Individual dopaminergic neurons induce unique, yet overlapping combinations of behavioural modulations, including safety learning, memory retrieval, and acute locomotion
- Leibniz Institute for Neurobiology, Germany
- Hokkaido University, Institute for Academic Innovation, Japan
- Hokkaido University, Graduate School of Science, Japan
- RWTH Aachen University, Imaging and Computer Vision, Germany
- Hokkaido University, Graduate School of Life Science, Japan
Figures
Figure 1 with 7 supplements
Whole-body expression patterns of dopaminergic neuron (DAN) driver strains.
All driver strains were crossed to an MB247-mCherry-CAAX, UAS-mIFP double effector that will show a mCherry signal (magenta) in the mushroom body and an mIFP signal (yellow) controlled by the driver strain. In addition, autofluorescence in the green range is shown in white. The insert shows a volume-restricted magnification of the CNS region. For images of each separate channel, as well as for genetic controls, see Figure 1—figure supplements 1–6. (A) MB328B, (B) SS02180, (C) SS01716, and (D) MB054B each drive expression in one to three cells per hemisphere (arrowheads) innervating the mushroom body as described previously (Eschbach et al., 2020; Weber et al., 2023). No additional cells were found in the body. (E) TH-Gal4 drives additionally to the expression in the central nervous system (CNS) (arrowheads indicate the dorso-lateral cluster 1 [DL1] cluster) also outside of the brain, most prominently in the melanin-producing cells at the segment barriers and the proventriculus (blue arrows). (F) R58E02-Gal4 drives expression in three cells per hemisphere (arrowheads) innervating the medial lobe of the mushroom body, as previously described (Rohwedder et al., 2016; Schleyer et al., 2020). Scale bars represent 250 µm for the whole body and 50 µm for the inserts.
Figure 1—figure supplement 1
Full body expression of MB328B>MB247-mCherry-CAAX, UAS-mIFP, and the genetic controls.
(A) The three channels (autofluorescence, mCherry, mIFP) for the experimental genotype (MB328B crossed to MB247-mCherry-CAAX, UAS-mIFP) that are shown as merged image in Figure 1A. (B) Driver control lacking the UAS construct. (C) Effector control crossed to yw. Note that this effector control is shared for all driver strains displayed in the following figure supplements. Next to the whole body, the insert shows a volume-restricted magnification of the CNS region. Blue arrowheads indicate dopaminergic neuron (DAN) cell bodies. Scale bars represent 250 µm for the full body and 50 µm for the inserts.
Figure 1—figure supplement 2
Full body expression of SS02180>MB247-mCherry-CAAX, UAS-mIFP, and the genetic controls.
(A) The three channels (autofluorescence, mCherry, mIFP) for the experimental genotype (SS02180 crossed to MB247-mCherry-CAAX, UAS-mIFP) that are shown as merged image in Figure 1B. (B) Driver control lacking the UAS construct. Next to the whole body, the insert shows a volume-restricted magnification of the CNS region. Blue arrowheads indicate dopaminergic neuron (DAN) cell bodies. Scale bars represent 250 µm for the full body and 50 µm for the inserts.
Figure 1—figure supplement 3
Full body expression of SS01716>MB247-mCherry-CAAX, UAS-mIFP, and the genetic controls.
(A) The three channels (autofluorescence, mCherry, mIFP) for the experimental genotype (SS01716 crossed to MB247-mCherry-CAAX, UAS-mIFP) that are shown as merged image in Figure 1C. (B) Driver control lacking the UAS construct. Next to the whole body, the insert shows a volume-restricted magnification of the CNS region. Blue arrowheads indicate dopaminergic neuron (DAN) cell bodies. Scale bars represent 250 µm for the full body and 50 µm for the inserts.
Figure 1—figure supplement 4
Full body expression of MB054B>MB247-mCherry-CAAX, UAS-mIFP, and the genetic controls.
(A) The three channels (autofluorescence, mCherry, mIFP) for the experimental genotype (MB054B crossed to MB247-mCherry-CAAX, UAS-mIFP) that are shown as merged image in Figure 1D. (B) Driver control lacking the UAS construct. Next to the whole body, the insert shows a volume-restricted magnification of the CNS region. Blue arrowheads indicate dopaminergic neuron (DAN) cell bodies. Scale bars represent 250 µm for the full body and 50 µm for the inserts.
Figure 1—figure supplement 5
Full body expression of TH-Gal4>MB247-mCherry-CAAX, UAS-mIFP, and the genetic controls.
(A) The three channels (autofluorescence, mCherry, mIFP) for the experimental genotype (TH-Gal4 crossed to MB247-mCherry-CAAX, UAS-mIFP) that are shown as merged image in Figure 1E. (B) Driver control lacking the UAS construct. Next to the whole body, the insert shows a volume-restricted magnification of the CNS region. Blue arrowheads indicate dopaminergic neuron (DAN) cell bodies of the dorso-lateral cluster 1 (DL1) cluster. Scale bars represent 250 µm for the full body and 50 µm for the inserts.
Figure 1—figure supplement 6
Full body expression of R58E02-Gal4>MB247-mCherry-CAAX, UAS-mIFP, and the genetic controls.
(A) The three channels (autofluorescence, mCherry, mIFP) for the experimental genotype (R58E02-Gal4 crossed to MB247-mCherry-CAAX, UAS-mIFP) that are shown as merged image in Figure 1F. (B) Driver control lacking the UAS construct. Next to the whole body, the insert shows a volume-restricted magnification of the CNS region. Blue arrowheads indicate dopaminergic neuron (DAN) cell bodies. Scale bars represent 250 µm for the full body and 50 µm for the inserts.
Figure 1—video 1
An animation through the data set used in Figure 1E.
TH-Gal4 was crossed to MB247-mCherry-CAAX, UAS-mIFP, and both the mCherry-CAAX (magenta) and the mIFP (yellow) signals are shown. The video starts with the larva’s head towards the front left. The second half shows the animal from top. At the end, the z-layers covered by the proventriculus and the melanin-producing cells were removed to allow the cells of the CNS to become visible. Grid spacing: 200 µm.
Figure 2 with 3 supplements
Individual dopaminergic neurons (DANs) are sufficient to establish and retrieve aversive memory.
(A) After training wild-type larvae with quinine as unconditioned stimuli (US), associative memory is observed only in the presence of the US but not in its absence. (B) When animals were trained with DAN-d1 activation as US and tested in darkness, memory was retrieved in neither genotype. When tested in the presence of blue light, negative memory scores only in the experimental genotype indicated aversive associative memory. For independent repetitions, see Figure 2—figure supplement 2A–D. (C) As in (B), but with DAN-f1 activation. For an independent repetition, see Figure 2—figure supplement 2E. (D) In the case of DAN-g1, aversive memory scores were observed in the experimental genotype, even in darkness. When tested in blue light, aversive memory scores were observed in all genotypes, but significantly stronger in the experimental genotype. (E) As in (D), but with a combination of DAN-f1 and DAN-g1. For independent repetitions, see Figure 2—figure supplement 2F and G. (F) As in (D), but with the broad TH-Gal4 pattern. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]), * below boxes indicate memory scores significantly different from zero (Wilcoxon signed-rank tests [WSR]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 2—figure supplement 1
Innate odour preferences.
(A) Groups of about 20 larvae were tested for 3 min for their odour preference, either in darkness or blue light. (B) Innate odour preferences of the genotypes used in Figure 2B, tested in darkness or blue light. No differences were found. (C) Innate odour preferences of the genotypes used in Figure 2C. The experimental genotypes displayed a higher odour preference than the controls, independent of the testing condition. Note that this cannot explain the difference in memory score between testing conditions found in Figure 2C. (D) Innate odour preferences of the genotypes used in Figure 2D. No differences were found. (E) Innate odour preferences of the genotypes used in Figure 2E. When tested in light, the experimental genotype displayed a higher odour preference than the effector. Note that this cannot explain the difference in memory score between genotypes tested in darkness as seen in Figure 2E. (F) Innate odour preferences of the genotypes used in Figure 2F. When tested in light, the experimental genotype displayed a higher odour preference than the controls. Note that this cannot explain the difference in memory score between genotypes tested in darkness as seen in Figure 2F. ns above a line indicates non-significance across the whole data set (Kruskal-Wallis test [KW]), * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 2—figure supplement 2
Replications of the experiments shown in Figure 2.
(A) Larvae were trained with odour presented either paired or unpaired with blue light activation and tested either in darkness or in blue light. (B) Larvae of the experimental genotype, expressing ChR2-XXL in DAN-d1, showed associative memory only when tested in the presence but not in the absence of the blue light. (C) Larvae of the experimental genotype showed associative memory only when trained with the standard weak light and tested in the presence of light (of the same strength). When they were trained and tested with a higher light intensity, no memories were observed. (D) Larvae of the experimental genotype showed associative memory only when raised in standard food and tested in the presence of light. When they were raised in food with all-trans-retinal added, which has been shown to increase the light sensitivity of ChR2-XXL (von Essen et al., 2011), no memories were observed. (E) Larvae expressing ChR2-XXL in DAN-f1 showed associative memory only when tested in the presence but not in the absence of the blue light. (F) Only the larvae of the experimental genotype, expressing ChR2-XXL in DAN-f1 and DAN-g1, showed associative memory independent of the presence or absence of the unconditioned stimuli (US). The genetic controls displayed no significant associative memory. (G) Larvae of the experimental genotype showed associative memory only when tested in the presence but not in the absence of the blue light. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]), * below boxes indicate memory scores significantly different from zero (Wilcoxon signed-rank tests [WSR]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 2—figure supplement 3
Dopaminergic neuron (DAN) activation with CsChrimson establishes memories that are retrieved independent of DAN activation during the test.
(A) Larvae were trained with the odour presented either paired or unpaired with blue (UAS-ChR2-XXL) or red (UAS-CsChrimson) light activation and tested either in darkness or the respective light. (B) In a replication of the experiment shown in Figure 2B, larvae displayed an associative memory only if DAN-d1 was activated also during the test (left). However, if instead of ChR2-XXL the red-shifted channel CsChrimson was used as effector, associative memory scores were observed independent of the activation of DAN-d1 during the test. (C) As in (B), but for DAN-f1. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]), * below boxes indicate memory scores significantly different from zero (Wilcoxon signed-rank tests [WSR]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 3
Individual dopaminergic neurons (DANs) are sufficient to establish safety memory.
(A) With quinine as unconditioned stimuli (US), odour preferences after paired training were more negative in the presence than in the absence of the US (left, indicating punishment memory), and after unpaired training were more positive (right, indicating safety memory). (B) Left: after paired training, the animals of the pooled genetic controls and the experimental genotype (DAN-d1>ChR2-XXL) showed the same odour preference, both when tested in darkness or light. Right: after unpaired training, the experimental genotype showed a more positive odour preference than the genetic controls only when tested in light, indicating safety memory. (C) As in (B), but with DAN-f1 activation. (D) Left: for DAN-g1, after paired training, the animals of both the controls and the experimental genotype showed lower odour preferences when tested in the presence than in the absence of light. Right: after unpaired training, animals of the experimental genotype showed higher odour preferences than the controls, irrespective of the testing condition (right). (E) For a combination of DAN-f1 and DAN-g1, the animals of the experimental genotype showed more negative odour preferences than the controls after paired training (left, indicating punishment memory) and more positive preferences after unpaired training (right, indicating safety memory), irrespective of the testing condition. (F) For the TH-Gal4-DANs, similar results as in (E) were observed. ns above a line indicates non-significance across the whole data set (Kruskal-Wallis test [KW]), * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 4 with 5 supplements
Two sets of dopaminergic neurons (DANs) induce opposite effects on locomotion.
(A) Behaviour of cohorts of experimentally naïve larvae was recorded. After 30 s of darkness, blue light was activated for 30 s. The second halves of the dark and light periods are henceforth referred to as ‘baseline’ and ‘stimulation’, respectively. (B) Based on the data from five experiments involving TH>ChR2-XXL and genetic controls within this study, the cross-correlation of 17 behavioural attributes during the baseline period revealed three clusters of inter-correlated attributes. Displayed is the Spearman’s correlation coefficient R for each pair of attributes. Values of 1 (red) indicate perfect positive, values of –1 (blue) perfect negative correlations. (C) A random forest algorithm was trained to classify individuals of the experimental genotype and either genetic control, using their change in behaviour between the baseline and stimulation. Displayed is the relative importance of each behavioural attribute, scaled from 0 (least important) to 100 (most important). (D) Average bending and tail velocity over time. The black and blue bars at the bottom indicate the light regimen. (E) The change in bending upon light stimulation. (F) As in (E), but for the tail velocity. (G) The average relative importance of each attribute cluster, as provided by the random forest. (H–L) As in (D–G), but using R58E02-Gal4 as driver. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 4—figure supplement 1
Random forest analysis of five independent repetitions of the same locomotion experiment.
(A) Summary of the experimental conditions of five independent replications of the locomotion experiment with TH-Gal4 and ChR2-XXL. For the right-most experiment, the larvae were placed on a yeast paste for 4 hr before the experiment (see Materials and methods for details). (B–D) Each column shows the relative importance based on the random forest algorithm of a given experimental replication. (B), (C), and (D) show the attributes of clusters 1, 2, and 3, respectively.
Figure 4—figure supplement 2
Light responses of genetic controls.
(A) Bending over time. Black and blue bars at the x-axis indicate the light regimen. Each line represents the average bending of one genetic control of one of this study’s experiments using strong blue light (specifically, the experiments shown in Figures 4 and 9). The curve of each control was normalised to its bending during the time period between 15 and 30 s to allow comparisons of the shape of the curves independently from the baseline behaviour. Please note that some experiments included only one cycle of light stimulations (these curves end at time point 120 s), others included two cycles. (B) As in (A), but for experiments using weak blue light (shown in Figure 5—figure supplement 1, Figure 9—figure supplement 2). (C) The highest value during the first stimulation of each curve was determined. This peak value was higher for strong than for weak light. (D) The time until the peak of the first stimulation of each curve. No differences were seen between the light intensities. (E–H) As for (A–D), but for the tail velocity. As ‘peak’ value, the lowest tail velocity of each curve during the first stimulation was used. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 4—figure supplement 3
Replication of the experiment shown in Figure 4D–G with three stimulation cycles.
(A) Average bending and tail velocity over time. Larvae of all genotypes increased bending and decreased tail velocity upon light stimulation. Both effects were enhanced in the experimental genotype. The black and blue bars at the bottom indicate the light regimen. (B) The average relative importance of all behavioural attributes within each cluster, as provided by the random forest algorithm. (C) A ∆ bending value was calculated in each individual to quantify the change in bending upon light stimulation. This change was significantly increased in the experimental genotype for each light stimulation. (D) As in (C), but only including individuals that were recorded during all three light stimulations. Differences across light stimulations are compared within individuals. The change in bending was reduced for the second and third stimulations in all genotypes. (E–G) As in (C–D), but for the tail velocity. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). In (D) and (F), # and ns above brackets indicate pairwise within-animal significance or non-significance, respectively (Wilcoxon signed-rank tests [WSR]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 4—video 1
A sample video of the experiment presented in Figure 4D-G with TH/ChR2-XXL larvae.
The video is played in double speed. Blue light (not visible in the video) was switched on at timestamp 15 s (30 s in real time) and off at timestamp 30 s (60 s in real time). Larval heads are marked in green, tails in red.
Figure 4—video 2
A sample video of the experiment presented in Figure 4H-L with R58E02/ChR2-XXL larvae.
The video is played in double speed. Blue light (not visible in the video) was switched on at timestamp 15 s (30 s in real time) and off at timestamp 30 s (60 s in real time).
Figure 5 with 3 supplements
Dopaminergic neuron (DAN) activations modulate locomotion independently of light.
(A) Average difference in bending and tail velocity of the experimental group from the control groups over time. Black and blue bars at the x-axis indicate the light regimen. Each curve represents the behaviour of the experimental group minus the pooled controls of a given experiment to indicate the effect of DAN activation, ‘purified’ from the light responses. Compared are experiments using strong blue light (dark blue, Figure 4D) and weak blue light (light blue, Figure 5—figure supplement 1A) with ChR2-XXL as effector and red light (red, Figure 5—figure supplement 2A) with CsChrimson. (B) As in (A), but for the experiments shown in Figure 4H, Figure 4—figure supplements 1E and 2E. (C) For each of the six experiments, the average ∆ bending (left) or ∆ tail velocity (right) of the pooled control groups was taken as the effect size of the light response (x-axis). The ∆ bending or ∆ tail velocity of the experimental group minus those of the pooled control groups was taken as the effect size of DAN activation (y-axis). Note that the left and right graphs use different axes and scaling. The colours match those of (A–B). All experiments using the same driver resulted in similar effect sizes of DAN activation, despite strong differences in effect sizes of the light response.
Figure 5—figure supplement 1
Dopaminergic neuron (DAN) activations modulate locomotion independently of light intensity.
Replication of the experiments and analyses of the experiments displayed in Figure 4D–L, but using weak blue light during the stimulations. (A, E) Average bending and tail velocity over time. (B, F) Change in bending upon light stimulation. (C, G) Change in tail velocity upon light stimulation. (D, H) Average relative importance of the behavioural attributes within each cluster, as provided by the random forest algorithm. For TH-Gal4, cluster 1 was less important than in the case of strong light, but the change in bending was nevertheless significant. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 5—figure supplement 2
Dopaminergic neuron (DAN) activations modulate locomotion independently of effector.
Replication of the experiments and analyses of the experiments displayed in Figure 4D–L, but using CsChrimson as effector and red light stimulation. The experimental groups were larvae expressing CsChrimson in TH-Gal4-DANs (A–D) or R58E02-Gal4-DANs (E–H), fed with all-trans-retinal. The control groups were of the same genotype in standard food without all-trans-retinal. Note that these control animals behaved comparably to an effector control (Figure 5—figure supplement 3), indicating that the DANs were not activated in the controls. (A, E) Average bending and tail velocity over time. (B, F) Change in bending upon light stimulation. (C, G) Change in tail velocity upon light stimulation. (D, H) Average relative importance of the behavioural attributes within each cluster, as provided by the random forest algorithm. Cluster 1 was less important than in the case of blue light, but the changes in bending were nevertheless significant. Strikingly, activating R58E02-Gal4-DANs decreased bending below baseline and increased velocity above baseline rather than just reverting the behaviour back to baseline. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]), * below boxes indicate memory scores significantly different from zero (Wilcoxon signed-rank tests [WSR]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 5—figure supplement 3
Comparison of controls for the experiments shown in Figure 5—figure supplement 2.
The change in (A) bending and (B) tail velocity of larvae expressing CsChrimson either under the control of TH-Gal4 or R58E02-Gal4 was compared with the heterozygous effector control. None of the genotypes were raised with all-trans retinal. Overall, the controls showed almost no reaction to the light. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 6
Dopaminergic neuron (DAN) activation during innate odour preference and learning experiments causes weak locomotion modulations.
(A) Larvae were video-recorded and their behaviour analysed during the experiment displayed in (A–C) Figure 2—figure supplement 1F (innate odour preference), (D–F) Figure 3F (paired training), and (G–I) Figure 3F (unpaired training). (A) Larvae of all genotypes and conditions approached the odour over time, with the experimental genotype (TH>ChR2-XXL) tested in light (blue) having the strongest preference (see Figure 2—figure supplement 1F). (B) The larvae of the experimental genotype had a higher average bending in blue light (blue) than in darkness (black). The driver control, too, had a higher bending in light (pink) than in darkness (dark grey) for the first third of the experiment. (C) The larvae of the experimental genotype showed a lower tail velocity in light than in darkness during the first half of the experiment. (D) After paired training, the larvae avoided the odour over time, with the experimental genotype showing the strongest aversion, especially when tested in light (see Figure 3F, left). (E) Only in the first 10 s of the experiment, the larvae of the experimental genotype showed stronger bending in light than in darkness. (F) The larvae of all genotypes and conditions had a similar tail velocity, with a small peak in the first 20 s. (G) After unpaired training, the larvae approached the odour, with the larvae of the experimental genotype having the highest preference, especially when tested in light (see Figure 3F, right). (H–I) As in (E–F), but after unpaired training. (K) No correlation between the bending of individual animals during the first 60 s of all three experiments and their odour preference in the following 60 s. (L) As in (K), but for the tail velocity. Displayed is Spearman’s correlation coefficient R as an indicator of the correlation. For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 7 with 3 supplements
Different subsets of dopaminergic neurons (DANs) convey opposite acute valence when activated.
(A) Larvae were given the choice between a dark and an illuminated (weak blue light) half of a Petri dish and their behaviour was recorded for 3 min. Animals were placed into the dark half at the dish. Displayed are two sample tracks for animals of TH>ChR2-XXL. (B) The light preference after 3 min. All genotypes showed light avoidance, which was enhanced in the experimental genotype. (C) As in (B), but for R58E02-Gal4 as driver. The experimental genotype showed less light avoidance than the controls. (D) Average bending and tail velocity of TH>ChR2-XXL and genetic controls over the X coordinate of the Petri dish, with 0 indicating the midline. The black and blue bars at the bottom indicate which half was illuminated. (E) The average bending on the illuminated side of the dish. We limited the analysis to an area up to 15 mm from the midline (indicated as stippled line in A) to capture the behaviour in the choice zone. (F) As in (E), but for the tail velocity. (G–I) As in (D–F), but for R58E02-Gal4 as driver. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 7—figure supplement 1
Independent repetition of the experiment of the experiment shown in Figure 7C with CsChrimson as effector.
(A) The light preference after 3 min. Larvae raised on food with ATR showed a preference for the light, whereas control larvae raised without ATR did not. This indicates that the larvae preferred to have the neurons covered by R58E02-Gal4 activated. (B) Average bending and tail velocity of R58E02>CsChrimson raised with or without ATR over the X coordinate of the Petri dish, with 0 indicating the midline. The black and red bars at the bottom indicate which half was illuminated. (C) The average bending on the illuminated side of the dish. We limited the analysis to an area up to 15 mm from the midline to capture the behaviour in the choice zone. (D) As in (C), but for the tail velocity. Overall, the effects on bending and tail velocity were qualitatively comparable to the experiments with ChR2-XXL, but with a reduced magnitude (please note the truncated y-axis). * above brackets indicate pairwise significance (Mann-Whitney U-tests [MW]), * below boxes indicate significant differences from zero (Wilcoxon signed-rank tests [WSR]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 7—video 1
A sample video of the experiment presented in Figure 7B with TH/ChR2-XXL larvae.
The video is played in double speed. The right half of the dish was illuminated with blue light (not visible in the video) throughout the experiment.
Figure 7—video 2
A sample video of the experiment presented in Figure 7C with R58E02/ChR2-XXL larvae.
The video is played in double speed. The right half of the dish was illuminated with blue light (not visible in the video) throughout the experiment.
Figure 8 with 1 supplement
The effects on locomotion upon dopaminergic neuron (DAN) activation are dopamine-dependent.
(A) Average difference in bending and tail velocity of the experimental groups from the control groups over time. Black and blue bars at the x-axis indicate the light regimen. (B) The animals’ average bending during the baseline period (15–30 s) in darkness. Both genotypes including the ple-dsRNA (TH-RNAi) construct displayed increased bending, suggesting a genetic background effect. (C) As for (B), but for the tail velocity. Animals expressing ple-dsRNA had an increased velocity compared to all other genotypes. (D) The change in bending upon the first light stimulation. This change was significantly increased in the experimental genotype and slightly reduced when in addition expressing ple-dsRNA. (E) As in (D), but for the tail velocity. (F) The average relative importance of all behavioural attributes within each cluster, as provided by the random forest algorithm when comparing TH>ChR2-XXL with the genetic controls. (G) The average relative importance of each attribute cluster for a comparison between animals with and without TH-RNAi. (H–O) As for (A–G) but feeding the TH inhibitor 3IY instead of inducing TH-RNAi. The changes in both bending and tail velocity upon light stimulation for animals fed the TH inhibitor 3IY were statistically different from animals without 3IY feeding and not statistically different from the genetic controls. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 8—figure supplement 1
Two independent replications of the experiments shown in Figure 8H–O.
(A, G) Average bending and tail velocity over time. The black and blue bars at the bottom indicate the light regimen. (B, H) The average relative importance of all behavioural attributes within each cluster, as provided by the random forest algorithm. (C, I) The animals’ average bending during the baseline period (15–30 s) in darkness. (D, K) As for (C,I), but for the tail velocity. Feeding 3IY reduced the tail velocity. (E, L) The change in bending upon the first light stimulation. This change was significantly decreased in animals fed with 3IY. (F, M) As in (E, L), but for the tail velocity. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 9 with 2 supplements
Individual dopaminergic neurons (DANs) are sufficient to modulate bending and tail velocity.
(A) Average bending and tail velocity over time. The black and blue bars at the bottom indicate the light regimen. Note that the second light stimulation is shown. For the full time course and a quantification of the first stimulation, see Figure 9—figure supplement 1. (B) The change in bending upon the second light stimulation was significantly increased when DAN-d1 was activated. (C) As in (B), but for the tail velocity. (D) The average relative importance of each attribute cluster, as provided by the random forest algorithm. Cluster 2 was clearly most important. (E–G) As in (A–C), but for DAN-f1. As no effects of DAN-f1 activation were seen, no random forest was applied. (H–L) As in (A–D), but for DAN-g1. (M–P) As in (A–D), but for a combination of DAN-f1 and DAN-g1. ns above a line indicates non-significance across the whole data set (Kruskal-Wallis test [KW]), * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 9—figure supplement 1
Individual dopaminergic neurons (DANs) are sufficient to modulate bending and tail velocity.
(A) Average bending and tail velocity over time. Notably, the difference between controls and experimental genotype (DAN-d1>ChR2-XXL) was more pronounced at the second stimulation. Partially, this was due to a more transient light response of the controls. The black and blue bars at the bottom indicate the light regimen. (B) The change in bending upon the first light stimulation. (C) As in (B), but for the tail velocity. (D–F) As for (A–C), but for DAN-f1. (G–I) As for (A–C) but for DAN-g1. (K–M) As for (A–C) but for a combination of DAN-f1 and DAN-g1. ns above a line indicates non-significance across the whole data set (Kruskal-Wallis test [KW]), * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 9—figure supplement 2
Activation of individual dopaminergic neurons (DANs) with weak light is not sufficient to modulate locomotion.
(A) Average bending and tail velocity over time. The black and blue bars at the bottom indicate the light regimen. (B) The change in bending upon the second light stimulation. There was no difference between the genotypes. (C) As in (B), but for the tail velocity. (D–F) As for (A–C), but for DAN-f1. (G–I) As for (A–C) but for DAN-g1. (K–M) As for (A–C) but for a combination of DAN-f1 and DAN-g1. ns above a line indicates non-significance across the whole data set (Kruskal-Wallis test [KW]), * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1.
Figure 10 with 7 supplements
Summary and working hypothesis.
(A) A table summarising the findings of this study as well as of a related recent study (Schleyer et al., 2020) for each dopaminergic neuron (DAN). (B) Aversive and appetitive memories are established via synapses between aversive US-signalling DANs (DAN-AV) and appetitive US-signalling DANs (DAN-AP) with Kenyon cells (KCs). Memories are stored in the strength of the synapses between the KCs and the respective mushroom body output neurons (MBONs), and memory retrieval is thought to be controlled via the DAN→MBON synapses. The balance of the MBON signals determines the resulting behaviour (appetitive or aversive). (C) The synapses on the DAN are organised in clusters with DAN→KC and DAN→MBON in the centre, surrounded by KC→DAN synapses. One sample cluster is schematically displayed. For more details, see Figure 10—figure supplement 1. (D–G) For clarity, only DAN-AV is shown. Thickness of arrows indicate relative strength of synapses and MBON activity, respectively. (D) Paired training with DAN-AV activation depresses the KC→MBON-AP synapses, shifting the balance towards aversion. (E) Unpaired training, in turn, potentiates the KC→MBON-AP synapses, shifting the balance towards approach. (F) DAN-AV activation may directly inhibit the MBON-AP, inducing acute aversive locomotor changes. (G) Alternatively, DAN-AV activation may acutely depress signals from the KC to the MBON-AP.
Figure 10—figure supplement 1
Synaptic organisation of the dorso-lateral cluster 1 (DL1) cluster dopaminergic neurons (DANs).
(A) Top: conditioned stimulus (CS) information is signalled towards the mushroom body Kenyon cells (KCs). DAN-d1 and most KCs on both hemispheres establish mutual synapses, and both DAN-d1 and KCs provide output to four mushroom body output neurons (MBONs). For simplicity, only synapses with the left DAN-d1 are shown. Middle: truncated dendrogram of the left DAN-d1 based on EM microscopy of stage 1 larvae (Eichler et al., 2017; Saumweber et al., 2018). The positions of all synapses with MBONs are indicated in the colours shown in the top sketch. Bottom: the positions of all synapses with KCs are indicated in purple. (B–C) As in (A), but for DAN-f1 and DAN-g1, respectively. Note that DAN-g1 only innervates the contralateral mushroom body. *: this MBON has no synapses with the respective DAN in stage 1, but innervates the compartment in stage 3; #: this MBON establishes synapses with the respective DAN in stage 1 but is not found in stage 3 (Eichler et al., 2017; Saumweber et al., 2018). (D) The synapses are organised in clusters. Displayed are six sample clusters of the left DAN-f1. (E) Distances of all synapses within each cluster from the cluster centre. For all six DANs, KC→DAN synapses are further away from the centre than the other synapse types. * and ns above brackets indicate pairwise significance or non-significance, respectively (Mann-Whitney U-tests [MW]). For the underlying source data and the results of the statistical tests, see Source data 1. For high-resolution dendrograms of both the left and right DANs, see Figure 10—figure supplements 2–7.
Figure 10—figure supplement 2
High-resolution dendrograms of the left DAN-d1.
For clarity, the synapses with Kenyon cells (KCs) and other synapses are displayed separately. (A) Only synapses with mushroom body output neurons (MBONs); (B) only synapses with KCs. All other synapses are represented with transparent grey symbols. Circles display synapses from the given dopaminergic neuron (DAN) to other neurons, triangles synapses from other neurons to that DAN. The colour code is given on the top left.
Figure 10—figure supplement 3
High-resolution dendrograms of the right DAN-d1.
For clarity, the synapses with Kenyon cells (KCs) and other synapses are displayed separately. (A) Only synapses with mushroom body output neurons (MBONs); (B) only synapses with KCs. All other synapses are represented with transparent grey symbols. Circles display synapses from the given dopaminergic neuron (DAN) to other neurons, triangles synapses from other neurons to that DAN. The colour code is given on the top left.
Figure 10—figure supplement 4
High-resolution dendrograms of the left DAN-f1.
For clarity, the synapses with Kenyon cells (KCs) and other synapses are displayed separately. (A) Only synapses with mushroom body output neurons (MBONs); (B) only synapses with KCs. All other synapses are represented with transparent grey symbols. Circles display synapses from the given dopaminergic neuron (DAN) to other neurons, triangles synapses from other neurons to that DAN. The colour code is given on the top left.
Figure 10—figure supplement 5
High-resolution dendrograms of the right DAN-f1.
For clarity, the synapses with Kenyon cells (KCs) and other synapses are displayed separately. (A) Only synapses with mushroom body output neurons (MBONs); (B) only synapses with KCs. All other synapses are represented with transparent grey symbols. Circles display synapses from the given dopaminergic neuron (DAN) to other neurons, triangles synapses from other neurons to that DAN. The colour code is given on the top left.
Figure 10—figure supplement 6
High-resolution dendrograms of the left DAN-g1.
For clarity, the synapses with Kenyon cells (KCs) and other synapses are displayed separately. (A) Only synapses with mushroom body output neurons (MBONs); (B) only synapses with KCs. All other synapses are represented with transparent grey symbols. Circles display synapses from the given dopaminergic neuron (DAN) to other neurons, triangles synapses from other neurons to that DAN. The colour code is given on the top left.
Figure 10—figure supplement 7
High-resolution dendrograms of the right DAN-g1.
For clarity, the synapses with Kenyon cells (KCs) and other synapses are displayed separately. (A) Only synapses with mushroom body output neurons (MBONs); (B) only synapses with KCs. All other synapses are represented with transparent grey symbols. Circles display synapses from the given dopaminergic neuron (DAN) to other neurons, triangles synapses from other neurons to that DAN. The colour code is given on the top left.
Tables
Table 1
A list and description of all behavioural attributes used for locomotion analysis in this study.
| Name | Unit | Description | Cluster |
|---|---|---|---|
| Bending | The bending angle (BA) is the angle between the larva’s tail vector and a vector from the middle of the larva to its head. Positive values indicate the larva bending to the left, negative values bending to the right. ‘Bending’ in this study refers to the absolute, i.e., direction-independent BA. | 1 | |
| Bending bias | The absolute of the average of all BA values of a larva, divided by the average of all absolute BA values: . The bending bias is constrained between 0 and 1, with 0 indicating a larva was bending equally to left and right, and 1 indicating a larva was only bending into one direction (no matter which direction). | 3 | |
| HV angular speed | The head vector angular speed (HVAS) is the angular speed of the head vector (HV) through the head of the larva. Positive values indicate movement of the head to the right, negative values to the left. ‘HV angular speed’ in this study refers to the absolute, i.e., direction-independent HVAS. | 1 | |
| HV angular speed bias | Calculated analogous to the bending bias. Constrained between 0 and 1, with 0 indicating a larva was moving its head equally fast to left and right, and 1 indicating a larva was moving its head only into one direction. | 3 | |
| HV angular acceleration | The absolute angular acceleration of the head vector (HV). | 1 | |
| TV angular speed | The tail vector angular speed (TVAS) is the angular speed of the tail vector (TV) through the rear half of the larva. Positive values indicate movement to the right, negative values to the left. ‘TV angular speed’ in this study refers to the absolute, i.e., direction-independent TVAS. | 1 | |
| TV angular speed bias | Calculated analogous to the bending bias. Constrained between 0 and 1, with 0 indicating a larva was moving its tail equally fast to left and right, and 1 indicating a larva was moving its tail only into one direction. | 3 | |
| TV angular acceleration | The absolute angular acceleration of the tail vector (TV). | 1 | |
| HC rate | The number of head casts (HC) per second. HCs were defined as events of an HVAS larger than 35 °/s or smaller than –35 °/s, respectively. | 1 | |
| HC rate bias | The HC rate of HCs to the left minus the HC rate of HCs to the right, divided by the total HC rate. Constrained between 0 and 1, with 0 indicating a larva had equal HC rates to left and right, and 1 indicating a larva was making HCs in only one direction. | 3 | |
| HC angle | The head cast angle (HCA) indicates how much the orientation of the animal changes upon each HC. It is defined as the difference between the BA at the end and the beginning of the HC. Positive values indicate that the larva changes its orientation to the left, negative values that it changes its orientation to the right. ‘HC angle’ in this study refers to the absolute, i.e., direction-independent HCA. | 1 | |
| HC angle bias | Calculated analogous to the bending bias. Constrained between 0 and 1, with 0 indicating a larva was making equally large HCs to left and right, and 1 indicating a larva was making HCs in only one direction. | 3 | |
| Distance travelled | The distance of the complete path a larva travels during the observation period (measured in body lengths [bl]). | 2 | |
| Midpoint speed | The speed of the larva’s midpoint, irrespective of direction. | 2 | |
| Midpoint acceleration | The absolute acceleration of the larva’s midpoint. | 2 | |
| Tail velocity | The velocity of the larva’s tail point in a forward direction, i.e., in the direction of the tail vector. | 2 | |
| Tail acceleration | The absolute acceleration of the larva’s tail in the direction of the tail vector. | 2 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | UAS-ChR2-XXL | Bloomington Stock Center | BDSC:58374 | |
| Strain, strain background (D. melanogaster) | 20xUAS-CsChrimson-mVenus | Bloomington Stock Center | BDSC:55134 | |
| Strain, strain background (D. melanogaster) | TH-Gal4 | Bloomington Stock Center | BDSC:8848 | |
| Strain, strain background (D. melanogaster)(D. melanogaster) | R58E02-Gal4 | Bloomington Stock Center | BDSC:41347 | |
| Strain, strain background (D. melanogaster) | w1118 | Bloomington Stock Center | BDSC:3605; BDSC:5905; BDSC:6326 | |
| Strain, strain background (D. melanogaster) | y[1] w* | Bloomington Stock Center | BDSC:1495 | |
| Strain, strain background (D. melanogaster) | y[1] v[1];P {TRiP.JF01813} attP2 | Bloomington Stock Center | BDSC:25796 | |
| Strain, strain background (D. melanogaster) | MB328B | Eschbach et al., 2020 | Covers DAN-d1 | |
| Strain, strain background (D. melanogaster) | MB054B | Eschbach et al., 2020 | Covers DAN-f1 and DAN-g1 | |
| Strain, strain background (D. melanogaster) | SS02180 | Eschbach et al., 2020 | Covers DAN-f1 | |
| Strain, strain background (D. melanogaster) | SS01716 | Eschbach et al., 2020 | Covers DAN-g1 | |
| Strain, strain background (D. melanogaster) | Empty-Gal4 (attP40/attP2) | Pfeiffer et al., 2010 | ||
| Strain, strain background (D. melanogaster) | y[1] w*;;MB247-mCherry-CAAX, UAS-mIFP-T2A-HO1; MB247-mCherry-CAAX, UAS-mIFP | Kobler et al., 2021 | ||
| Strain, strain background (D. melanogaster) | y[1] w*;;MB247-mCherry-CAAX | Kobler et al., 2021 | ||
| Chemical compound, drug | Sodium hypochlorite | Roth | Order # 9062.1 | |
| Chemical compound, drug | Ethyl cinnamate; ethyl 3-phenyl-2-propenoate | Sigma-Aldrich | Order # 112372-100G | |
| Software, algorithm | Imaris | Bitplane | Version 10.1 | |
| Software, algorithm | Adobe Premiere Pro 2020 | Adobe | Version 14.9.0 | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | ||
| Software, algorithm | IMBA (Individual Maggot Behaviour Analyser) | Thane et al., 2023 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/106148/elife-106148-mdarchecklist1-v1.pdf
-
Source data 1
Raw data and results of statistical tests.
This Excel file provides all raw data underlying the figures. Each tab shows the data of one figure or figure supplement, with the results of all statistical tests to the left.
- https://cdn.elifesciences.org/articles/106148/elife-106148-data1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
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Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Individual dopaminergic neurons induce unique, yet overlapping combinations of behavioural modulations, including safety learning, memory retrieval, and acute locomotion
eLife 14:RP106148.
https://doi.org/10.7554/eLife.106148.3
