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Dopamine neurons projecting to the posterior striatum reinforce avoidance of threatening stimuli

Abstract

Midbrain dopamine neurons are well known for their role in reward-based reinforcement learning. We found that the activity of dopamine axons in the posterior tail of the striatum (TS) scaled with the novelty and intensity of external stimuli, but did not encode reward value. We demonstrated that the ablation of TS-projecting dopamine neurons specifically inhibited avoidance of novel or high-intensity stimuli without affecting animals’ initial avoidance responses, suggesting a role in reinforcement rather than simply in avoidance itself. Furthermore, we found that animals avoided optogenetic activation of dopamine axons in TS during a choice task and that this stimulation could partially reinstate avoidance of a familiar object. These results suggest that TS-projecting dopamine neurons reinforce avoidance of threatening stimuli. More generally, our results indicate that there are at least two axes of reinforcement learning using dopamine in the striatum: one based on value and one based on external threat.

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Fig. 1: Signals from dopamine axons in TS scale with external stimulus intensity but not with reward volume.
Fig. 2: TS-projecting dopamine neurons are mainly localized in SNL and do not send substantial collaterals to other regions.
Fig. 3: Choice bias against stimulation of dopamine axons in TS.
Fig. 4: Ablation of TS-projecting dopamine neurons eliminates choice bias against a threatening stimulus.
Fig. 5: Ablation of TS-projecting dopamine neurons reduces avoidance without affecting initial responses to high-intensity external stimuli.
Fig. 6: Dopamine axons in TS are active as animals retreat from novel objects but not as they approach novel objects.
Fig. 7: Ablation of TS-projecting dopamine neurons reduces retreat from novel objects and stimulation partially reinstates avoidance of novel objects.
Fig. 8: Separate axes for dopamine-based reinforcement learning.

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Acknowledgements

We thank F. Morgese, M. Tripodi, and E. Ciabatti at MRC Laboratory of Molecular Biology in Cambridge, UK, for the self-inactivating rabies virus amplification kit. We thank B. Lowell at Harvard University in Boston, MA, for the Vglut2flox mouse. We thank E. Boyden at MIT in Cambridge, MA, for pAAV-FLEX-GFP, K. Deisseroth at Stanford University in Stanford, CA, for pAAV-DIO-ChR2 and pAAV-fDIO-EYFP, and V. Jayaraman, R. Kerr, D. Kim, L. Looger, and K. Svoboda from the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute for pGP-GCaMP6f and AAV-FLEX-GCaMP6m. We also appreciate C. Dulac and J. Assad for critically reading the manuscript. Finally, we thank H. Kim for help setting up the closed-loop optogenetic stimulation system. This work was supported by National Institutes of Health grants R01MH095953 (N.U.), R01MH101207 (N.U.), R01MH110404 (N.U.), Harvard Mind Brain and Behavior faculty grant (N.U.) and JSPS Overseas Research Fellowships (R.A.).

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Contributions

W.M.: conceptualization, investigation, formal analysis, writing (original draft); K.A.: methodology (novel object); R.A.: methodology (rabies virus); N.U.: conceptualization, supervision, writing (review and editing); M.W.-U.: conceptualization, supervision, writing (original draft).

Corresponding author

Correspondence to Mitsuko Watabe-Uchida.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Summary of GCaMP recording fiber implant sites.

Location of optic fibers used to collect GCaMP signals in VS (top) and TS (bottom) in head fixed (Fig. 1) and freely moving (Fig. 7) animals. Reference slices are depicted with 0.5 mm spacing, and fiber positions are plotted on the nearest reference slice. The approximate location of the tip of each 200 µm diameter fiber is indicated (green). Light emitted by axons directly below these sites could be detected.

Supplementary Figure 2 GCaMP normalization and GFP control signals.

(a) Left: schematic of strategy for expressing GCaMP in dopamine axons (green) and collecting signal using an implanted optic fiber. Middle: example raw voltage traces (green), running median (blue), and normalized “dF/F” signal (black). Right: Comparison of dF/F signals (one line per session) sorted from smallest to largest dF/F value. (b) GFP control schematic (left), example signals (middle), and comparison across sessions (right). (c) Average signals following unexpected water delivery (left) or tone delivery (right) in GFP expressing control mice.

Supplementary Figure 3 Responses in dopamine axons in TS scale with external stimulus novelty and intensity.

Left: average responses to different tone sizes in dopamine axons in TS (top). Data was plotted in bins of 15 trials to demonstrate the effect of novelty (middle) and displayed as a heat map (bottom). Right: average responses to different air puff sizes in dopamine axons in TS (top). Data was plotted in bins of 15 trials (middle) and displayed as a heat map (bottom). Note that “trial number” refers to the total number of each stimulus rather than the number of stimuli of that intensity prior to that point. * p < 0.05, 1-way ANOVA. For tone: f(8) = 3.05, p = 0.0275, 1-way ANOVA. For air puff: f(10) = 4.93, p = 0.00081, 1-way ANOVA.

Suppplementary Figure 4 Comparison of reward-omission signals in dopamine axons in VS and TS, and responses to external stimuli.

(a) Top: trial structure for classical conditioning. Odor A is 90% followed by water (green, expected water) and 10% followed by nothing (red, omitted water). Odor B (black) is 100% followed by nothing. A small number ( < 5%) of all trials consisted of unexpected water (blue). Bottom: example single trial responses to unexpected water of different sizes simultaneously recorded from VS (cyan) and TS (magenta). (b) Example recording sites in VS (top) and TS (bottom). (c) Average responses across animals recorded from dopamine axons in VS (top) and TS (bottom). (d) Average difference (mean ± s.e.m. across n = 11 animals) between responses to expected and omitted water (expected minus omitted) across animals in dopamine axons in VS (top; t = 4.98, p = 0.00076, n = 11 animals, paired t-test) and TS (bottom; t = -0.83, p = 0.43, n = 11 animals, paired t-test). TS dopamine axon responses (mean ± s.e.m. across n = 11 animals) are also shown for (e) odor: t = 6.49, p = 0.0029, paired t-test, (f) tone: t = 5.057, p = 0.0072, paired t-test, (g) light: t = 2.83, p = 0.018, paired t-test, and (h) air puff: t = 5.82, p = 0.00025, paired t-test. For statistical tests, peak signals in a 1 second window during the inter-trial interval (“Baseline”) is compared to the peak signal in the 1 second window following stimulus presentation (red). Solid dots indicate mean and transparent dots indicate each animal. * P< 0.01, two-sided t-test.

Supplementary Figure 5 Distribution of TS-projecting dopamine neurons in the midbrain.

TS-projecting dopamine neurons labelled with rabies-GFP (Fig. 4). (a) Example midbrain section (approximately Bregma −3.10) from each injected animal. Left: dopamine neurons labelled with anti-TH antibody (magenta) and TS-projecting dopamine neurons labelled with rabies-GFP (green). Right: TS-projecting dopamine neurons labelled with rabies-GFP (green) and anterior striatum projecting dopamine neurons labeled with rabies-mCherry. (b) GFP-labelled neurons (TS-projecting dopamine neurons) from all animals plotted to summarize their distribution.

Supplementary Figure 6 TS-projecting dopamine neurons in SNL respond to novel and high-intensity external stimuli.

(a) Responses to a novel odor (black, first 5 presentations) compared to all presentations of that odor in TS-projecting dopamine neurons in SNL. (b) Responses to first 5 presentations of sucrose (black) compared to all presentations of sucrose (grey). (c) Responses to a loud tone (black, also see Fig. 1d) and a quiet tone (grey, also see Fig. 1d). (d) Responses to a high concentration of sucrose (black, 10%) compared to a low concentration of sucrose (grey, 2%). Solid dots indicate mean and transparent dots indicate each animal. Transparent error bars depict s.e.m. n= 6 animals.

Supplementary Figure 7 Summary of ChR2 stimulation fiber sites.

(a) Location of optic fiber implants used to stimulate ChR2-expressing dopamine axons in VS (top) and TS (bottom) in freely moving animals (Fig. 3). Reference slices are depicted with 0.5 mm spacing, and fiber positions are plotted on the nearest reference slice. The approximate location of the tip of each 200 µm diameter fiber is indicated (green). (b) Schematic of light spread from fiber. (c) Position of fibers relative to dorsal tip of amygdala. (d) Correlation between distance away from tip of amygdala and effect size (Fig. 3).

Supplementary Figure 8 Example 6-OHDA histology.

Example slices stained with anti-TH (green) antibody to label dopamine cell bodies and axons. (a) Axons of dopamine neurons (green) in an example intact animal (top) and lesioned animal (bottom) in VS, DMS, DLS, TS, and Ce. (b) Cell bodies of dopamine neurons (green) in an example intact animal (top) and lesioned animal (bottom) in VTA, SNC, SNL, and RRF. Also shown are TH-labelled noradrenaline neurons in LC (right). (c) Average fluorescence in dopamine axon target regions in intact (blue) and lesion (red) animals (mean ± s.e.m. across n = 20 per condition, each hemisphere quantified separately). For anterior TS: t = 3.86, p = 0.00054, n = 20 hemispheres per condition, unpaired t-test. For posterior TS: t = 7.20, p = 0.00000091, n = 20 hemispheres per condition, unpaired t-test. (d) Average cell body counts per section in intact (blue) and lesion (red) animals (mean ± s.e.m. across n = 20 per condition, each hemisphere quantified separately). For SNL: t = 4.61, p = 0.00049, n = 20 hemispheres per condition, unpaired t-test. VS: ventral striatum, DMS: dorsomedial striatum, DLS: dorsolateral striatum, aTS: anterior tail of striatum, pTS: posterior tail of striatum, Ce: central amygdala, BLA: basolateral amygdala, LHb: lateral habenula, VTA: ventral tegmental area, SNC: substantia nigra pars compacta, SNL: substantia nigra pars lateralis, RRF: retrorubral field, LC: locus coeruleus. ** P < 0.005, unpaired t-test.

Supplementary Figure 9 Average velocity is not affected by 6-OHDA lesion.

(a) Left: example cumulative center of mass traces for saline (blue) and lesion (red) mice during a 10-minute session. Right: example velocity plot for saline (blue) and lesion (red) mice during one minute of free movement. (b) Average time spent moving (top) and average velocity (bottom) of saline and lesion mice (mean + s.e.m. across n = 10 animals per condition) for first ten minutes of free movement For average velocity: Saline x 6OHDA: t = -0.45, p = 0.66, n = 10 animals per condition. Solid dots indicate mean and transparent dots indicate each animal. (c) Time spend in center (top) and maximum velocity (bottom) of saline and lesion mice (mean + s.e.m. across n = 10 animals per condition) during the first ten minutes of free movement. For maximum velocity: Saline x 6OHDA: t = -0.31, p = 0.76, n = 10 animals per condition). Solid dots indicate mean and transparent dots indicate each animal. All panels in this figure use data from mice not performing any choice task or engaging in novel object exploration.

Supplementary Figure 10 Time-course of choice-bias, retreat, and TS dopamine axons signals elicited by airpuff in saline control and 6-OHDA lesion animals.

(a) Time course of choice of water + puff in saline (blue) and lesion (red) animals. Here, trial number indicates the total trial number (note that Fig. 4 shows the time course of retreats based on the water + air puff choice number). (b) All choices from saline (blue) and lesion (red) animals are plotted by trial number. (c) Left: average stay (%) in saline and 6OHDA mice when choosing between ports of equal value (i.e. water only). Stay (%) defined as the fraction of trials in which mice did not switch choice ports on the next trial. Saline x 6-OHDA: t = 0.010, p = 0.99, n = 10 animals in each group, unpaired t-test. Right: average stay (%) in saline and 6OHDA mice when choosing between water (cyan) and water + air puff (magenta). Saline: t = 3.57, p = 0.0022, n = 10 animals, paired t-test. 6-OHDA: t = -0.54, p = 0.60, n = 10 animals, paired t-test. ** P < 0.005, paired t-test. n = 10 animals for each condition. (d) Retreats from air puff plotted as a function of total trial number. (e) Peak GCaMP responses to air puff in dopamine axons in TS as a function of total trial number. In all panels, solid dots indicate mean and transparent dots indicate each animal. Asterisks indicate that several animals did not choose air puff at all within this time bin, so an average could not be defined. For all time courses in this figure, data was analyzed in bins of 5 trials.

Supplementary Figure 11 Example video clips of airpuff responses in saline control and 6-OHDA lesion animals.

(a) Example of a control (saline) animal’s air puff response in early (top) and later (bottom) trials. (b) Example of a lesion (6OHDA) animal’s air puff response in early (top) and later (bottom) trials.

Supplementary Figure 12 D1-antagonist in TS eliminates choice bias and modulates airpuff responses.

(a) Average choice bias (mean ± s.e.m. across n = 6 animals) on days with saline injection with no air puff trials (black), with saline injection with air puff on one port (blue), and with D1 antagonist injection with air puff on one port (red) in the same set of cannula-implanted mice. Saline x D1 antagonist: t = 4.44, p = 0.0068, paired t-test. Solid dots indicate mean and transparent dots indicate each animal. (b) Examples of retreat distance over time for each condition. (c) Retreat distance (mean ± s.e.m. across n = 6 animals) on trial 1 (left; Saline x Saline + puff: f = 4.67, p = 0.00088, n = 6, paired t-test; Saline x D1 + puff: f = -2.88, p = 0.016, n = 6, paired t-test) and trial 1-10 (right; Saline x Saline + puff: f = 2.51, p = 0.031, n = 6, paired t-test, Saline + puff x D1 + puff: f = 2.23, p = 0.045, n = 6, paired t-test) for each condition. Solid dots indicate mean and transparent dots indicate each animal. (d) Schematic of choice task. (e) Average velocity (left; Saline x D1: t = 1.72, p = 0.12, n = 6, paired t-test) and time spent moving (right; Saline x D1: t = 1.09, p = 0.30, n = 6, paired t-test) while mice engage in the choice task (mean + s.e.m. across n = 6 animals). Solid dots indicate mean and transparent dots indicate each animal. (f) Example center of mass traces from mice engaged in the choice task. * P < 0.01, two-tailed t-test.

Supplementary Figure 13 Example video clips of novel object investigation in saline control and 6-OHDA lesion animals.

(a) Example of a control (saline) animal’s early (top) and later (bottom) investigation of a novel object. (b) Example of a lesion (6OHDA) animal’s early (top) and later (bottom) investigation of a novel object.

Supplementary Figure 14 Responses in dopamine axons in TS are not locked to initiation of movement.

(a) Example velocity traces (grey) and GCaMP traces (green) as animals move freely within a behavior box (not performing a task or interacting with a novel object). Three 10 second example traces are shown. (b) Animal velocity was aligned to movement initiation and separated based on the minimum gain of velocity (top). Corresponding GCaMP traces aligned to movement initiation (middle). Quantification of peak GCaMP signal for movements of each size (bottom). (c) Top: bouts of novel object exploration were aligned to the nearest point of investigation. Middle: average GCaMP (green) and GFP control (grey) traces locked to retreat from novel object. Bottom: GCaMP signals (green) and GFP signals (grey) locked to retreat from novel object (average + s.e.m. across n = 6 animals).

Supplementary Figure 15 Time-course of responses to novel objects in dopamine axons in TS; removing Vglut2 from dopamine neurons does not diminish avoidance of novel objects.

(a) Responses in dopamine axons in TS plotted by bout (i.e. trial) for the first 30 interactions with a novel object. Solid dots indicate mean and transparent dots indicate each animal. (b) Control animal that does not express DAT-cre, meaning that Vglut2 is not knocked out in any neurons (black), and knock out animal expressing DAT-cre to specifically knock out Vglut2 in dopamine neurons (magenta). (c) Example center of mass traces of control (black) and knock-out (magenta) mice not performing any task or engaging in novel object interaction. Right: average velocity (Control x Vglut2 -/-: t = 1.16, p = 0.26, n = 8 animals per group, unpaired t-test). Solid dots indicate mean and transparent dots indicate each animal. (d) Example center of mass traces of control (black) and knock-out (magenta) mice interacting with a novel object. Right: average fraction of time spent near object in a session (Control x Vglut2 -/-: t = -0.31, p = 0.76, n = 8 animals per group, unpaired t-test). Solid dots indicate mean and transparent dots indicate each animal.

Supplementary Information

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Supplementary Figures 1–15

Reporting Summary

Example control mouse – early interaction with a novel object

Supplementary Video 1 . A video (60 fps) of a saline-injected control mouse interacting with a novel object. This video is taken from the first minute of the first session that the mouse interacted with the object.

6-OHDA lesion mouse – early interaction with a novel object

Supplementary Video 2 . A video (60 fps) of a 6-OHDA-injected mouse with a lesion of TS-projecting dopamine neurons interacting with a novel object. This video is taken from the first minute of the first session that the mouse interacted with the object.

Example control mouse – late interaction with a novel object

Supplementary Video 3 . A video (60 fps) of a saline-injected control mouse interacting with a novel object. This video is taken from ~ 5 min into the first session that the mouse interacted with the object.

6-OHDA lesion mouse – late interaction with a novel object

Supplementary Video 4 . A video (60 fps) of a 6-OHDA-injected mouse with a lesion of TS-projecting dopamine neurons interacting with a novel object. This video is taken from ~5 min into the first session that the mouse interacted with the object.

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Menegas, W., Akiti, K., Amo, R. et al. Dopamine neurons projecting to the posterior striatum reinforce avoidance of threatening stimuli. Nat Neurosci 21, 1421–1430 (2018). https://doi.org/10.1038/s41593-018-0222-1

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