Wall-following behaviour (WAFO) and total walking activity (TOWA) in the OFT upon exposure to multiple stressors.

A) Typical examples of walking trajectories in the glass-covered arena before experiencing multiple stressors (red), directly after exposure to multiple stressors (blue, one day of social isolation, starvation and sleep deprivation) and after restoration (green, 1 day on food together with other flies of mixed sex and without shaking) as automatically tracked by Ctrax. A’) WAFO and TOWA values increase significantly after experiencing multiple stressors and decrease back to pre-stress levels after restoration. A’’) The median values of the data in A) were normalised to the median pre-stress value (red dot) and plotted as fold change compared to the pre-stress levels as the fold change of - WAFO on the X-axis, and fold change in TOWA on the Y-axis.

Wall-following behaviour (WAFO) and total walking activity (TOWA) with different stressors.

Raw data are shown in X), while X’) shows the median after normalisation to the pre-treatment condition, plotted as fold changes. A-A’) Effect of social isolation. A) Flies were tested in the OFT, then socially isolated and re-tested on day two and four, and then regrouped and tested again. Both WAFO and TOWA are strongly affected by social isolation. A’) With increasing social isolation, flies walk increasingly more and closer to the wall. After restoration of social contact, data points shifted back in direction to pre-stress levels. B-B’) Effect of starvation. B) Flies were tested in the OFT, then starved and tested after one and two days in starvation and then retested again after one day on food. Both WAFO and TOWA are strongly affected by starvation. B’) Starvation for one and two days increasingly leads to higher TOWA and WAFO levels. After re-feeding, levels shift back towards pre-starvation levels. C-C’) Effect of sleep deprivation. C) Flies were tested in the OFT, then mechanically sleep deprived and tested in the middle of the light phase (ZT7) and at the beginning of the dark phase (ZT13) of day 1 during sleep interruption. Following a day of mechanically undisturbed sleep, flies were then retested. WAFO but not TOWA was strongly affected by sleep deprivation. C’) Sleep deprivation for the first half or the whole photophase lead to increased WAFO but had negligible effect on TOWA. After restoration of sleep, WAFO levels approached pre-stress levels with strongly increased locomotor activity. D-D’) Effect of mating rejection on unmated males. D) Unmated male flies were tested in the OFT and then paired with mated females. After being mating-rejected from these females for an hour each on day 1-2, male flies increasingly shifted towards higher TOWA and WAFO levels. After restoration by a successful mating on day 3, a further increase in TOWA but a partial decrease in WAFO was observed. D’) After being rejected by females on day 1 and 2, males become increasingly active and show increased WAFO compared to unmated males. After restoration of courtship, the males showed a further increase in locomotor activity and a decrease in WAFO levels towards the pre-stress condition. E-E’) Effect of sexual deprivation on mated males. E) Male flies were mated at day 1 and afterwards tested in the OFT, then sexually deprived and tested on day 2 and 3 of sexual deprivation. E’) This treatment led to a strong and increasing shift towards higher TOWA levels, while WAFO did not change significantly. After restoration by a successful mating on day 3, TOWA levels dropped back and WAFO increased compared to 2 days of sexual deprivation. F-F’) Effect of age. F) Different groups of male flies were tested in the OFT at an age of either 2-3, 5-6 and 10-11 days. F’)This treatment led to a strong and increasing shift towards lower WAFO levels, while TOWA did not change significantly.

Aversive stimulation and pharmaceutical treatment consistently alter open field behaviour.

Raw data are shown in X), while X’) shows the median after normalisation to the pre-treatment condition, plotted as fold changes. A-A’) Effect of mechanical shocks (tapping). B-B’) Effect of a 10 min heat-shock of 37°C during the OFT and C-C’) an electric foot shock (EFS) prior to the OFT increased TOWA and WAFO compared to pre-stimulus conditions. D-D’) The effect of an EFS given on two consecutive days confirmed the increase of WAFO and TOWA after the first shock, while the second shock had a reduced effect on WAFO and no effect on TOWA. E-E’) Effect of 10% ethanol. Feeding on 10% ethanol in sucrose induced a mild increase in WAFO compared to sucrose alone, while TOWA was weakly reduced. F-F’) Effect of 5 mM diazepam. Feeding on 5 mM diazepam plus sucrose induced a stronger increase in WAFO compared to sucrose alone, while TOWA was unaffected.

Genetic manipulation of aminergic and peptidergic signalling affects open field behaviour.

A) Flies with temperature-induced (29 °C, red) conditionally reduced serotonin transporter expression (nSyb tubGal80ts x SerT RNAi) or UAS-mediated SerT overexpression (nSyb tubGal80ts x SerT) showed significantly reduced WAFO compared to flies at the non-inducing condition (18 °C, green), while WAFO was unaffected by temperature in the genetic controls. B) Similar experiment as in A) but after an electric foot shock (EFS) showed a reduced but still significant decrease in WAFO after manipulation of SerT expression. C) Thermogenetic suppression of dopaminergic reward neurons of the PAM cluster significantly increased WAFO compared to their temperature control, while thermogenetic activation of the same PAM neurons decreased WAFO value compared to their temperature control. Genetic controls were not significantly affected by temperature. D) Thermogenetic activation of reward-mediating NPF-positive neurons significantly decreased WAFO compared to temperature controls, while thermogenetic silencing had no effect. Temperature did not significantly affect WAFO in the genetic controls.

Comparison of WAFO and TOWA and spontaneous activity between naïve laboratory (OrR, CS, CS110) and wild-caught Drosophila melanogaster wild-type strains.

A) Heat map for WAFO values obtained in 6h intervals during the day. WAFO appeared to be much lower in the inbred lab strains (CS, OrR) compared to an outcrossed lab strain (CS110) and wild-caught strains. B) Heat map for TOWA values obtained in 6h intervals during the day. TOWA appeared to be much lower in the inbred lab strains compared to an outcrossed lab strain (CS110) and wild-caught strains. C) Heat map of spontaneous locomotor activity as measured in the TriKinetics DAM system, obtained in 6h intervals during the day. Spontaneous locomotor activity appeared lower in the inbred lab strains compared to an outcrossed lab strain (CS110) and wild-caught strains. . All strains show higher activity during the morning (ZT0) and evening (ZT12), in line with a circadian morning and evening peak in locomotor activity. D) Scatter plot of the WAFO and TOWA values obtained in the laboratory strains (CS, CS110, OrR) and the wild-caught strains. Very low WAFO values were only observed in the laboratory strains. E) The inbred laboratory strains CS and OrR but not the outcrossed CS110 strain showed a weak linear relationship and positive correlation between TOWA and WAFO. F) In the wild-caught strains, WAFO and TOWA were not (Finland, Munich) or only weakly (Hubland) positively correlated. G) Spontaneous locomotor activity in the DAM monitors was generally higher in the wild-caught strains than in the laboratory-derived strains. H) The spontaneous locomotor activity in the DAM monitors was only weakly correlated with the TOWA in the OFT. I) The spontaneous locomotor activity in the DAM monitors was only weakly and non-monotonously correlated with WAFO in the OFT.

TOWA but not WAFO depends on sex and mating status.

A) Sex and mating status does not influence wall following in naïve CantonS flies. A’) Mating has opposite effects on locomotor activity in the OFT between the sexes. While mated females show significantly reduced TOWA compared to virgins, mated males are significantly more active than unmated males. The active and inactive states between the sexes show similar activity levels. B) Mated females show a WAFO response only after repeated mechanical shock (tapping), while TOWA is increased directly after the first mechanical stimulus (B’).

Summary of the results from all treatments plotted analogously to the circumplex model of valence and arousal.

The median values from all treatments were normalized to the median control value: for post-stress, the pre-stress median served as the control, and for post-restoration, the post-stress median served as the control. These normalized values were then plotted as fold changes relative to the control state, with the fold change in -WAFO represented on the X-axis and the fold change in TOWA represented on the Y-axis. It is important to note that the ratio of the positive and negative axes on the XY plot is not consistent.

Open field behaviour of CantonS flies in circular arenas with a diameter of 1.0, 2.2 or 5.8 cm.

A) Mean TOWA per minute in naïve flies was quite constant during the first 10 min of recording. B-B’’) Statistics for the TOWA data in A) for the arena diameter of B) 1 cm, B’) 2.2 cm and B’’) 5.8 cm. Although there was a trend towards higher activity in the first minute in the 1 cm arena, the difference is not significant.C) Accumulated TOWA during the 10 min in B-B ’) did not reveal statistically significant differences between the arena sizes. D-D’’) WAFO and TOWA responses to mechanical stress (shake by tapping) in the arenas with different diameters. In all arena sizes, flies showed significantly increased WAFO after tapping. TOWA was similarly increased, yet the increase is not statisticially significant for the 2.2 cm arena. E-E’) WAFO and TOWA in 10 min bins did not vary significantly during a 60 min recording of naïve untreated flies. F-F’) The same set of naïve flies were tested once per day in the OFT on three consecutive days. WAFO and TOWA was similar on all three days, suggesting that flies do not habituate to the situation.

Open field behaviour of CantonS flies after mechanical stress (shake) in the 1cm arena.

A) Mean WAFO directly after mechanical stimulus (tapping, orange dots post-stress), 30 min or 60 min later. It is visible that the stress-induced WAFO is only temporary and has significantly declined to the level of naïve flies after 30 and 60 min. A’) Mean TOWA directly after mechanical shake (orange dots, post-stress), 30 min or 60 min later. It is visible that the stress-induced increase in TOWA is only temporary and has significantly declined to the level of naïve flies after 30 min which remains constant until 60 min post-stress.

Effects of genetic manipulation of biogenic amine and neuropeptide signalling on open field behaviour.

A) Flies with temperature-induced (29 °C, red) conditionally reduced serotonin transporter expression (nSyb tubGal80ts x SerT RNAi) or UAS-mediated SerT overexpression (nSyb tubGal80ts x SerT) showed no change in TOWA compared to flies at the non-inducing condition (18 °C, green). B) Thermogenetic suppression of dopaminergic reward neurons of the PAM cluster by a temperature-sensitive shibire allele did not alter TOWA compared to the controls at 18°C. Thermogenetic activation of the same PAM neurons by TRPA1 decreased TOWA compared to the temperature controls. C) Flies with reduced serotonin transporter expression (nSyb tubGal80ts x SerT RNAi) cultured at 18°C or 29°C before receiving EFS (green) and after receiving EFS (red) showed a significant difference in WAFO. Flies with UAS-mediated SerT overexpression (nSyb tubGal80ts x SerT) showed a trend of increased WAFO after receiving EFS. D) Flies with reduced serotonin transporter expression (nSyb tubGal80ts x SerT RNAi) and flies overexpressing serotonin transporter (nSyb tubGal80ts x SerT) cultured at 18°C and 29°C all showed a significant increased TOWA after receiving EFS (red) compared to the state before receiving EFS (green). E) WAFO and E’) TOWA in flies with conditionally blocked neuropeptide production (svrPG33 mutants) compared to controls (FM7). Blocked neuropeptide production for 1-3 days resulted in decreased WAFO (E), while TOWA remained unaffected (E’). F)Thermogenetic suppression and activation of NPF-positive neurons had no effect on TOWA compared to the respective temperature controls.

Statistics for Figure 5A-B.

The data for the different ZT times were analysed by a Kruskal-Wallis test. If significant differences were found, Dunn’s multiple comparison test was used for pairwise comparison between ZT times.