials from two replicates for IPC activation in fed flies (detailed information as in D). H) Average FV of all flies while activating IPCs in fed flies (detailed information as in E). I) Average FV of flies representing one replicate during optogenetic activation of IPCs in starved flies. J) Average of FV across all trials from two replicates for IPC activation in starved flies (detailed information as in D). K) Average FV of all flies while activating IPCs in starved flies (detailed information as in K). L) and M) Average FV pooled across all activation trials (1-5) and post activation windows (P1-P5), respectively. Median and IQR are presented. p-values from Wilcoxon rank-sum test. Where no detailed p-value is stated, asterisks represent statistical significance. See also Table S1 and S2.
IPCs are not sensitive to glucose perfusion but DH44PINs are.
A) Schematic showing the experimental paradigm. IPC and DH44PIN baseline spike rates were recorded in glucose-free extracellular saline followed by recordings in glucose-rich saline. B) IPC spike rate and delta spike rate in glucose-free and glucose-rich extracellular saline, in starved flies. Spike rates were averaged within a five minute window. Delta spike rate was calculated by subtracting the baseline (Pre) from each trial for each IPC. Pre: 5-minute recording in glucose-free extracellular saline. Glucose-rich saline was allowed to perfuse for about eight minutes before analyzing IPC activity. Glu1 and Glu2: Two subsequent, 5-minute-long recordings in glucose-rich extracellular saline, starting eight minutes after onset of glucose perfusion. Each circle represents an individual IPC from a different fly, the thick line represents the grand mean of all recordings. p-values were calculated via Wilcoxon signed-rank test. C) Comparison of IPC baseline spike rate between starved, glucose-refed, and glucose-perfused flies highlighting the ‘incretin effect’. Median and IQR are indicated. D) Staining showing Drosophila brain with IPCs (magenta) and DH44Ns (green). GFP was enhanced with anti-GFP (green), brain neuropils were stained with anti-nc82 (cyan), and IPCs were labelled using a DILP2 antibody (magenta). E) Example membrane potential of a DH44PIN recorded in a fed (black) and a starved fly (orange). F) Baseline spike rate in fed (black) and starved flies (orange), each circle represents an individual DH44PIN. G) Comparison of the membrane potential of DH44PINs in fed and starved flies. Median and IQR are indicated. p-values calculated via Wilcoxon rank-sum test. H) Spike rate and delta spike rate of DH44PINs in glucose-free and glucose-rich extracellular saline, in starved flies. Thick line represents grand mean. p-value from Wilcoxon signed-rank test. I) Schematic showing the regulation of IPCs, DH44PINs and DH44Ns outside PI.
DH44Ns outside the PI inhibit IPCs and drive increases in locomotor activity
A) Immunolabelling showing DH44 expression in the brain and the VNC from broad DH44-GAL4 driver line. GFP was enhanced with anti-GFP (green), brain 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 overlapped and the median of all trials (brown 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 a sparse line which labels only DH44PINs. Plot details as in B). G) Spike density and baseline subtracted medians of individual IPCs while activating DH44PI line. Plot details as in C). H) Pre and post delta Vm of IPCs before and after optogenetic activation of the DH44PI line. Plot details as in in D). I) Average FV of 20 flies during optogenetic activation of the DH44Ns using the broad driver line (A). Empty-GAL4 was used as control for all experiments (black). Red shading shows 300s activation windows. J) Average FV across all DH44N activation trials based on two independent replications of the experiment in I. K) Average FV of all flies during each stimulus trial (1-5) and post-stimulus trial (300 s window immediately after activation seized, 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 also Table S3 and S4.
Mating state and aging affect IPC activity, and dietary restriction impairs survival in Drosophila.
A) Comparison of baseline spike rate of IPCs between virgin females, mated females, and males. B) Comparison of baseline spike rate between Drosophila of different age groups. d = days, n = number of individual IPC recordings. Each circle represents an individual IPC, error bars indicate median (circle) and interquartile range (IQR, bars). p-values from Wilcoxon rank sum test. C) Percentage survival of flies on different diets. After 24 h of starvation, flies were kept on a high glucose (HG), high protein (HP) or standard diet (SD). Data points represent sum of two replicates of 20 flies per condition, in total we used N = 40 flies per condition.
Starvation duration and OAN activation affect foraging behavior.
A) Average forward velocity of flies during different periods of starvation. N = 20 flies per condition, each circle represents an individual fly, gray circles and lines represent means. B) Upper panel: Forward velocities of five example flies displaying stopping behavior during OAN activation. Examples are shown for the fifth activation cycle. Lower panel: Average forward velocity of all flies from one replicate (N = 20). Red bar represents OAN activation.
Glucose perfusion does not affect the activity of IPCs and DH44PINs in fed flies but shifts spike activity patterns in DH44PINs.
Spike rate and delta spike rate of IPCs and DH44PINs in different conditions. A) IPCs in fed flies under 40 mM glucose perfusion. B) DH44PINs in fed flies under 40 mM glucose perfusion. Pre, five-minute recording in glucose-free extracellular saline, Glu1 and Glu2, two subsequent five-minute recordings in glucose-rich extracellular saline, starting eight minutes after onset of glucose perfusion. Each circle represents an individual fly, thick line represents the grand mean of all flies, p-values were calculated via Wilcoxon signed-rank test. Spike rates were averaged for each fly within a five-minute window, delta spike rates were calculated by subtracting the mean spike rate during the Pre window from all subsequent means. C) DH44PIN spike activity patterns change in fed and starved flies. Cumulative probability distribution of the ISI in DH44PINs and IPCs. F = fed flies, S = starved flies. Distributions were compared using the two-sample Kolgomorov-Smirnov test (DH44PI_F vs IPCs_F: p = 1.5e-216; DH44PI_F vs DH44PI_S: p = 2.5e-86; DH44PI_S vs IPCs_F: p = 7.4e-51). D) Cumulative probability distribution of the ISI in Pre, Glu 1 and Glu 2 windows reveals that DH44PIN activity became more bursty during glucose perfusion. Distributions were compared using the two-sample Kolgomorov-Smirnov test (DH44PI_pre vs DH44PI_Glu1: p = 6.5e-30; DH44PI_pre vs DH44PI_Glu2: p = 2.3e-21; DH44PI_Glu1 vs DH44PI_Glu2: p = 0.05). E) DH44PIN spike activity patterns change during glucose perfusion. Inter-spike-interval (ISI) for DH44PINs before (Pre) and during (Glu) 40 mM glucose perfusion.
Effects of differential activation of DH44Ns and DH44PINs on IPCs and behavior.
A) and B) Labeling of DH44Ns in the brain using a broad (DH44N, A) and a sparse (DH44PIN, B) GAL4 driver line and a UAS-GFPp10 reporter, respectively. GFP was enhanced with anti-GFP (green), brain neuropils were stained with anti-nc82 (magenta). C) Example recording of an IPC during optogenetic activation of the broad DH44N driver line. In this one example, we observed strong activation of the IPC (marked by asterisk) during DH44N activation. However, about ∼100 ms after activation, the IPC was inhibited similar to all other recordings (see also Fig 4B). D) and E) Number of proboscis extensions (PE) before (Pre 1), during (S1), and after the first LED pulse (Post 1), while activating DH44Ns (D) or DH44PINs (E), respectively, in freely walking flies in the UFO. PEs were counted manually. Thick, black lines represents the grand mean.
p-values values for statistical comparisons in Figure 2.
p-values were determined using the Wilcoxon rank-sum test. 1-5 represent the five activation cycles in Figure 2E, H and K. ‘Activation’ shows the p-values comparing average forward velocity pooled across activation trials (Figure 2L).
p-values values for statistical comparisons in Figure 2.
p-values were determined using Wilcoxon rank-sum test. P1-P5 represent the ‘post activation’ windows in Figure 2E, H and K. ‘Post activation’ contains the p-values comparing average forward velocity pooled across all trials (Figure 2M).
